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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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Variant-specific PCRs.A, Locations of the amplicons are shown in reference to the 5′-ETS. The large arrow depicts transcription start site and direction; asterisk, the first cleavage site in processing the pre-rRNA; small arrows, PCR primers; and the Roman numerals, PCR specificity. B, The specificity of each variant-specific PCR was tested with cloned v-rDNAs. The PCR was also adjusted, using cloned v-rDNA, to have similar amplification efficiency. Lowercase letters on top of the gel image indicate cloned templates, and uppercase letters to the left of image, the specificity of primers, m, molecular weight marker. C, An example of the specificity of qPCR for variant IV is shown (SYBG fluorescence). The delta Ct between specific and non-specific reactions is greater than 10.
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pone-0001843-g005: Variant-specific PCRs.A, Locations of the amplicons are shown in reference to the 5′-ETS. The large arrow depicts transcription start site and direction; asterisk, the first cleavage site in processing the pre-rRNA; small arrows, PCR primers; and the Roman numerals, PCR specificity. B, The specificity of each variant-specific PCR was tested with cloned v-rDNAs. The PCR was also adjusted, using cloned v-rDNA, to have similar amplification efficiency. Lowercase letters on top of the gel image indicate cloned templates, and uppercase letters to the left of image, the specificity of primers, m, molecular weight marker. C, An example of the specificity of qPCR for variant IV is shown (SYBG fluorescence). The delta Ct between specific and non-specific reactions is greater than 10.

Mentions: Multi-sequence alignment showed variant-specific SNPs in the transcribed region of v-rDNA (Fig. 3B). Statistical analysis of the SNPs in the 26 v-rDNA clones confirmed the specificity of these SNPs and suggested that by employing two such SNPs, highly specific PCR assays could be developed for each v-rDNA type (Table 2). Based on these observations, we developed a set of variant-specific PCR assays. The variant-specific SNPs were examined for their specificity and location within the transcribed region. Six sets of SNPs were chosen to design the PCR primers (Table 2, Fig. 5A and Methods S1). In these PCR primers, the SNPs were the 3′ nucleotide matching to the sequence of its targeted v-rDNA. An exception was that for the 3′ primer of v-rDNA III, the specific nucleotide was in the middle of the primer (Methods S1). The primers were also selected to flank the first excision site of the pre-rRNA [34] to ensure amplification of only the full-length transcript (Fig. 5A). Using the cloned v-rDNA as templates, the PCR conditions were optimized so that each reaction amplified its intended target v-rDNA with high specificity (Fig. 5B). The sequences of v-rDNA I and II were very closely related and no primer could distinguish them and these two v-rDNA types were assayed together. Using the cloned v-rDNA as templates, the efficiencies of the variant-specific PCRs were adjusted to be similar. Some of these reactions (i.e., I+II, IV, V, VI) were adapted for a real-time PCR assay by adding SYBR green to the PCR reaction (Fig. 5C). Other reactions (i.e., III and VII) could not be adapted because their specificity deteriorated in the presence of SYBR, i.e., delta-Ct value for specific and non-specific templates was less than 10.


Mouse ribosomal RNA genes contain multiple differentially regulated variants.

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

Variant-specific PCRs.A, Locations of the amplicons are shown in reference to the 5′-ETS. The large arrow depicts transcription start site and direction; asterisk, the first cleavage site in processing the pre-rRNA; small arrows, PCR primers; and the Roman numerals, PCR specificity. B, The specificity of each variant-specific PCR was tested with cloned v-rDNAs. The PCR was also adjusted, using cloned v-rDNA, to have similar amplification efficiency. Lowercase letters on top of the gel image indicate cloned templates, and uppercase letters to the left of image, the specificity of primers, m, molecular weight marker. C, An example of the specificity of qPCR for variant IV is shown (SYBG fluorescence). The delta Ct between specific and non-specific reactions is greater than 10.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001843-g005: Variant-specific PCRs.A, Locations of the amplicons are shown in reference to the 5′-ETS. The large arrow depicts transcription start site and direction; asterisk, the first cleavage site in processing the pre-rRNA; small arrows, PCR primers; and the Roman numerals, PCR specificity. B, The specificity of each variant-specific PCR was tested with cloned v-rDNAs. The PCR was also adjusted, using cloned v-rDNA, to have similar amplification efficiency. Lowercase letters on top of the gel image indicate cloned templates, and uppercase letters to the left of image, the specificity of primers, m, molecular weight marker. C, An example of the specificity of qPCR for variant IV is shown (SYBG fluorescence). The delta Ct between specific and non-specific reactions is greater than 10.
Mentions: Multi-sequence alignment showed variant-specific SNPs in the transcribed region of v-rDNA (Fig. 3B). Statistical analysis of the SNPs in the 26 v-rDNA clones confirmed the specificity of these SNPs and suggested that by employing two such SNPs, highly specific PCR assays could be developed for each v-rDNA type (Table 2). Based on these observations, we developed a set of variant-specific PCR assays. The variant-specific SNPs were examined for their specificity and location within the transcribed region. Six sets of SNPs were chosen to design the PCR primers (Table 2, Fig. 5A and Methods S1). In these PCR primers, the SNPs were the 3′ nucleotide matching to the sequence of its targeted v-rDNA. An exception was that for the 3′ primer of v-rDNA III, the specific nucleotide was in the middle of the primer (Methods S1). The primers were also selected to flank the first excision site of the pre-rRNA [34] to ensure amplification of only the full-length transcript (Fig. 5A). Using the cloned v-rDNA as templates, the PCR conditions were optimized so that each reaction amplified its intended target v-rDNA with high specificity (Fig. 5B). The sequences of v-rDNA I and II were very closely related and no primer could distinguish them and these two v-rDNA types were assayed together. Using the cloned v-rDNA as templates, the efficiencies of the variant-specific PCRs were adjusted to be similar. Some of these reactions (i.e., I+II, IV, V, VI) were adapted for a real-time PCR assay by adding SYBR green to the PCR reaction (Fig. 5C). Other reactions (i.e., III and VII) could not be adapted because their specificity deteriorated in the presence of SYBR, i.e., delta-Ct value for specific and non-specific templates was less than 10.

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