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Mouse ribosomal RNA genes contain multiple differentially regulated variants.

Tseng H, Chou W, Wang J, Zhang X, Zhang S, Schultz RM - PLoS ONE (2008)

Bottom Line: However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful.These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent).Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America. htsengpe@mail.med.upenn.edu

ABSTRACT
Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA.

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Molecular cloning of rDNA variants from CF1 mouse.A, An example of rDNA RFLPs revealed by Southern analyses of CF1 liver genomic DNA. The Southern blot was probed with a DNA fragment from the promoter-leader region of rDNA. A molecular weight marker (mw) is included (lane 5) and the molecular weights indicated to the left. Bands of size predicted by the published rDNA sequence (BX000964) are indicated by arrowheads. A serial copy number marker is indicated by the copy numbers each band contains (i.e., 100, 10, 1). The copy number was calculated as described in Tian et al., 2001, using 2.7×109 bp for the haploid mouse genome. The fragments, whose copy numbers were assessed and described in the text, are alphabetically labeled (lanes 10 and 11). The restriction enzymes used to generate A are the most informative among the thirty enzymes tested. They are listed as they appear in B (i.e., lane 1, BamHI, lane 2, Bgl II, with the exception of lane 5). B, A summary of the RFLPs detected by the Southern analyses. A generic rDNA (partial) is depicted in the middle, with transcription start site (bent arrow), the coding sequence (18S and 28S) and the hybridization probe (hammers) indicated. The polymorphism or lack thereof is depicted by circle-ended grey lines and black lines, respectively. The number of polymorphisms for a given restriction fragment is shown in parentheses. The gray area indicates the variable region around the transcription start site. C, The promoter region RFLP is stable during organ development. Genomic DNA was isolated from tissues of five unrelated mice, three males (M1, 2 and 3) and two females (F1 and 2) and analyzed by Southern blots. Tissue genomic DNAs were digested with one of the four enzymes (Bsp E1, Stu I, Sty I and NgoM IV) as indicated to the left of the gel image. The blots were probed with the same rDNA promoter fragment as shown in A. SP, spleen, K, kidney, T, testis, L, liver, I, intestine, SK, skin. Note that no variation is detected among the tissues of an individual. D, A restriction map indicates the cutting sites of relevant enzymes. The thicker line depicts the target fragment for cloning. The indicated size (∼6 kb) is that predicted by the published sequence. E, Liver genomic DNA was double-digested with Bcl I and another restriction enzyme and probed with the promoter-leader probe. Bcl I-Pst I digestion yielded three groups of bands, which were purified individually by size-fractionation and used to generate libraries designated A, B and C, respectively. F, Seven variant rDNAs (v-rDNA) were cloned and each contained an internal BspE1 fragment (lanes 1 to 7) that matched a BspE1 RFLP of the genomic DNA (lane G). When mixed as a group, the BspE1 digestion pattern of these v-rDNA clones (lane 8) resembles that of the genomic digestion (lane G). Note one v-rDNA RFLP was not isolated (band E, arrow head in lane G).
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pone-0001843-g001: Molecular cloning of rDNA variants from CF1 mouse.A, An example of rDNA RFLPs revealed by Southern analyses of CF1 liver genomic DNA. The Southern blot was probed with a DNA fragment from the promoter-leader region of rDNA. A molecular weight marker (mw) is included (lane 5) and the molecular weights indicated to the left. Bands of size predicted by the published rDNA sequence (BX000964) are indicated by arrowheads. A serial copy number marker is indicated by the copy numbers each band contains (i.e., 100, 10, 1). The copy number was calculated as described in Tian et al., 2001, using 2.7×109 bp for the haploid mouse genome. The fragments, whose copy numbers were assessed and described in the text, are alphabetically labeled (lanes 10 and 11). The restriction enzymes used to generate A are the most informative among the thirty enzymes tested. They are listed as they appear in B (i.e., lane 1, BamHI, lane 2, Bgl II, with the exception of lane 5). B, A summary of the RFLPs detected by the Southern analyses. A generic rDNA (partial) is depicted in the middle, with transcription start site (bent arrow), the coding sequence (18S and 28S) and the hybridization probe (hammers) indicated. The polymorphism or lack thereof is depicted by circle-ended grey lines and black lines, respectively. The number of polymorphisms for a given restriction fragment is shown in parentheses. The gray area indicates the variable region around the transcription start site. C, The promoter region RFLP is stable during organ development. Genomic DNA was isolated from tissues of five unrelated mice, three males (M1, 2 and 3) and two females (F1 and 2) and analyzed by Southern blots. Tissue genomic DNAs were digested with one of the four enzymes (Bsp E1, Stu I, Sty I and NgoM IV) as indicated to the left of the gel image. The blots were probed with the same rDNA promoter fragment as shown in A. SP, spleen, K, kidney, T, testis, L, liver, I, intestine, SK, skin. Note that no variation is detected among the tissues of an individual. D, A restriction map indicates the cutting sites of relevant enzymes. The thicker line depicts the target fragment for cloning. The indicated size (∼6 kb) is that predicted by the published sequence. E, Liver genomic DNA was double-digested with Bcl I and another restriction enzyme and probed with the promoter-leader probe. Bcl I-Pst I digestion yielded three groups of bands, which were purified individually by size-fractionation and used to generate libraries designated A, B and C, respectively. F, Seven variant rDNAs (v-rDNA) were cloned and each contained an internal BspE1 fragment (lanes 1 to 7) that matched a BspE1 RFLP of the genomic DNA (lane G). When mixed as a group, the BspE1 digestion pattern of these v-rDNA clones (lane 8) resembles that of the genomic digestion (lane G). Note one v-rDNA RFLP was not isolated (band E, arrow head in lane G).

