The Effect of Magnesium Ions on Chromosome Structure: Insights from G-Banding

Magnesium ion (Mg 2+ ) plays a fundamental role in the chromosome condensation which is important for genetic material segregation. Studies about the effects of Mg 2+ on the overall chromosome structure have been reported. Nevertheless, its effects on the distribution of heterochromatin and euchromatin region have yet to be investigated. This study was aimed to evaluate the effects of Mg 2+ on the banding pattern of the chromosome structure. Chromosome analysis was performed using the GTL-banding technique on synchronized HeLa cells. The effect of Mg 2+ was evaluated by subjecting the chromosomes to three different solutions, namely XBE5 (5 mM Mg 2+ ) as a control, XBE (0 mM Mg 2+ ), and 1 mM EDTA as cations chelator. The results showed a condensed chromosome structure with a clear banding pattern when it was treated with a buffer containing 5 mM Mg 2+ . In contrast, chromosomes treated with a buffer containing no Mg 2+ and those treated with an ions chelator showed an expanded and brous structure with the lower intensity of the banding pattern. Elongation of the chromosome caused by decondensation resulted in the band splitting. The results of this study further emphasized the role of Mg 2+ on chromosome structure and gave insights into Mg 2+ effects on the banding distribution.


Introduction
Chromosome structure is the result of the condensation of chromatin that consists of DNA and histone proteins. The compact structure of the chromosome is required for sister chromatid segregation during the mitotic phase in the cell cycle. One of the essential factors contribute to chromosome condensation is divalent cations, especially Mg 2 + 1 . Magnesium ions play a role to construct and maintain the structure of the chromatin in a condense state. A previous study showed that Mg 2+ concentration is high during metaphase and remains high during anaphase in the cell cycle, which promotes sister chromatid condensation 2 . In addition, several studies showed that the chromatin was more condensed when the Mg 2+ is present [2][3][4][5] . Inside the cells, concentration of Mg 2+ ranges from 5 mM until 30 mM 3 .
The evaluation of the Mg 2+ effects on the overall chromosome structure has been conducted using several microscopes such as uorescence microscope (FM), scanning electron microscope (SEM), and scanning transmission electron microscope (STEM). The chromosome showed a less condense structure after treated with buffer lacking Mg 2 + 4 . Furthermore, the structure of the chromosome was expanded after treated with a 1 mM ethylenediaminetetraacetic acid (EDTA), an ions chelator 5 . The decondensed chromosomes showed a brous structure around the chromatid 6 . All of these results showed the important role of the Mg 2+ for chromosome condensation.
Chromosome structure consists of heterochromatin and euchromatin regions. These regions re ect the degree of chromatin condensation, that affect the DNA accessibility for gene expression. The heterochromatin region is more condensed compared to the euchromatin region which consists of a transcriptionally active gene 7 . The more loosen structure of euchromatin enabling the transcription factor to access the DNA more easily in an open chromatin ber. These differences can be visualized by the banding technique, where G-banding is one of the conventional techniques used in cytogenetics laboratory to identify chromosome structure related to abnormalities. This technique produces G-band that contains a dark band represent heterochromatin and euchromatin as a light band.
A previous study using high resolution scanning ion microprobe (SIM) reported that Mg 2+ is correlated to the heterochromatin region of the chromosome, where it interacts with DNA and protein that contribute to its condensation 8 . Furthermore, the existence of 5 mM Mg 2+ makes the heterochromatin more compact 9 .
Evaluation of the effects of Mg 2+ on the overall chromosome structure observed by various microscopes has been reported. However, to date, how the Mg 2+ affects the chromosome structure correlates to the banding patterns has not been investigated yet. The information about the Mg 2+ effects on chromosome banding pattern would give better insights into the detailed mechanism of Mg 2+ roles on chromosome condensation and further related to the gene expression. Thus, in this study, we analyzed the effects of Mg 2+ on chromosome structure as observed by G-banding.

Results
In this study, we investigated the effects of Mg 2+ on the morphological changes of chromosome structure and its G-band patterns. Figure   The results (Table 1) showed that the addition of a buffer containing 5 mM Mg 2+ resulted in the condensed chromosome structure with a clear banding pattern ( Fig. 1a; blue arrow). Meanwhile, the chromosome treated with 0 mM Mg 2+ showed a less condensed and more dispersed structure ( Fig. 2; green arrow). The intensity of the banding pattern was lower than those in control. The addition of EDTA also resulted in the decondensed chromosome structure with an unclear banding pattern ( Fig. 1c; green arrow). The chromosomes were well-spread in 5 mM Mg 2+ compared to those in 0 mM Mg 2+ and 1 mM EDTA that had more overlapped chromosomes ( Fig. 1b and 1c; red arrow). The chromosome value in 5 mM Mg 2+ range from 2 until 3, where the 0 mM Mg 2+ reached the value of 4. Meanwhile, the value of the chromosome in 1 mM EDTA was higher, which range from 3 until 6.
Chromosome structure and banding pattern To further analyze the effect of the Mg 2+ , chromosome 1 was chosen as a representative. The results showed that the differences in Mg 2+ treatment caused changes in the chromosome structure. Chelating Mg 2+ affected the chromosome structure, length, and banding pattern. As shown in Fig. 2, the chromosome is highly condensed and has a compact structure in 5 mM Mg 2+ (Fig. 2a). In contrast, chromosomes treated with buffer without Mg 2+ showed a less condensed structure with the expansion of the chromosome area ( Fig. 2b). Similar results were also obtained from the chromosomes treated with 1 mM EDTA (Fig. 2c), which shows that the chromosome area was more expanded than the control. The brous structure was observed when the chromosomes were treated with a buffer containing no Mg 2+ and EDTA (red arrow shown in Fig. 2b' and Fig. 2c').
In regards to the banding pattern, the treatment of Mg 2+ in uenced the quality of the banding pattern's intensity (red box). It is shown that the chromosome in 5 mM Mg 2+ had a good quality of the banding pattern qualitatively. The dark band of the chromosome has a clear band appearance (Fig. 2a). In contrast, the chromosome in 0 mM Mg 2+ (Fig. 2b)

