In this study, we evaluate the effects of Mg2+ on chromosome structure by subjecting the chromosomes into three different treatments, i.e. buffer containing 5 mM Mg2+ as the control, buffer containing no Mg2+, and EDTA as an ions chelator according to the previous report4,5. The results of this study demonstrated the changes in chromosome structure with the appearance of fibrous chromatin and disrupted G-band as a consequence of lacking Mg2+. The more decondensed structure, longer arms, and fibrous structure of the chromosomes resulted from the treatment of 0 mM Mg2+ and EDTA were also established from the ultrastructure visualization using electron microscopy2,5 which showed the obvious structural alteration of the chromosomes upon Mg2+ concentration chelation. The chromosomes were less condensed and showed the fibrous structure once they were treated with buffer without Mg2+. Furthermore, when the cations were removed by using EDTA, the large expansion of the chromosome structure was evident2,5.
Chelating Mg2+ from the chromosome, followed by enzymatic treatment, resulted in the structural collapse of chromatin10. In contrast, the chromosome in XBE5 which contained 5 mM Mg2+ was remained condensed. Magnesium ions maintain the structure of the chromosome more condensed. There was no fibrous structure that appeared in the edges of the chromosome treated with 5 mM Mg2+. This observation exhibit that lacking Mg2+ makes the structure of the chromosome undergo decondensation. This result implies that Mg2+ has an important role in the structural maintenance of the chromosome. Magnesium ions stabilize the charge of DNA and proteins in the nucleosome, inducing chromatin folding11. The depletion of the Mg2+ caused instability of the nucleosome interaction and unraveling chromatin which further resulted in the structural alteration. The fibrous structure in this research was shown along the chromosome arm, indicating the chromatin fibers radiated out of the decondensed chromosome12. Besides, divalent cation used to protect nucleosome by forming a layer outside the nucleosome13. Consequently, chelating Mg2+ using EDTA as a cation chelator caused chromosome decondensation, yield a fibrous structure. Therefore, chelating cation using EDTA indicates that divalent cation plays an important role to serve the chromatin in its condense state. This change suggested the importance of these divalent cations for compaction of chromatin and maintenance of the chromosome structure.
The changes in chromosome length are consistent with the study reported by Martonfiova14, which stated that chromosome length was affected by the condensation of the chromosome. The condensation resulted in a shorter chromosome, as shown in the results of chromosomes treated with 5 mM Mg2+ because Mg2+ induced nucleosome compaction15,16. Lacking Mg2+ allows chromosome structure to be stretched and induced the changes of its length, as depicted by the results of chromosomes treated with buffer containing 0 mM Mg2+ and those treated with EDTA. The elongation indicated the unwinding chromatid that reflected different condensation of chromatin17.
Chromosome banding has been used extensively to delineates euchromatin from heterochromatin visually, which can be seen from the appearance of G-bands. In the chromosome banding, the level of condensation is reflected by the band that was produced and the intensity of the band correlated with GC content18. Heterochromatin has a low GC content with a more condensed structure compared to the euchromatin. The less condense region, euchromatin, stained less intensely. The produced bands were a result of the formation of complex dye that interacts with the DNA in the chromosome19. The condensed structure which was maintained by the presence of Mg2+ produced a clear and high intensity of the dark band. Furthermore, the G-bands produced in the compact chromosome are easily distinguishable. Previous studies reported that a high concentration of Mg2+ (≥ 2 mM) reserved heterochromatin in a condense state9. The presence of Mg2+ leads the DNA to maintain their position link to each other that facilitates their interaction with the dye, mediates the construction of the complex20. It indicates that the presence of Mg2+ maintains the heterochromatin in its condense state. Another study demonstrated that the presence of Mg2+ modulated SIR heterochromatin folding became compact21,22. The more loosen structure as a result of the absence of Mg2+ resulted in the low intensity of the dark band, it showed that the dye could not intercalate well which prohibit the dye interaction with the DNA, so the complex dye would not be achieved in adjacent conformation19,23. The dispersed band in 0 mM Mg2+ showed that the heterochromatin of the chromosome underwent the decondensation causing decreases width among the DNA that allowed the dye to bond to the enable DNA to promote the complex formation. Moreover, the bands in the chromosome treated with EDTA were still visible, although the produced G-bands were unclear with low intensity. As a consequence of chromosome decondensation, the complex dye was not adequately formed because the dye could not intercalate into the chromosome structure which prevents the binding of the dye, where this condition led to poorly stained bands24. These results implied that different degrees of chromatin condensation affected the accessibility of DNA to the binding of the dye25.
The collapse of the chromosome structure increases the chromosome length, and in turn, increases their value according to ISCN10. The band resolution that was produced depends on the chromosome condensation26. Elongation of the chromosome due to the decondensation affects the reproducibility of the band number. The chromosome elongation induced the band splitting, where this phenomenon enhanced the observable number of the band. Band splitting occurred of the dark band into subband26–28. As chromosome size decreased, a lower number of the band was visualized. In chromosomes treated with 0 mM Mg2+ and 1 mM EDTA, new bands were formed as compared to those treated with 5 mM Mg2+. The qualitative evaluation of the band level was done by identifying the landmark of the chromosome that can be seen, later it was converted to the estimation of quantitative scale classification based on the assessement criteria29. The identification of the optimum band number in chromosomes treated with 1 mM EDTA was relatively more difficult due to the poor banding.
In conclusion, the condensation of chromatin forming chromosome structure was affected by Mg2+. This is the first report revealing the effects of Mg2+ on the chromosome banding pattern. Magnesium ions are important for the maintenance of the chromosome structure. In addition, this study also provides new insight into the correlation of chromosome condensation with the production of G-bands.