Mentions: The RFLPs in the non-transcribed spacer region of rDNA were noted three decades ago [8], [9], [28]. To identify rDNA RFLPs that could be used in isolating potential rDNA variants (v-rDNA), we examined the RFLPs of thirty restriction enzymes for their size distribution and stability during organogenesis. For size distribution analysis, mouse liver genomic DNA (Strain CF1) was cut in single or double restriction digestions and subjected to Southern analyses (Fig. 1A). Three hybridization probes were used to examine 1) the promoter and the transcript-leader region (referred to as promoter-leader), 2) the 18S, and 3) 28S rRNA coding regions (Fig. 1B). Some restriction digestions were not informative (i.e., they cut at either too many or too few sites). Only the informative digestions are shown in Fig. 1. In general, the promoter-leader region contained many RFLPs (Fig. 1A) and the number of RFLPs varied from two to seven (e.g., Fig. 1A lanes 4 and 8, respectively). In contrast, the rRNA coding regions (18S and 28S) contained no RFLP, i.e., the fragment size predicted by the known sequence (BK000964) agreed well with the size of the bands (Southern data not shown but summarized in Fig. 1B, black lines). These Southern analyses confirmed previous conclusions that most of the RFLPs were near the transcription start site or up-stream from it (Fig. 1B, gray area).


Mouse ribosomal RNA genes contain multiple differentially regulated variants.

Tseng H, Chou W, Wang J, Zhang X, Zhang S, Schultz RM - PLoS ONE (2008)

Molecular cloning of rDNA variants from CF1 mouse.A, An example of rDNA RFLPs revealed by Southern analyses of CF1 liver genomic DNA. The Southern blot was probed with a DNA fragment from the promoter-leader region of rDNA. A molecular weight marker (mw) is included (lane 5) and the molecular weights indicated to the left. Bands of size predicted by the published rDNA sequence (BX000964) are indicated by arrowheads. A serial copy number marker is indicated by the copy numbers each band contains (i.e., 100, 10, 1). The copy number was calculated as described in Tian et al., 2001, using 2.7×109 bp for the haploid mouse genome. The fragments, whose copy numbers were assessed and described in the text, are alphabetically labeled (lanes 10 and 11). The restriction enzymes used to generate A are the most informative among the thirty enzymes tested. They are listed as they appear in B (i.e., lane 1, BamHI, lane 2, Bgl II, with the exception of lane 5). B, A summary of the RFLPs detected by the Southern analyses. A generic rDNA (partial) is depicted in the middle, with transcription start site (bent arrow), the coding sequence (18S and 28S) and the hybridization probe (hammers) indicated. The polymorphism or lack thereof is depicted by circle-ended grey lines and black lines, respectively. The number of polymorphisms for a given restriction fragment is shown in parentheses. The gray area indicates the variable region around the transcription start site. C, The promoter region RFLP is stable during organ development. Genomic DNA was isolated from tissues of five unrelated mice, three males (M1, 2 and 3) and two females (F1 and 2) and analyzed by Southern blots. Tissue genomic DNAs were digested with one of the four enzymes (Bsp E1, Stu I, Sty I and NgoM IV) as indicated to the left of the gel image. The blots were probed with the same rDNA promoter fragment as shown in A. SP, spleen, K, kidney, T, testis, L, liver, I, intestine, SK, skin. Note that no variation is detected among the tissues of an individual. D, A restriction map indicates the cutting sites of relevant enzymes. The thicker line depicts the target fragment for cloning. The indicated size (∼6 kb) is that predicted by the published sequence. E, Liver genomic DNA was double-digested with Bcl I and another restriction enzyme and probed with the promoter-leader probe. Bcl I-Pst I digestion yielded three groups of bands, which were purified individually by size-fractionation and used to generate libraries designated A, B and C, respectively. F, Seven variant rDNAs (v-rDNA) were cloned and each contained an internal BspE1 fragment (lanes 1 to 7) that matched a BspE1 RFLP of the genomic DNA (lane G). When mixed as a group, the BspE1 digestion pattern of these v-rDNA clones (lane 8) resembles that of the genomic digestion (lane G). Note one v-rDNA RFLP was not isolated (band E, arrow head in lane G).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266999&req=5

