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DNA moves sequentially towards the nuclear matrix during DNA replication in vivo.

Rivera-Mulia JC, Hernández-Muñoz R, Martínez F, Aranda-Anzaldo A - BMC Cell Biol. (2011)

Bottom Line: We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated.These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, CP 50000 Toluca, Edo Méx, México.

ABSTRACT

Background: In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results: In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions: Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

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Positional mapping relative to the NM of unrelated gene sequences. Nucleoids from rat hepatocytes were treated with DNase I (0.5 U/ml) for different times. The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of small target sequences (Table 2) located in the 5' ends of the corresponding genes (Fyn, CD23, GFAP and MPZ). Each of such genes is located in a different chromosome and thus they represent separate chromosome territories within the nucleus. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). CT, control G0 hepatocytes; PHx-24 h, hepatocytes 24 h after partial hepatectomy; PHx-7 D, hepatocytes 7 days after partial hepatectomy C, 0' digestion-time control. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). The amplification patterns were consistently reproduced in separate experiments with samples from independent animals (n ≥ 3).
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Figure 7: Positional mapping relative to the NM of unrelated gene sequences. Nucleoids from rat hepatocytes were treated with DNase I (0.5 U/ml) for different times. The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of small target sequences (Table 2) located in the 5' ends of the corresponding genes (Fyn, CD23, GFAP and MPZ). Each of such genes is located in a different chromosome and thus they represent separate chromosome territories within the nucleus. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). CT, control G0 hepatocytes; PHx-24 h, hepatocytes 24 h after partial hepatectomy; PHx-7 D, hepatocytes 7 days after partial hepatectomy C, 0' digestion-time control. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). The amplification patterns were consistently reproduced in separate experiments with samples from independent animals (n ≥ 3).

Mentions: The fifteen sequences analyzed so far belong to a genomic region that comprises three genes and one pseudo-gene of the albumin gene family (Figure 3) and are located within five consecutive structural DNA loops corresponding to 162 kb of chromosome 14 in the rat (Figure 5A). Thus, we also mapped the position relative to the NM of four target- sequences corresponding to four unrelated genes each located in a different rat chromosome (Table 5) in order to corroborate whether nuclear DNA moves towards the NM during DNA replication. Thus amplicons belonging to the 5' ends of the genes GFAP, CD23 (also known as Feer2), MPZ and Fyn were positionally mapped relative to the NM in G0, PHx-24 and PHx-7D samples. The results indicate that all target sequences shift from their original locations to a topological zone closer to the NM at 24 h after partial-hepatectomy when the peak of DNA synthesis occurs, and then recover their original positions relative to the NM as shown in PHx-7D samples obtained when the liver regeneration is complete (Figure 7 and Table 6). This suggests that movement of DNA towards the NM is a general nuclear phenomenon during DNA synthesis in vivo and supports the notion that during replication it is the DNA template that moves towards the replication factories on the NM.


DNA moves sequentially towards the nuclear matrix during DNA replication in vivo.

Rivera-Mulia JC, Hernández-Muñoz R, Martínez F, Aranda-Anzaldo A - BMC Cell Biol. (2011)

Positional mapping relative to the NM of unrelated gene sequences. Nucleoids from rat hepatocytes were treated with DNase I (0.5 U/ml) for different times. The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of small target sequences (Table 2) located in the 5' ends of the corresponding genes (Fyn, CD23, GFAP and MPZ). Each of such genes is located in a different chromosome and thus they represent separate chromosome territories within the nucleus. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). CT, control G0 hepatocytes; PHx-24 h, hepatocytes 24 h after partial hepatectomy; PHx-7 D, hepatocytes 7 days after partial hepatectomy C, 0' digestion-time control. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). The amplification patterns were consistently reproduced in separate experiments with samples from independent animals (n ≥ 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037911&req=5

Figure 7: Positional mapping relative to the NM of unrelated gene sequences. Nucleoids from rat hepatocytes were treated with DNase I (0.5 U/ml) for different times. The residual NM-bound DNA in the partially digested samples was directly used as template for PCR amplification of small target sequences (Table 2) located in the 5' ends of the corresponding genes (Fyn, CD23, GFAP and MPZ). Each of such genes is located in a different chromosome and thus they represent separate chromosome territories within the nucleus. The specific amplicons were resolved in 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). CT, control G0 hepatocytes; PHx-24 h, hepatocytes 24 h after partial hepatectomy; PHx-7 D, hepatocytes 7 days after partial hepatectomy C, 0' digestion-time control. Topological zones relative to the NM: D, distal; P, proximal; VC, very close; E, embedded within the NM. (-) Negative control (no template); (+) positive control (pure genomic DNA as template). The amplification patterns were consistently reproduced in separate experiments with samples from independent animals (n ≥ 3).
Mentions: The fifteen sequences analyzed so far belong to a genomic region that comprises three genes and one pseudo-gene of the albumin gene family (Figure 3) and are located within five consecutive structural DNA loops corresponding to 162 kb of chromosome 14 in the rat (Figure 5A). Thus, we also mapped the position relative to the NM of four target- sequences corresponding to four unrelated genes each located in a different rat chromosome (Table 5) in order to corroborate whether nuclear DNA moves towards the NM during DNA replication. Thus amplicons belonging to the 5' ends of the genes GFAP, CD23 (also known as Feer2), MPZ and Fyn were positionally mapped relative to the NM in G0, PHx-24 and PHx-7D samples. The results indicate that all target sequences shift from their original locations to a topological zone closer to the NM at 24 h after partial-hepatectomy when the peak of DNA synthesis occurs, and then recover their original positions relative to the NM as shown in PHx-7D samples obtained when the liver regeneration is complete (Figure 7 and Table 6). This suggests that movement of DNA towards the NM is a general nuclear phenomenon during DNA synthesis in vivo and supports the notion that during replication it is the DNA template that moves towards the replication factories on the NM.

Bottom Line: We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated.These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, CP 50000 Toluca, Edo Méx, México.

ABSTRACT

Background: In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results: In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions: Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

Show MeSH
Related in: MedlinePlus