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DNA loop domain organization in nucleoids from cells of different types
Abstract
The loop domain organization of chromatin plays an important role in transcription regulation and thus may be assumed to vary in cells of different types. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) for nucleoids obtained from human lymphocytes, lymphoblasts and glioblastoma T98G cells. The results confirm our previous observation that there are three parts of DNA in nucleoids: DNA on the nucleoid surface, loops up to ∼150 kb inside the nucleoid, and larger loops that cannot migrate. However, the relative amounts of the three parts were found to be very different for different cell types. The distributions of the loop length up to 150 kb were shown to be exponential, with the distribution parameter, the loop density, to be dependent on the cell type.
... Analysis of the electrophoretic track (comet tail) formation allows us to evaluate a number of parameters of the loop organization in the cell nuclei. In particular, it enables to determine the amount of DNA in the loops localized in different regions of the nucleus (inside or on the surface) as well as the contour length and the linear density of these loops [12][13][14]. It was shown that the loop organization in activated lymphocytes and malignant lymphoid cells is similar. ...
... The comet assay was performed as described earlier [11][12][13]. Briefly, cell suspension in Hanks' salt solution was mixed with 1% low-melting point agarose and an aliquote of the mixture was used to prepare a microscope slide covered with 1% high-melting point agarose. ...
... The dependences of the relative amount of DNA in the tail f on the contour length l m of the longest loops in the tail were fitted with the equation [13]: ...
Background:
The loop domain organization of chromatin, which plays an important role in transcription regulation, may depend on the cell functional state. The aim of this work was to investigate DNA loop reorganization upon functional transitions in two cell lines ‒ glioblastoma multiforme T98G and glioblastoma astrocytoma U373.
Materials and methods:
Single cell gel electrophoresis (the comet assay) was used to analyze the kinetics of the DNA loop migration from the nucleoids obtained from the lysed cells.
Results:
The DNA fraction in the surface loops and the size of these loops were found to be similar in two glioblastoma cell lines. When synthetic processes were inhibited, the migration of a small portion of inner loops was observed in T98G but not U373 cells. In T98G cells, stimulation of cell proliferation and transcription was accompanied by an increase in DNA fraction in the inner loops and an essential increase in the size of these loops. The effect of stimulation was practically absent in U373 cells. However, the linear density of the loops resolved by the comet assay was found to decrease upon stimulation of proliferation in both cell lines.
Conclusion:
A decrease in the loop density appears to be associated with an intensification of the synthetic processes in cells upon their stimulation.
... The approach is based on an analysis of the electrophoretic track (the comet tail) formation in single cell gel electrophoresis (the comet assay). Measuring the kinetics of DNA exit during the co met assay we have shown that DNA loops in the nucleoids (cells lysed in the presence of detergents and high salt) correspond to chromatin loops in living cells [19][20][21]. We have shown also that the kinetic behavior of the two parameters, the relative amount of DNA in the comet tail and the tail length, depend on the cell type and/or the cell functional state [20,22,23]. ...
... Measuring the kinetics of DNA exit during the co met assay we have shown that DNA loops in the nucleoids (cells lysed in the presence of detergents and high salt) correspond to chromatin loops in living cells [19][20][21]. We have shown also that the kinetic behavior of the two parameters, the relative amount of DNA in the comet tail and the tail length, depend on the cell type and/or the cell functional state [20,22,23]. ...
... The comet assay was performed as described earlier [19,20,25]. Briefly, neurons were embedded in the 0.67 % agarose gel on the surface of a microscope slide. ...
... In other words, large-scale features of the loop organization are preserved in nucleoids obtained after cell lysis [20,21]. Our analysis of the two parameters that change during electrophoresis, the relative amount of DNA in the comet tail and the tail length, makes it possible to obtain two quantitative characteristics: (i) to divide all the loops into three subsets -loops on the nucleoid surface, inner loops inside the nucleoid, and very large loops (longer than ~200 kb) that cannot migrate [20,21]; (ii) to estimate the loop size distribution and the average linear density of the loops as its main parameter [20,22]. In our previous work we have shown that the two characteristics depend on the cell type and/or the cell functional state: in particular, a loop redistribution was observed in human lymphocytes after their activation (blast transformation) with interleukin 2 [22]. ...
... Our analysis of the two parameters that change during electrophoresis, the relative amount of DNA in the comet tail and the tail length, makes it possible to obtain two quantitative characteristics: (i) to divide all the loops into three subsets -loops on the nucleoid surface, inner loops inside the nucleoid, and very large loops (longer than ~200 kb) that cannot migrate [20,21]; (ii) to estimate the loop size distribution and the average linear density of the loops as its main parameter [20,22]. In our previous work we have shown that the two characteristics depend on the cell type and/or the cell functional state: in particular, a loop redistribution was observed in human lymphocytes after their activation (blast transformation) with interleukin 2 [22]. ...
... Kinetic plots (the relative amount f of DNA in the tail versus electrophoresis time t) were analyzed as before (see [20][21][22] for details): the plots were fitted with the equation f = A 1 f 1 + A 2 f 2 , where A 1 and A 2 are the maximum amplitudes of two components (A 2 may be equal to zero). The functions f 1 and f 2 are described by a standard equation of monomolecular kinetics ...