Discussion
In this study, we evaluate the effects of Mg 2+ on chromosome structure by subjecting the chromosomes into three different treatments, i.e. buffer containing 5 mM Mg 2+ as the control, buffer containing no Mg 2+ , and EDTA as an ions chelator according to the previous report 4, 5 . The results of this study demonstrated the changes in chromosome structure with the appearance of brous chromatin and disrupted G-band as a consequence of lacking Mg 2+ . The more decondensed structure, longer arms, and brous structure of the chromosomes resulted from the treatment of 0 mM Mg 2+ and EDTA were also established from the ultrastructure visualization using electron microscopy 2,5 which showed the obvious structural alteration of the chromosomes upon Mg 2+ concentration chelation. The chromosomes were less condensed and showed the brous structure once they were treated with buffer without Mg 2+ . Furthermore, when the cations were removed by using EDTA, the large expansion of the chromosome structure was evident 2,5 .
Chelating Mg 2+ from the chromosome, followed by enzymatic treatment, resulted in the structural collapse of chromatin 10 . In contrast, the chromosome in XBE5 which contained 5 mM Mg 2+ was remained condensed. Magnesium ions maintain the structure of the chromosome more condensed.
There was no brous structure that appeared in the edges of the chromosome treated with 5 mM Mg 2+ .
This observation exhibit that lacking Mg 2+ makes the structure of the chromosome undergo decondensation. This result implies that Mg 2+ 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 folding 11 . The depletion of the Mg 2+ caused instability of the nucleosome interaction and unraveling chromatin which further resulted in the structural alteration. The brous structure in this research was shown along the chromosome arm, indicating the chromatin bers radiated out of the decondensed chromosome 12 . Besides, divalent cation used to protect nucleosome by forming a layer outside the nucleosome 13 . Consequently, chelating Mg 2+ using EDTA as a cation chelator caused chromosome decondensation, yield a brous 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 Marton ova 14  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 re ected by the band that was produced and the intensity of the band correlated with GC content 18 . 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 chromosome 19 . The condensed structure which was maintained by the presence of Mg 2+ 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 Mg 2+ (≥ 2 mM) reserved heterochromatin in a condense state 9 . The presence of Mg 2+ leads the DNA to maintain their position link to each other that facilitates their interaction with the dye, mediates the construction of the complex 20 . It indicates that the presence of Mg 2+ maintains the heterochromatin in its condense state. Another study demonstrated that the presence of Mg 2+ modulated SIR heterochromatin folding became compact 21,22 . The more loosen structure as a result of the absence of Mg 2+ 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 conformation 19,23 . The dispersed band in 0 mM Mg 2+ 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 bands 24 . These results implied that different degrees of chromatin condensation affected the accessibility of DNA to the binding of the dye 25 .
The collapse of the chromosome structure increases the chromosome length, and in turn, increases their value according to ISCN 10 . The band resolution that was produced depends on the chromosome condensation 26 . 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 subband [26][27][28] . As chromosome size decreased, a lower number of the band was visualized. In chromosomes treated with 0 mM Mg 2+ and 1 mM EDTA, new bands were formed as compared to those treated with 5 mM Mg 2+ . 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 classi cation based on the assessement criteria 29 . The identi cation of the optimum band number in chromosomes treated with 1 mM EDTA was relatively more di cult due to the poor banding.
In conclusion, the condensation of chromatin forming chromosome structure was affected by Mg 2+ . This is the rst report revealing the effects of Mg 2+ 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.

Methods
Cell culture and chromosome harvest. The HeLa wildtype cells were cultured on a coverslip in a petri dish. The culture medium consisted of Rosewell Park Memorial Institute (RPMI) 1640 (Gibco), 10% Fetal Bovine Serum (FBS) (Gibco), and 1,000 U/mL Penicillin-Streptomycin (Gibco). The culture was incubated at 37 o C with 5% CO 2 for 72 hours. The cell culture was synchronized using bromodeoxyuridine (BrdU) followed by incubation for 14-16 hours at 37 o C with 5% CO 2 . After that, the medium was changed by the new culture medium, following the addition of colchicine. The hypotonic solution (RPMI: ddH 2 O) was added followed by 20 minutes incubation. The treatment for evaluating the effects of Mg 2+ was carried out after the hypotonic solution, using three different buffer solutions according to Dwiranti et al. 5 , namely XBE5 (10 mM HEPES pH 7.7, 100 mM KCl, 5 mM EGTA, and 5 mM MgCl 2 ) as the control, XBE (10 mM HEPES pH 7.7, 100 mM KCl, 5 mM EGTA) which contains no Mg 2+ , and 1 mM ethylenediaminetetraacetic acid (EDTA) as an ions chelator. Cells were subjected to these three different treatments for 30 minutes. After the treatment, the chromosome was xed in methanol: acetic acid (3:1).
Chromosome Banding. GTL banding (G bands by trypsin using Leishman) was performed after the aging process. The chromosomes were banded using 0.25% trypsin (Gibco) for 16 seconds, washed in Phosphate Buffer Solution (PBS), and then followed with staining using Leishman solution (Sigma) for 4 minutes.
Chromosome Imaging. The slides were observed using a light microscope [Nikon] with 1000x magni cation. The spreads of the metaphase chromosome were captured using VideoTesTKaryo 3.1 System software (VideoTesT ltd.). Declarations