pone-0001843-g001: Molecular cloning of rDNA variants from CF1 mouse.A, An example of rDNA RFLPs revealed by Southern analyses of CF1 liver genomic DNA. The Southern blot was probed with a DNA fragment from the promoter-leader region of rDNA. A molecular weight marker (mw) is included (lane 5) and the molecular weights indicated to the left. Bands of size predicted by the published rDNA sequence (BX000964) are indicated by arrowheads. A serial copy number marker is indicated by the copy numbers each band contains (i.e., 100, 10, 1). The copy number was calculated as described in Tian et al., 2001, using 2.7×109 bp for the haploid mouse genome. The fragments, whose copy numbers were assessed and described in the text, are alphabetically labeled (lanes 10 and 11). The restriction enzymes used to generate A are the most informative among the thirty enzymes tested. They are listed as they appear in B (i.e., lane 1, BamHI, lane 2, Bgl II, with the exception of lane 5). B, A summary of the RFLPs detected by the Southern analyses. A generic rDNA (partial) is depicted in the middle, with transcription start site (bent arrow), the coding sequence (18S and 28S) and the hybridization probe (hammers) indicated. The polymorphism or lack thereof is depicted by circle-ended grey lines and black lines, respectively. The number of polymorphisms for a given restriction fragment is shown in parentheses. The gray area indicates the variable region around the transcription start site. C, The promoter region RFLP is stable during organ development. Genomic DNA was isolated from tissues of five unrelated mice, three males (M1, 2 and 3) and two females (F1 and 2) and analyzed by Southern blots. Tissue genomic DNAs were digested with one of the four enzymes (Bsp E1, Stu I, Sty I and NgoM IV) as indicated to the left of the gel image. The blots were probed with the same rDNA promoter fragment as shown in A. SP, spleen, K, kidney, T, testis, L, liver, I, intestine, SK, skin. Note that no variation is detected among the tissues of an individual. D, A restriction map indicates the cutting sites of relevant enzymes. The thicker line depicts the target fragment for cloning. The indicated size (∼6 kb) is that predicted by the published sequence. E, Liver genomic DNA was double-digested with Bcl I and another restriction enzyme and probed with the promoter-leader probe. Bcl I-Pst I digestion yielded three groups of bands, which were purified individually by size-fractionation and used to generate libraries designated A, B and C, respectively. F, Seven variant rDNAs (v-rDNA) were cloned and each contained an internal BspE1 fragment (lanes 1 to 7) that matched a BspE1 RFLP of the genomic DNA (lane G). When mixed as a group, the BspE1 digestion pattern of these v-rDNA clones (lane 8) resembles that of the genomic digestion (lane G). Note one v-rDNA RFLP was not isolated (band E, arrow head in lane G).
Mentions: The RFLPs in the non-transcribed spacer region of rDNA were noted three decades ago [8], [9], [28]. To identify rDNA RFLPs that could be used in isolating potential rDNA variants (v-rDNA), we examined the RFLPs of thirty restriction enzymes for their size distribution and stability during organogenesis. For size distribution analysis, mouse liver genomic DNA (Strain CF1) was cut in single or double restriction digestions and subjected to Southern analyses (Fig. 1A). Three hybridization probes were used to examine 1) the promoter and the transcript-leader region (referred to as promoter-leader), 2) the 18S, and 3) 28S rRNA coding regions (Fig. 1B). Some restriction digestions were not informative (i.e., they cut at either too many or too few sites). Only the informative digestions are shown in Fig. 1. In general, the promoter-leader region contained many RFLPs (Fig. 1A) and the number of RFLPs varied from two to seven (e.g., Fig. 1A lanes 4 and 8, respectively). In contrast, the rRNA coding regions (18S and 28S) contained no RFLP, i.e., the fragment size predicted by the known sequence (BK000964) agreed well with the size of the bands (Southern data not shown but summarized in Fig. 1B, black lines). These Southern analyses confirmed previous conclusions that most of the RFLPs were near the transcription start site or up-stream from it (Fig. 1B, gray area).

Bottom Line: However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful.These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent).Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America. htsengpe@mail.med.upenn.edu

ABSTRACT
Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA.

Show MeSH
Related in: MedlinePlus