Chromatin loops are important elements of both chromatin higher-order structure and transcription regulation system. Our previous works have shown that several features of the loop domain organization could be investigated by single cell gel electrophoresis (the comet assay) using the kinetic approach. In this study we applied this technique to study DNA loop domain organization in lymphoid cells: human lymphocytes, lymphoblasts cultivated during 24 h and 44 h, and T cells of Jurkat cell line. Two features of the loop domain organization were found to depend on the cell functional state. First, DNA fraction in the loops of large sizes (more than ~200 kb) was essentially increased in proliferating (de-differentiated) cells in comparison with terminally differentiated lymphocytes. Second, the linear density of the loops not larger than ~200 kb was decreased in transcriptionally active cells and was increased upon their inactivation.
... In the electric field the nucleoid DNA ISSN 1993-6842 (on- migrates towards the anode forming an electrophoretic track, which resembles a comet tail and can be visualized by fluorescent microscopy [13,14]. In our previous works [15][16][17][18][19][20][21], measuring the kinetics of DNA exit during electrophoresis, we have shown that the nucleoid structure reflects some important features of the loop organization in vivo. Moreover, we have argued that some large-scale features of the loop domain organization (and re-organization) are preserved in nucleoids after cell lysis and hence may be detected due to a relatively simple technique, the comet assay [20,21]. ...
... In our previous works [15][16][17][18][19][20][21], measuring the kinetics of DNA exit during electrophoresis, we have shown that the nucleoid structure reflects some important features of the loop organization in vivo. Moreover, we have argued that some large-scale features of the loop domain organization (and re-organization) are preserved in nucleoids after cell lysis and hence may be detected due to a relatively simple technique, the comet assay [20,21]. ...
... Most of our previous experiments were done using intact human lymphocytes. However, in our recent work [20] we have shown that the reorganization of DNA loops occurs upon the lymphocyte activation by interleukin 2, and that the loops in glioblastoma T98G cells are organized differently with respect to lymphocytes. In this article we studied these cancer cells in more details. ...
The loop domain organization of chromatin, which plays an important role in transcription regulation, may depend on the cell functional state. The aim of this work was to investigate DNA loop reorganization upon functional transitions in the glioblastoma T98G cells. Methods. Single cell gel electrophoresis (a comet assay) was used to analyze the kinetics of the DNA loop migration from the nucleoids obtained from the lysed cells. Results. The cells arrested in the G1 phase of the cell cycle were characterized by a relatively low amount of DNA in the comet tails due to a low content of DNA in the loops which may be resolved by the comet assay (up to ~300 kb). After cell reactivation, the contour length of the loops essentially increased in parallel with a decrease in the linear loop density along the genome. Conclusions. An increase in the loop size and a respective decrease in the loop density may be a general feature of activated cells as we earlier observed similar effects upon activation of human lymphocytes.
... Two steps of the DNA exit from nucleoids appeared to be a universal characteristic of DNA migration during the comet assay. The two-step shape of the kinetic plots was observed for nucleoids prepared not only from human lymphocytes (see above) but also from lymphoblasts (lymphocytes that were activated by interleukin 2) and glioblastoma T98G cells [58]. However, the relative contributions of the "rapid" (surface) and "slow" (inner) loops were found to be dependent on the cell type. ...
... Another kind of the loop distributions that can be tested by the comet assay is the size distribution [52,58]. As it was observed in our experiments, for all cell types studied, the tail length (which represents the contour length of the longest loops in the tail) changed approximately in parallel with the DNA amount in the tails (Fig. 3, see also [58]). ...
... Another kind of the loop distributions that can be tested by the comet assay is the size distribution [52,58]. As it was observed in our experiments, for all cell types studied, the tail length (which represents the contour length of the longest loops in the tail) changed approximately in parallel with the DNA amount in the tails (Fig. 3, see also [58]). The fraction f of DNA in the tails (at given time of electrophoresis) can be obviously written as ...
The comet assay is a sensitive method to assess DNA damages in single cells. The approach consists of an analysis of electrophoretic migration of DNA from nucleoids obtained after cell lysis in a thin layer of agarose. Although the method is widely used the physical mechanisms of DNA track formation remained to be rather elusive for a long time. This review is devoted to our recent results pertaining to this subject, using an original approach based on the kinetic measurements of the comet formation. We argue that linear DNA fragments give an essential contribution into the tail formation in the alkaline conditions and, at neutral pH, when the level of DNA damages is very high. On the other hand, in the neutral comet assay at low levels of DNA damages (and also in the case of undamaged cells) the tail is formed by extended DNA loops. These loops are about the same as chromatin loops in the cell nuclei. Kinetic measurements in the comet assay give an opportunity to investigate the topology of the loops and large-scale features of the loop domain organization (and re-organization) in nucleoids obtained from different cell types.
... Next, we wanted to further evaluate whether the detection of 8-oxodG in nuclear DNA was influenced by a different accessibility to the reagents used in the two procedures. To this end, untreated and KBrO3-treated HaCaT cells were embedded in agarose and exposed to high salt extraction in order to obtained nucleoids, i.e., nuclei devoid of most nuclear proteins, such as those used for the comet assay [36,37]. The extent of 8-oxodG labeling obtained with N45.1 antibody or with avidin-AF488 is shown in Figure 5A,B, respectively. ...
