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Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach.

Rivera-Mulia JC, Aranda-Anzaldo A - DNA Res. (2010)

Bottom Line: Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence.The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced.We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

View Article: PubMed Central - PubMed

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

ABSTRACT
Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

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The 162-kb genomic region containing members of the rat albumin gene family (chromosome 14): Alb (albumin), Afp (alpha-fetoprotein), Afm (afamin), Afp-L (pseudo-gene similar to Afp). The letters indicate the location of the target DNA sequences to be positionally mapped relative to the NM.
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DSP027F4: The 162-kb genomic region containing members of the rat albumin gene family (chromosome 14): Alb (albumin), Afp (alpha-fetoprotein), Afm (afamin), Afp-L (pseudo-gene similar to Afp). The letters indicate the location of the target DNA sequences to be positionally mapped relative to the NM.

Mentions: On the basis of the above considerations and results, we used the general strategy depicted in Fig. 3 in order to map the position relative to the NM of 15 small DNA sequences located along a region corresponding to 162 kb of chromosome 14 in the rat, which includes the loci of the following gene members of the albumin gene family: albumin (Alb), alpha-fetoprotein (Afp), afamin (Afm) and the pseudo-gene similar to Afp (Afp-L). Such small DNA sequences were spaced some 10 kb apart (Fig. 4). Therefore, nucleoids prepared from freshly isolated hepatocytes or naïve B-lymphocytes (Fig. 3A) were incubated with DNase I (0.5 U/ml) for different times so as to obtain nucleoid samples with differential amounts of DNA associated with the NM (Fig. 3B and C), and such samples with partially digested NM-bound DNA were used for PCR amplification of the chosen target sequences located along the 162-kb genomic region under study (Fig. 3A). By correlating the amplification data (Fig. 3A) with the kinetics of the nucleoid-DNA digestion (Fig. 3B), it is possible to locate each target sequence within a topological zone relative to the NM (Fig. 2). Previous studies have shown that the average size of the nuclear DNA fragments liberated by non-specific nucleases in rat hepatocytes is 0.8 kb.12 Thus, the DNA sequences to be mapped are <550 bp in length (Table 1), and so likely to be cut as whole units by the endonuclease instead of being progressively eroded by partial digestions. Therefore, in our mapping protocol, we score the specific templates as either present (amplifiable) or absent (non-amplifiable) as a function of endonuclease-digestion time (Fig. 5 and Supplementary Fig. S1). This was scored without considering the intensity of the amplicon signals, but just whether such signals are detected or not by an image-analysis program (Kodak 1D Image Analysis Software 3.5), using the default settings. We established these criteria because in our topological-mapping approach, it is the average relative position to the NM anchoring point and not the actual template length the critical parameter that determines the average sensitivity to DNase I of each sequence mapped. Nevertheless, the chosen amplicons were also selected on the basis that all of them may be amplified with similar efficiency using the same amplification program (see Section 2). Thus, the absence of amplified product at a given digestion time-point indicates that the relative abundance of the target template has fallen to a non-amplifiable level within the large nucleoid population analysed in each sample.6


Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach.

Rivera-Mulia JC, Aranda-Anzaldo A - DNA Res. (2010)

The 162-kb genomic region containing members of the rat albumin gene family (chromosome 14): Alb (albumin), Afp (alpha-fetoprotein), Afm (afamin), Afp-L (pseudo-gene similar to Afp). The letters indicate the location of the target DNA sequences to be positionally mapped relative to the NM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2818189&req=5

DSP027F4: The 162-kb genomic region containing members of the rat albumin gene family (chromosome 14): Alb (albumin), Afp (alpha-fetoprotein), Afm (afamin), Afp-L (pseudo-gene similar to Afp). The letters indicate the location of the target DNA sequences to be positionally mapped relative to the NM.
Mentions: On the basis of the above considerations and results, we used the general strategy depicted in Fig. 3 in order to map the position relative to the NM of 15 small DNA sequences located along a region corresponding to 162 kb of chromosome 14 in the rat, which includes the loci of the following gene members of the albumin gene family: albumin (Alb), alpha-fetoprotein (Afp), afamin (Afm) and the pseudo-gene similar to Afp (Afp-L). Such small DNA sequences were spaced some 10 kb apart (Fig. 4). Therefore, nucleoids prepared from freshly isolated hepatocytes or naïve B-lymphocytes (Fig. 3A) were incubated with DNase I (0.5 U/ml) for different times so as to obtain nucleoid samples with differential amounts of DNA associated with the NM (Fig. 3B and C), and such samples with partially digested NM-bound DNA were used for PCR amplification of the chosen target sequences located along the 162-kb genomic region under study (Fig. 3A). By correlating the amplification data (Fig. 3A) with the kinetics of the nucleoid-DNA digestion (Fig. 3B), it is possible to locate each target sequence within a topological zone relative to the NM (Fig. 2). Previous studies have shown that the average size of the nuclear DNA fragments liberated by non-specific nucleases in rat hepatocytes is 0.8 kb.12 Thus, the DNA sequences to be mapped are <550 bp in length (Table 1), and so likely to be cut as whole units by the endonuclease instead of being progressively eroded by partial digestions. Therefore, in our mapping protocol, we score the specific templates as either present (amplifiable) or absent (non-amplifiable) as a function of endonuclease-digestion time (Fig. 5 and Supplementary Fig. S1). This was scored without considering the intensity of the amplicon signals, but just whether such signals are detected or not by an image-analysis program (Kodak 1D Image Analysis Software 3.5), using the default settings. We established these criteria because in our topological-mapping approach, it is the average relative position to the NM anchoring point and not the actual template length the critical parameter that determines the average sensitivity to DNase I of each sequence mapped. Nevertheless, the chosen amplicons were also selected on the basis that all of them may be amplified with similar efficiency using the same amplification program (see Section 2). Thus, the absence of amplified product at a given digestion time-point indicates that the relative abundance of the target template has fallen to a non-amplifiable level within the large nucleoid population analysed in each sample.6

Bottom Line: Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence.The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced.We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

View Article: PubMed Central - PubMed

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

ABSTRACT
Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

Show MeSH
Related in: MedlinePlus