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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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Quantitative analysis of v-rDNA expression.A, RNA samples from two hbyrids (C57BL/129Sv) were assayed with qPCR, the expression level was normalized with the transcript of beta-actin. (n = 2). Note that because V was not detected, only three bars are visible. B, In another experiment, the v-rDNA expression level was normalized with the 47S pre-rRNA (represented by the full circle) to show the contribution of each v-rDNA to the pre-rRNA pool. The values are average of two measurements. The blank sector is the portion of pre-rRNA not accounted for by the v-rDNA transcripts. C, A comparison of RNA and DNA quantity ratios. Ct values of RNA and DNA are superimposed on the standard curves of the two qPCRs for v-rDNA IV and V. Ct values of serial dilutions of cloned DNA (IV, black triangles; and V, open circles) are shown with the standard curve. RNA (transcript) Ct values are shown by squares (IV, black). The value of v-rDNA V is out of range, thus not plotted, but the average and standard deviation are indicated. Genomic DNA measurements are depicted as diamonds (IV, black and V, open). The average and standard deviation of 2 samples, each measured twice, are indicated by pins; circle-ended pins for variant IV and diamond-ended ones, V.
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pone-0001843-g007: Quantitative analysis of v-rDNA expression.A, RNA samples from two hbyrids (C57BL/129Sv) were assayed with qPCR, the expression level was normalized with the transcript of beta-actin. (n = 2). Note that because V was not detected, only three bars are visible. B, In another experiment, the v-rDNA expression level was normalized with the 47S pre-rRNA (represented by the full circle) to show the contribution of each v-rDNA to the pre-rRNA pool. The values are average of two measurements. The blank sector is the portion of pre-rRNA not accounted for by the v-rDNA transcripts. C, A comparison of RNA and DNA quantity ratios. Ct values of RNA and DNA are superimposed on the standard curves of the two qPCRs for v-rDNA IV and V. Ct values of serial dilutions of cloned DNA (IV, black triangles; and V, open circles) are shown with the standard curve. RNA (transcript) Ct values are shown by squares (IV, black). The value of v-rDNA V is out of range, thus not plotted, but the average and standard deviation are indicated. Genomic DNA measurements are depicted as diamonds (IV, black and V, open). The average and standard deviation of 2 samples, each measured twice, are indicated by pins; circle-ended pins for variant IV and diamond-ended ones, V.

Mentions: Using quantitative PCR (qPCR), we investigated the relative transcript level of each v-rDNA in brain, skin, and testis (Fig. 7A). The relative level of rDNA expression was higher in the brain and testis than that of skin. This study also suggested that in these three tissues, each v-rDNA contributed similarly to the pre-rRNA pool. This conclusion was confirmed when the level of v-rDNA transcripts was normalized against the level of the 47S pre-rRNA. Interestingly, the sum of the five v-rDNAs (i.e., I, II, IV, V and VI) could account for no more than 75% of the 47S pre-rRNA. Of the two v-rDNAs (i.e., III and VII) that were not measured because of technical difficulties, III was expressed at a low level and VII was not expressed at all (Fig. 6). Thus neither v-rDNA could make up the missing 25% of the pre-rRNA transcript. This observation suggested contributions from yet identified v-rDNAs.


Mouse ribosomal RNA genes contain multiple differentially regulated variants.

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

Quantitative analysis of v-rDNA expression.A, RNA samples from two hbyrids (C57BL/129Sv) were assayed with qPCR, the expression level was normalized with the transcript of beta-actin. (n = 2). Note that because V was not detected, only three bars are visible. B, In another experiment, the v-rDNA expression level was normalized with the 47S pre-rRNA (represented by the full circle) to show the contribution of each v-rDNA to the pre-rRNA pool. The values are average of two measurements. The blank sector is the portion of pre-rRNA not accounted for by the v-rDNA transcripts. C, A comparison of RNA and DNA quantity ratios. Ct values of RNA and DNA are superimposed on the standard curves of the two qPCRs for v-rDNA IV and V. Ct values of serial dilutions of cloned DNA (IV, black triangles; and V, open circles) are shown with the standard curve. RNA (transcript) Ct values are shown by squares (IV, black). The value of v-rDNA V is out of range, thus not plotted, but the average and standard deviation are indicated. Genomic DNA measurements are depicted as diamonds (IV, black and V, open). The average and standard deviation of 2 samples, each measured twice, are indicated by pins; circle-ended pins for variant IV and diamond-ended ones, V.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266999&req=5

pone-0001843-g007: Quantitative analysis of v-rDNA expression.A, RNA samples from two hbyrids (C57BL/129Sv) were assayed with qPCR, the expression level was normalized with the transcript of beta-actin. (n = 2). Note that because V was not detected, only three bars are visible. B, In another experiment, the v-rDNA expression level was normalized with the 47S pre-rRNA (represented by the full circle) to show the contribution of each v-rDNA to the pre-rRNA pool. The values are average of two measurements. The blank sector is the portion of pre-rRNA not accounted for by the v-rDNA transcripts. C, A comparison of RNA and DNA quantity ratios. Ct values of RNA and DNA are superimposed on the standard curves of the two qPCRs for v-rDNA IV and V. Ct values of serial dilutions of cloned DNA (IV, black triangles; and V, open circles) are shown with the standard curve. RNA (transcript) Ct values are shown by squares (IV, black). The value of v-rDNA V is out of range, thus not plotted, but the average and standard deviation are indicated. Genomic DNA measurements are depicted as diamonds (IV, black and V, open). The average and standard deviation of 2 samples, each measured twice, are indicated by pins; circle-ended pins for variant IV and diamond-ended ones, V.
Mentions: Using quantitative PCR (qPCR), we investigated the relative transcript level of each v-rDNA in brain, skin, and testis (Fig. 7A). The relative level of rDNA expression was higher in the brain and testis than that of skin. This study also suggested that in these three tissues, each v-rDNA contributed similarly to the pre-rRNA pool. This conclusion was confirmed when the level of v-rDNA transcripts was normalized against the level of the 47S pre-rRNA. Interestingly, the sum of the five v-rDNAs (i.e., I, II, IV, V and VI) could account for no more than 75% of the 47S pre-rRNA. Of the two v-rDNAs (i.e., III and VII) that were not measured because of technical difficulties, III was expressed at a low level and VII was not expressed at all (Fig. 6). Thus neither v-rDNA could make up the missing 25% of the pre-rRNA transcript. This observation suggested contributions from yet identified v-rDNAs.

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