... It can be observed that the fluorescence intensity images related Next, we wanted to further evaluate whether the detection of 8-oxodG in nuclear DNA was influenced by a different accessibility to the reagents used in the two procedures. To this end, untreated and KBrO 3 -treated HaCaT cells were embedded in agarose and exposed to high salt extraction in order to obtained nucleoids, i.e., nuclei devoid of most nuclear proteins, such as those used for the comet assay [36,37]. The extent of 8-oxodG labeling obtained with N45.1 antibody or with avidin-AF488 is shown in Figure 5A,B, respectively. ...
An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.
... Результати дослідження кінетики формування електрофоретичного треку свідчать, що у випадку інтактних нуклеоїдів за нейтральних значень рН міграція ДНК при електрофорезі забезпечується витягуванням до аноду надспіралізованих петельних доменів [8,9]. Аналіз кінетичних параметрів дозволяє встановити ряд важливих характеристик петель ДНК та дослідити особливості їх організації у клітинах різних типів та при різних функціональних станах [9,12,13]. ...
... пар основ) петельних доме-нів. У попередніх дослідженнях нами було показано, що двоступеневість виходу ДНК у хвіст комети відображає особливості організації петель у нуклеоїді: перший швидкий рух здійснюють петлі, розміщені на поверхні нуклеоїду [9,12]. Їх міграція не залежить від рівня надспіралізації, що може свідчити про наявність одноабо дволанцюгових розривів у таких поверхневих петлях. ...
Aim. Aim was to investigate possible changes in the DNA loop domain organization upon activation of human lymphocytes. The rational for this task is the knowledge that the chromatin looping plays an important role in transcription regulation and thus may vary depending on cell functional state. Methods. The kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) was studied for nucleoids obtained from human lymphocytes and lymphoblasts activated to proliferation by interleukin 2. Results. Three part of DNA were observed in nucleoids: DNA on the nucleoid surface, loops up to ~150 kb inside the nucleoid, and larger loops that cannot migrate. An essential redistribution of the loop domains between the inside and surface fractions occurs upon activation (at G1 phase). Later on (at the end of S phase) the inside fraction becomes lower in favor of the large loops. Conclusions. Changes in the cell functional state are accompanied by large-scale changes in the loop domain organization that can be detected by the comet assay.Keywords: DNA loops, nucleoid, comet assay, lymphocytes, lymphoblasts.
... Images were examined using the ImageJ image-processing program (imagej.nih.gov) with the OpenComet plugin [20]. "Tail Moment" (TM) was used as a parameter for evaluation of relative level of DNA damages [21]. "Atypical comets" (AC), which formed from cells with high DNA fragmentation, were scored separately. ...
... This situation probably reflects not so much the availability in lymphocyte culture of the population of radio-resistant and/or fully recovered cells as the presence of heavily damaged cells in which the checkpoint has acted on the S-phase of the cell cycle, because, if the cells are in this phase, then significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [19,21]. This opinion is confirmed by the lack of increase in frequency of "comets" from the tenth group after radiation exposure at the 40th h of cultivation: most blasttransformed lymphocytes must be on the S phase, and cells with very high level of DNA damages cannot pass the S/G 2 checkpoint; as a result, they are delayed on this phase. ...
Peculiarities of Modification by Astaxanthin of Radiation-Induced
Damages in the Genome of Human Blood Lymphocytes Exposed
in vitro on Different Stages of the Mitotic Cycle1
D. A. Kurinnyia, *, S. R. Rushkovskyb, O. M. Demchenkoa, and M. A. Pilinskaa
aState Institution “National Research Center for Radiation Medicine,” National Academy of Medical Sciences of Ukraine,
Kyiv, 04050 Ukraine
bEducational and Research Center “Institute of Biology and Medicine,” Taras Shevchenko National University of Kyiv,
Kyiv, 01601 Ukraine
*e-mail: kurinnyi.d@gmail.com
Received
Abstract⎯The features of astaxanthin impact (20 μg/mL) in the culture of human blood lymphocytes
exposed to γ-radiation (1.0 Gy) on the G0, S, and G2 phases of the cell cycle were studied using Comet assay.
Decrease in the level of DNA damages (Tail Moment index) under astaxanthin influence on lymphocytes
irradiated in all stages of cell division was established, while, as a result of previous cytogenetic investigations,
lack of the modifying action of astaxanthin after irradiation of cells in the G2 stage and radioprotective effect
in the G0 stage of the mitotic cycle had been revealed. In G0 phase, the activation of the processes of apoptosis
by astaxanthin in irradiated cells with high levels of genomic damages was found. The obtained data demonstrate
that astaxanthin has a powerful radioprotective potential, mainly due to its apoptogenic properties.
Keywords: culture of human peripheral blood lymphocytes, gamma-irradiation, astaxanthin, Comet assay,
DNA injuries, apoptosis
... Images were examined using the ImageJ image-processing program (imagej.nih.gov) with the OpenComet plugin [20]. "Tail Moment" (TM) was used as a parameter for evaluation of relative level of DNA damages [21]. "Atypical comets" (AC), which formed from cells with high DNA fragmentation, were scored separately. ...
... This situation probably reflects not so much the availability in lymphocyte culture of the population of radio-resistant and/or fully recovered cells as the presence of heavily damaged cells in which the checkpoint has acted on the S-phase of the cell cycle, because, if the cells are in this phase, then significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [19,21]. This opinion is confirmed by the lack of increase in frequency of "comets" from the tenth group after radiation exposure at the 40th h of cultivation: most blasttransformed lymphocytes must be on the S phase, and cells with very high level of DNA damages cannot pass the S/G 2 checkpoint; as a result, they are delayed on this phase. ...
The features of astaxanthin impact (20 μg/mL) in the culture of human blood lymphocytes exposed to γ-radiation (1.0 Gy) on the G0, S, and G2 phases of the cell cycle were studied using Comet assay. Decrease in the level of DNA damages (Tail Moment index) under astaxanthin influence on lymphocytes irradiated in all stages of cell division was established, while, as a result of previous cytogenetic investigations, lack of the modifying action of astaxanthin after irradiation of cells in the G2 stage and radioprotective effect in the G0 stage of the mitotic cycle had been revealed. In G0 phase, the activation of the processes of apoptosis by astaxanthin in irradiated cells with high levels of genomic damages was found. The obtained data demonstrate that astaxanthin has a powerful radioprotective potential, mainly due to its apoptogenic properties.
... Images were examined using the ImageJ image-processing program (imagej.nih.gov) with the OpenComet plugin [20]. "Tail Moment" (TM) was used as a parameter for evaluation of relative level of DNA damages [21]. "Atypical comets" (AC), which formed from cells with high DNA fragmentation, were scored separately. ...
... This situation probably reflects not so much the availability in lymphocyte culture of the population of radio-resistant and/or fully recovered cells as the presence of heavily damaged cells in which the checkpoint has acted on the S-phase of the cell cycle, because, if the cells are in this phase, then significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [19,21]. This opinion is confirmed by the lack of increase in frequency of "comets" from the tenth group after radiation exposure at the 40th h of cultivation: most blasttransformed lymphocytes must be on the S phase, and cells with very high level of DNA damages cannot pass the S/G 2 checkpoint; as a result, they are delayed on this phase. ...
Objective:
To identify the possibility of modification by astaxanthin the level of genome damages induced by gamma quanta in the culture of human peripheral blood lymphocytes exposed in vitro on postsynthetic (G2) phase of the first mitotic cycle.
Materials and methods:
Peripheral blood lymphocytes from four apparently healthy volunteers 35-51 years old were cultivated using modified micromethod. To obtain genomic damages in G2 phase of the first mitotic cycle the part of cultures was irradiated by γ quanta in dose 1.0 Gy through 46 hours of cultivation. Astaxanthin in final con centration 20 μg/ml was exposed to lymphocytes' cultures before the irradiation. Cytogenetic analysis the uniform ly stained slides of metaphase chromosomes was carried out to determine the frequencies of chromosome and chro matid types of aberrations. Using the method of individual cells electrophoresis (Comet assay) the relative level of DNA damages (Tail Moment index) and the frequency of apoptotic cells with high level of DNA fragmentation were evaluated.
Results:
Mean group frequencies of chromosome aberrations after gamma irradiation of lymphocytes in vitro exceed ed those without radiation exposure and were 72.35 ± 1.17 and 2.46 ± 0.30 per 100 metaphases, respectively (p < 0.001), mainly due to chromatid type of aberrations (58.32 ± 1.29 per 100 metaphases). Adding of astaxanthin into culture medium before the irradiation did not result in changes as in the frequency of chromosomal damages (71.54 ± 1.34 per 100 metaphases) as in the spectrum of aberrations - also prevailed chromatid type of aberrations (58.47 ± 1.47 per 100 metaphases). The increase of Tail Moment index after radiation exposure (from 3.84 ± 0.36 to 12.06 ± 1.88, respectively, p < 0.001) and lack of significant impact of astaxanthin on this index in the irradiated lym phocytes (8.96 ± 2.39, p > 0.05) was established, ie astaxanthin didn't change the relative level of radiation induced DNA damages. Also apoptogenic effect of astaxanthin was not found: frequency of apoptotic cells were (2.25 ± 1.49) % in cultures of intact lymphocytes, (2.08 ± 1.54) % in irradiated cultures and (1.78 ± 1.25) % under joint action of gamma radiation and astaxanthin (p > 0.05).
Conclusions:
Noimpactofastaxanthinongenomicinstabilityinducedbygammairradiation invitroinculturesof human peripheral blood lymphocytes on postsynthetic (G2) phase of first mitotic cycle had been established.
... The chromatin comet assay is based on the SCGE and was designed to give a quick and broad view of the organisation of the cellular genome in chromatin [23]. It relies on the electric current pulling DNA fibers and chromatin loops from the nucleus [33,34]. After many attempts and applying chromatin structure sensing nucleases, we have designed conditions where the comet assay can accurately and reliably assess the chromatin general loop organisation and its dynamics in any cell population [23]. ...
... The method is based on an analysis of DNA migration from nucleoids immobilized in a thin layer of agarose on microscopic slides. In our previous works [7,9,10], measuring the kinetics of DNA exit during electrophoresis, we have shown that, in the case when intact undamaged cells are used for the lysis, the electrophoretic track (the comet tail) contains nothing but supercoiled DNA loops, which are extended to the anode. The exit of these loops occurs in two steps: the first rapid step corresponds to loops, migration of which is insensitive to supercoiling, whereas the migration at the second, delayed and cooperative, step is very sensitive to alteration of DNA supercoiling level. ...
The comet assay has proved itself to be not only a method of detection of DNA damages at the level of individual cells but also an approach for investigation of spatial organization of DNA loop domains in nucleoids. Usually, those nucleoids are obtained after cell lysis in high-salt buffer (e. g. 2.5 M NaCl) with a detergent: these conditions ensure the removal of cell membranes and most of the chromatin proteins, while supercoiled DNA loop domains remain untouched. In this work, we tested the comet assay applied to nucleoids obtained in low-salt solution (1 M NaCl). These nucleoids keep most of the histones and thus contain the loops resembling the chromatin loops to a greater extent. It was shown that, despite some quantitative differences, the most general features of the kinetics of DNA exit are about the same for nucleoids obtained in high- and low-salt conditions. It can be concluded that the DNA loops in high-salt nucleoids can be efficiently used to investigate the spatial DNA organization in chromatin.
... It is noteworthy that γ-radiation exposure at 40 and 46 h of incubation did not cause decrease in the frequency of group 1, which includes the "comets" with the smallest DNA release into the "comet" tail (TM from 0 to 0.81). Probably, this situation reflects not so much on the existence in lymphocyte cultures of the populations of radiation-resistant and/or fully recovered cells, as the presence of heavily damaged cells in which the checkpoint has acted on the S phase of the cell cycle, because if the cells are in this phase, then significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [35,36]. This opinion is confirmed by the lack of increase in frequency of "comets" from the tenth group after radiation exposure at 40 h of cultivation: most of blast-transformed lymphocytes must be on S phase, and cells with the very high level of DNA damages cannot pass S/G 2 checkpoint, and, as a result, they are delayed on this phase. ...
... It is noteworthy that γ-radiation exposure at 40 and 46 h of incubation did not cause decrease in the frequency of group 1, which includes the "comets" with the smallest DNA release into the "comet" tail (TM from 0 to 0.81). Probably, this situation reflects not so much on the existence in lymphocyte cultures of the populations of radiation-resistant and/or fully recovered cells, as the presence of heavily damaged cells in which the checkpoint has acted on the S phase of the cell cycle, because if the cells are in this phase, then significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [35,36]. This opinion is confirmed by the lack of increase in frequency of "comets" from the tenth group after radiation exposure at 40 h of cultivation: most of blast-transformed lymphocytes must be on S phase, and cells with the very high level of DNA damages cannot pass S/G 2 checkpoint, and, as a result, they are delayed on this phase. ...
ABSTRACT
Purpose: Astaxanthin (a carotenoid of xanthophyll group) satisfies all requirements demanded to potential radioprotectors. The purpose of our study was to investigate a modifying effect of astaxanthin on radiation induced genome damages in cultivating human peripheral blood lymphocytes (PBL).
Materials and methods: To establish astaxanthin radioprotective activity, we used method for scoring of structural chromosomal aberrations (ChA) in PBL and neutral single-cell gel electrophoresis (comet assay) for estimation of the level of DNA breaks in cells (expressed as tail moment (TM) for “classical comets” and rate of “atypical comets” (АС) for cells with greatly fragmented DNA). Blood samples were obtained by venepuncture from five conditionally healthy volunteers. Astaxanthin in final concentrations of 2.0, 10.0, 20.0 and 40.0 µg/ml had been added into the culture medium before cultivation began, prior to γ- irradiation of PBL (dose 1.0 Gy). Cultures were incubated at 37°C for 48-50 h. Chromosome slides were prepared after 50 h of cultivation. Comet assay was carried out before cultivation and on 48 h of culturing.
Results: Astaxanthin in all studied concentrations per se had no mutagenic activity and obviously reduced the level of radiation induced chromosomal aberrations. The most efficient concentration of astaxanthin was 20.0 µg/ml. The effect of astaxanthin in this concentration on exposed cells manifested in significant decreasing of the radiation-induced ChA level: from 26.06 ± 1.81 per 100 metaphases (phm) to
9.03 ± 0.73 phm (p<0,001). Comparative analysis of the ChA spectra indicated the most substantial reduction of classic unstable cytogenetic markers of radiation exposure (dicentric and ring chromosomes). Likewise, statistically significant decrease in TM in irradiated lymphocytes treated by astaxanthin compared with untreated irradiated samples was observed both before cultivation (3.21 ± 0.48 and 6.55 ± 1.82 respectively, p<0,05) and after 48 h of cultivation (5.27 ± 1.77 and 12.86 ± 0.74 respectively, p<0,05). The level of AC, which evidently arose from apoptotic cells, in irradiated cultures treated by astaxanthin was approximately two times higher as compared with irradiated samples (7.15 ± 1.13% and 3.57 ± 0,81% respectively, p<0,05).
Conclusions: Our study indicated that astaxanthin in concentration of 20.0 µg/ml demonstrated evident radioprotective properties by reducing the level of ChA, decreasing DNA damage and increasing the apoptotic rate.
Keywords Astaxanthin, human peripheral blood lymphocytes, chromosomal aberrations, DNA breaks, apoptosis, radioprotective effect
... This phenomenon could affect the relationship between the relaxation of the supercoiled DNA and the amount of breaks present in the molecule. An insufficient protein digestion is related to the differences in the generation and migration of the DNA loops [27]. ...
Comet assays or single cell gel electrophoresis for detection of DNA damage is a test that has been widely utilized to assess the effects of expositions to environmental genotoxicants. The test is also used to evaluate DNA damage related to chronic inflammation or preneoplastic and neoplastic conditions. The cells more frequently assessed in comet assay in humans are the peripheral blood lymphocytes, but there are other cell types that have been considered for that purpose. Among those, buccal cells have received attention for its suitability for comet assay, but there have been Prieto González et al.; JAMMR, 26(4): 1-14, 2018; Article no.JAMMR.38227 relatively few studies on comet assay in buccal epithelial cells. However, there are technical difficulties related to comet assay in buccal epithelial cells that justifies the attempts to develop or optimize protocols that could contribute to standardize the test allowing more widespread use of buccal cells in biomonitoring or clinical trials. In the present work, we compared three protocols: the standard technique of alkaline comet from Tice et al. 1999 and the protocols developed specifically for oral cavity cells from Valverde et al. 1997 and Szeto et al. 2005. We introduced modifications in the protocols related to, a device utilized to scrape the cells from the mucosa, the place and volume of sample enzymatic digestion, trypsin concentration, and also, the times for lysis incubation and unwinding. This modified protocol is a contribution to the optimization of comet assay for buccal cells and contributes to its utilization in biomonitoring human DNA damage.
Background. Medicinal plant extracts are widely used in traditional and non-conventional medicine. Special interest is given to extracts obtained from transgenic so-called "hairy" roots. Usually, such roots are characterized by a high content of biologically active components. However, such plant transformation can lead to the appearance of both undesirable effects of the obtained extracts (e.g., a genotoxic effect) and positive effects (e.g., antioxidant and UV-protective properties). In this work the content of flavonoids in extracts of roots of three species of the Artemisia genus, as well as their potential genotoxic and UV-protective properties, were studied. Methods. Transgenic roots of Artemisia annua, A. vulgaris and A. tilesii with the inserted roll gene were obtained using Agrobacterium rhizogenes A4 mediated transformation. Water extracts were prepared according to a standard procedure. Total flavonoid amount was estimated spectrophotometrically. Comet assay was used as an approach to evaluate extracts genotoxicity and UV-protective properties. Results. It was shown that the content of flavonoids in extracts obtained from the transgenic plants is more than 2 times higher than that for control plants. Extracts obtained from A. vulgaris and A. annua had no genotoxic effect, while extracts obtained from A. tilesii had a weak but statistically significant mutagenic effect. A. vulgaris extracts had pronounced UV-protective properties, which were correlated with the content of flavonoids in these extracts. Conclusions. Extracts of plants studied mostly did not show a genotoxic effect, but had UV-protective properties. The ability of extracts to reduce the negative effects of UV-irradiation depends on the concentration of flavonoids: the increased content of these compounds in transgenic plants led to almost 3-fold decrease in the relative amount of DNA in the comet tails.
Methylsensitive comet electrophoresis is based on the assessment of the level of DNA migration from individual lysed cells after treatment with methylsensitive restriction enzymes. Using model human lymphocytes, the optimal combination of restriction intensity and electrophoresis time was selected and a new approach for evaluating the relative level of DNA methylation was proposed. It was established that in the cells of the T98G culture, which are actively proliferating, the level of methylation is higher than in cells arrested at the G1 phase of the cell cycle. At the same time, the level of DNA methylation in G1 cells of the T98G line is significantly lower compared to lymphocytes.
At higher order levels chromatin is organized into loops, and this looping plays an important role in transcription regulation. In our previous works we investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) of nucleoids obtained from lysed cells. It was shown that there are three parts of DNA in nucleoids: DNA in rather small loops which migrate rapidly; DNA in the loops up to ~150 kb, the migration of which is retarded; and larger loops that cannot migrate. Here we applied, for the first time, the pulse-field electrophoresis in the comet assay. Our results show that the first rapid step during the usual comet assay can be attributed to loops on the nucleoid surface while the second slow component represents loops inside the nucleoid.
Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.
The interaction of proteins binding non-specifically to DNA, as well as the properties of many other interacting ligand-lattice systems important in molecular biology, requires a fundamentally different type of theoretical analysis than that provided by the classical Scatchard independent-binding-site treatment. Exact and relatively simple equations describing the binding of both non-interacting and interacting (co-operative) ligands to a homogeneous one-dimensional lattice are derived in terms of ligand site size, intrinsic binding constant and ligand-ligand co-operativity (equations (10) and (15) in the text). The mathematical approach is based on simple conditional probabilities, and reveals some largely unrecognized characteristics of such lattice binding systems. The results indicate that the binding of any non-interacting ligand covering more than one lattice residue results in non-linear (convex downward) Scatchard plots. The introduction of positive ligand-ligand co-operativity antagonizes this non-linearity, and eventually leads to plots of the opposite curvature. The maxima, limiting slopes, and intercepts of such plots can be used to estimate the required binding parameters. The method can be extended to systems involving heterogeneous ligands, and some types of heterogeneous lattices. Procedures for applying the method to a variety of interacting systems are presented, and a preliminary analysis is carried out for some selected sets of data from the literature.
Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations—including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments—and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes.
Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. Advanced ChIA-PET shows that CTCF/cohesion and RNA polymerase II arrange spatial organization for coordinated transcription. Haplotype variants exhibit allelic effects on chromatin topology and transcription that link disease susceptibility.
In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease. Ji et al. map the chromosome organizational structures that underlie gene regulation in human naive and primed pluripotent cells. Their framework of cohesin-associated CTCF loops, and the cohesin-associated enhancer-promoter loops within them, provides a reference map for future interrogation of regulatory interactions.
Significance
When the human genome folds up inside the cell nucleus, it is spatially partitioned into numerous loops and contact domains. How these structures form is unknown. Here, we show that data from high-resolution spatial proximity maps are consistent with a model in which a complex, including the proteins CCCTC-binding factor (CTCF) and cohesin, mediates the formation of loops by a process of extrusion. Contact domains form as a byproduct of this process. The model accurately predicts how the genome will fold, using only information about the locations at which CTCF is bound. We demonstrate the ability to reengineer loops and domains in a predictable manner by creating highly targeted mutations, some as small as a single base pair, at CTCF sites.
Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.
We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats. PAPERFLICK:
Copyright © 2014 Elsevier Inc. All rights reserved.
In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C) and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.
The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term 'topological domains', as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.
Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation. Here we present a high-resolution chromosomal contact map derived from a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of contact density and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, whereas active domains reach out of the territory to form remote intra- and interchromosomal contacts. Moreover, we systematically identify specific long-range intrachromosomal contacts between Polycomb-repressed domains. Together, these observations allow for quantitative prediction of the Drosophila chromosomal contact map, laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system.
We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation
with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of
1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes.
We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed
chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal
globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold
and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our
results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.
Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated.
Rho GTPases are key regulators of many cellular functions, including cytoskeleton organization which is important for cell morphology and mobility, gene expression, cell cycle progression, and cytokinesis. In addition, it has recently been recognized that Rho GTPase activity is required for development of the immune system, as well as for the specialized functions of the peripheral cells that act in the immune response such as antigen presenting cells and lymphocytes. Stimulation of T lymphocytes with interleukin-2 (IL-2) induces clonal expansion of antigen-specific populations and provides a model to study cell cycle entry and cell cycle progression. We have performed gene expression analysis in a model of human T lymphocytes, which proliferate in response to IL-2. In addition to changes in genes relevant to cell cycling and to the antiapoptotic effects of IL-2, we have analyzed expression and variations of more than 300 genes involved in Rho GTPase signaling pathways. We report here that IL-2 regulates the expression of a number of proteins, which participate in the Rho GTPase pathways, including some of the GTPases themselves, GDP/GTP exchange factors, GTPase activating proteins, as well as GDIs and effectors. Our results suggest that regulation of expression of components of the Rho GTPase pathways may be an important mechanism in assembling specific signal transduction cascades that need to be active at certain times during the cell cycle. Some of our findings may also be relevant to the roles of Rho GTPases in T lymphocyte functions and proliferation.
Interleukin 2 (IL-2) was one of the first cytokines to be discovered. However, the complex role of IL-2 and its receptor in the regulation of immune responses is only now emerging. This review explores the various signals triggered by IL-2 and discusses their translation into biological function. A model is outlined that accommodates the seemingly contradictory functions of IL-2, and explains how one cytokine can be an essential T-cell growth and differentiation factor and yet also be indispensable to maintain peripheral tolerance.
How eukaryotic chromosomes fold inside the nucleus is an age-old question that remains unanswered today. Early biochemical and microscopic studies revealed the existence of chromatin domains and loops as a pervasive feature of interphase chromosomes, but the biological implications of such organizational features were obscure. Genome-wide analysis of pair-wise chromatin interactions using chromatin conformation capture (3C)-based techniques has shed new light on the organization of chromosomes in interphase nuclei. Particularly, the finding of cell-type invariant, evolutionarily conserved topologically associating domains (TADs) in a broad spectrum of cell types has provided a new molecular framework for the study of animal development and human diseases. Here, we review recent progress in characterization of such chromatin domains and delineation of mechanisms of their formation in animal cells.
Proper expression of genes requires communication with their regulatory elements that can be located elsewhere along the chromosome. The physics of chromatin fibers imposes a range of constraints on such communication. The molecular and biophysical mechanisms by which chromosomal communication is established, or prevented, have become a topic of intense study, and important roles for the spatial organization of chromosomes are being discovered. Here we present a view of the interphase 3D genome characterized by extensive physical compartmentalization and insulation on the one hand and facilitated long-range interactions on the other. We propose the existence of topological machines dedicated to set up and to exploit a 3D genome organization to both promote and censor communication along and between chromosomes.
The genome must be highly compacted to fit within eukaryotic nuclei but must be accessible to the transcriptional machinery to allow appropriate expression of genes in different cell types and throughout developmental pathways. A growing body of work has shown that the genome, analogously to proteins, forms an ordered, hierarchical structure that closely correlates and may even be causally linked with regulation of functions such as transcription. This review describes our current understanding of how these functional genomic "secondary and tertiary structures" form a blueprint for global nuclear architecture and the potential they hold for understanding and manipulating genomic regulation.
Copyright © 2015 Elsevier Inc. All rights reserved.
Single-cell gel electrophoresis, or the comet assay, is usually performed with nucleoids prepared after a lysis of either whole cells (more often) or isolated cell nuclei (rarely). Electrophoretic properties of the second type of nucleoids have never been investigated carefully. We measured the kinetics of the DNA exit from nuclei-derived nucleoids in comparison with cell-derived nucleoids. The results show that general organization of the nuclei-derived nucleoids is not changed very much in comparison with nucleoids commonly obtained from whole cells. At the same time, in contrast to the cell-derived nucleoids, for which the exit is stepwise and cooperative, the DNA exit from the nuclei-derived nucleoids can be described by a simple monomolecular kinetics. This difference is probably due to agarose penetration into nuclei (but not into cells) before polymerization of the agarose gel. We suggest that single nucleus gel electrophoresis may be a way for the comet assay standardization.This article is protected by copyright. All rights reserved
At higher order levels chromatin is organized into loops. This looping, which plays an important role in transcription regulation and other processes, remains poorly understood. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay). The migration of a part of the loops was shown to be reversible after switching off electrophoresis and to be sensitive to intercalation-induced changes in supercoiling. Another group of the loops migrates rapidly, the rate being insensitive to the supercoiling level. The largest part of the loops cannot migrate at all, presumably because of their large size. The loop ends can be detached in the presence of high concentrations of intercalators or protein denaturants, thus increasing the fraction of DNA that cannot migrate in the gel. The distribution of the loop length up to 100kb appears to be consistent with the fractal globule organization.
How DNA is organized in three dimensions inside the cell nucleus and how this affects the ways in which cells access, read and interpret genetic information are among the longest standing questions in cell biology. Using newly developed molecular, genomic and computational approaches based on the chromosome conformation capture technology (such as 3C, 4C, 5C and Hi-C), the spatial organization of genomes is being explored at unprecedented resolution. Interpreting the increasingly large chromatin interaction data sets is now posing novel challenges. Here we describe several types of statistical and computational approaches that have recently been developed to analyse chromatin interaction data.
Mammalian genomes encode genetic information in their linear sequence, but appropriate expression of their genes requires chromosomes to fold into complex three-dimensional structures. Transcriptional control involves the establishment of physical connections among genes and regulatory elements, both along and between chromosomes. Recent technological innovations in probing the folding of chromosomes are providing new insights into the spatial organization of genomes and its role in gene regulation. It is emerging that folding of large complex chromosomes involves a hierarchy of structures, from chromatin loops that connect genes and enhancers to larger chromosomal domains and nuclear compartments. The larger these structures are along this hierarchy, the more stable they are within cells, while becoming more stochastic between cells. Here, we review the experimental and theoretical data on this hierarchy of structures and propose a key role for the recently discovered topologically associating domains.
Cohesin is a member of the Smc family of protein complexes that mediates higher-order chromosome structure by tethering different regions of chromatin. We present a new in vitro system that assembles cohesin-DNA complexes with in vivo properties. The assembly of these physiological salt-resistant complexes requires the cohesin holo-complex, its ability to bind ATP, the cohesin loader Scc2p and a closed DNA topology. Both the number of cohesin molecules bound to the DNA substrate and their distribution on the DNA substrate are limited. Cohesin and Scc2p bind preferentially to cohesin associated regions (CARs), DNA sequences with enriched cohesin binding in vivo. A subsequence of CARC1 promotes cohesin binding to neighboring sequences within CARC1. The enhancer-like function of this sequence is validated by in vivo deletion analysis. By demonstrating the physiological relevance of these in vitro assembled cohesin-DNA complexes, we establish our in vitro system as a powerful tool to elucidate the mechanism of cohesin and other Smc complexes.
We investigated the mechanisms of DNA exit during single-cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single-strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.
The most salient feature of the lymphokine interleukin-2 (IL-2), a hormone-like protein, is its ability to sustain the proliferation of immunocompetent T cells. Results of initial studies characterizing IL-2 in vitro led investigators to conclude that IL-2 had no known effects on lymphocytes that had not been previously activated by exposure to a mitogen or antigen. Several groups postulated that T cell growth required two signals. The first signal, delivered by a mitogen or antigen, induced T cell activation. Resting T cells, which were thought to lack the membrane receptor for interleukin-2 (IL-2R), progressed from the G0-G1 phase to the S phase, during which time they converted from IL-2R- to IL-2R+ cells. Thereafter, the second signal, served by IL-2, induced T cell proliferation of the IL-2R+ cells. Recently, a number of investigators have demonstrated that highly purified preparations of both natural and recombinant IL-2 induced high levels of T cell proliferation in the absence of any known mitogens or antigens. Presented herein is a review of these studies and an overview of the hypotheses of the mechanisms whereby IL-2 alone induces T cell activation and proliferation.
31P NMR spectra of tumor-infiltrating lymphocytes (TILs) were found to be significantly different from those of normal peripheral lymphocytes. The greatest difference was in the phosphodiester (PDE) region, mainly in the glycerophosphocholine (GPC) signal. Short-term activation of peripheral lymphocytes with interleukin-2 induced a small increase in ATP levels. In all lymphocytes the phosphomonoester (PME) region is dominated by phosphoethanolamine (PE), while there is an unusual absence of phosphocholine (PC). Perfusion of these cells with high concentrations of choline caused only a minimal increase in PC, indicating that choline kinase is not the rate limiting step of lecithin synthesis in lymphocytes.
Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides. The slides were after gelling at 0 degree C immersed in a neutral detergent solution which lysed the cells. A weak electric field (5 V/cm) was then applied over the gel for 5 minutes. The DNA in the gel was stained with the fluorescent dye acridine orange and gives a green emission in a microscope photometer. DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells. The differences in migration pattern were quantitatively measured. The lower detection limit was below 0.5 Gy and a plateau in the dose-effect curve was reached at about 3 Gy. In repair experiments residual DNA damage could be observed after postirradiation incubation for 60 minutes. The advantages of the method is: no radioactive labelling and only a few number of cells is required.
- P Nmr
P NMR studies, FEBS Lett. 258 (1989) 55e58.