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Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

Inácio V, Rocheta M, Morais-Cecílio L - PLoS ONE (2014)

Bottom Line: In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak.Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea.These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

View Article: PubMed Central - PubMed

Affiliation: Centre for Botany Applied to Agriculture (CBAA), Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal.

ABSTRACT
The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

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Structural organization of the nuclear-encoded 18-5.8-25S rDNA tandem repeats in F. sylvatica and Q. suber.IGS – intergenic spacer; * 3′-ETS - 3′ External transcribed spacer is 18 bp long; TTS - transcription termination site; NTS – non-transcribed spacer; TIS - transcription initiation site; 5′-ETS – 5′ external transcribed; ITS – internal transcribed spacer; B1, B2, and B3 - Bam HI restriction sites. IGS1 and IGS2 - primers used in the IGS amplification.
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pone-0098678-g001: Structural organization of the nuclear-encoded 18-5.8-25S rDNA tandem repeats in F. sylvatica and Q. suber.IGS – intergenic spacer; * 3′-ETS - 3′ External transcribed spacer is 18 bp long; TTS - transcription termination site; NTS – non-transcribed spacer; TIS - transcription initiation site; 5′-ETS – 5′ external transcribed; ITS – internal transcribed spacer; B1, B2, and B3 - Bam HI restriction sites. IGS1 and IGS2 - primers used in the IGS amplification.

Mentions: Genomic DNA (20 µg) from all species was digested with Bam HI (Roche, Switzerland) in order to analyze inter-specific IGS length variations. Digested DNA was separated by electrophoresis at 40 kV (1 kV/cm) overnight on a 0.8% agarose gel and then blotted into Hybond-N (GE Healthcare, UK). Dig High Prime DNA labelling and Detection Starter Kit I (Roche) was used to hybridize the membrane according to manufacturer's instructions. One of the IGS sequenced clones from F. sylvatica and Q. suber were used as probes. Bam HI activity is inhibited by the presence of 5- or 4-methylcytosine at the internal C residue indicated (*) in the recognition sequence GGATC*C. A methylation-sensitive quantitative PCR assay was performed according to [12] using primers flanking the 25S or the 18S Bam HI restriction sites (B2 and B3, Figure 1, Table S3). Briefly, 50 ng of genomic DNA from all species were digested in a volume of 30 µL with 5 units of Bam HI for 2 h while a mock digestion with no enzyme was performed in parallel. PCR reactions were performed in optical 96-well plates with an IQTM5 Real Time PCR (Bio-Rad, Hercules, CA). Two different samples of the same species were used, and each PCR reaction was done in triplicate. The 20 µL reaction mixture was composed of digested or undigested DNA diluted 1000 times, 0.2 mM gene-specific primers (Table S3), and 2× master mix SsoFastTM_EvaGreen Supermix, Bio-Rad, Hercules, CA). Amplification of PCR products was monitored via intercalation of Eva-Green (included in the master mix). The following program was applied: 95°C for 3 min; then 40 cycles at 95°C for 45 s, 62°C for 45 s, and 72°C for 1 min and a final extension at 72°C for 5 min. Each run was completed with a melting curve analysis to confirm the specificity of amplification and the lack of primer dimers. Changes in the Ct values (ΔCt) of the digested templates are expressed relatively to the undigested samples. Taking into account that about a 2-fold increase in the amount of product results from each successive round of PCR amplification, a ΔCt of 1, 2 and 3 refers to 50%, 75% and 87.5% of template cleavage, respectively. The relationship between the ΔCt and the percentage of methylation can then be described as %Methylation = 100(e−0.7(ΔCt)).


Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

Inácio V, Rocheta M, Morais-Cecílio L - PLoS ONE (2014)

Structural organization of the nuclear-encoded 18-5.8-25S rDNA tandem repeats in F. sylvatica and Q. suber.IGS – intergenic spacer; * 3′-ETS - 3′ External transcribed spacer is 18 bp long; TTS - transcription termination site; NTS – non-transcribed spacer; TIS - transcription initiation site; 5′-ETS – 5′ external transcribed; ITS – internal transcribed spacer; B1, B2, and B3 - Bam HI restriction sites. IGS1 and IGS2 - primers used in the IGS amplification.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043768&req=5

pone-0098678-g001: Structural organization of the nuclear-encoded 18-5.8-25S rDNA tandem repeats in F. sylvatica and Q. suber.IGS – intergenic spacer; * 3′-ETS - 3′ External transcribed spacer is 18 bp long; TTS - transcription termination site; NTS – non-transcribed spacer; TIS - transcription initiation site; 5′-ETS – 5′ external transcribed; ITS – internal transcribed spacer; B1, B2, and B3 - Bam HI restriction sites. IGS1 and IGS2 - primers used in the IGS amplification.
Mentions: Genomic DNA (20 µg) from all species was digested with Bam HI (Roche, Switzerland) in order to analyze inter-specific IGS length variations. Digested DNA was separated by electrophoresis at 40 kV (1 kV/cm) overnight on a 0.8% agarose gel and then blotted into Hybond-N (GE Healthcare, UK). Dig High Prime DNA labelling and Detection Starter Kit I (Roche) was used to hybridize the membrane according to manufacturer's instructions. One of the IGS sequenced clones from F. sylvatica and Q. suber were used as probes. Bam HI activity is inhibited by the presence of 5- or 4-methylcytosine at the internal C residue indicated (*) in the recognition sequence GGATC*C. A methylation-sensitive quantitative PCR assay was performed according to [12] using primers flanking the 25S or the 18S Bam HI restriction sites (B2 and B3, Figure 1, Table S3). Briefly, 50 ng of genomic DNA from all species were digested in a volume of 30 µL with 5 units of Bam HI for 2 h while a mock digestion with no enzyme was performed in parallel. PCR reactions were performed in optical 96-well plates with an IQTM5 Real Time PCR (Bio-Rad, Hercules, CA). Two different samples of the same species were used, and each PCR reaction was done in triplicate. The 20 µL reaction mixture was composed of digested or undigested DNA diluted 1000 times, 0.2 mM gene-specific primers (Table S3), and 2× master mix SsoFastTM_EvaGreen Supermix, Bio-Rad, Hercules, CA). Amplification of PCR products was monitored via intercalation of Eva-Green (included in the master mix). The following program was applied: 95°C for 3 min; then 40 cycles at 95°C for 45 s, 62°C for 45 s, and 72°C for 1 min and a final extension at 72°C for 5 min. Each run was completed with a melting curve analysis to confirm the specificity of amplification and the lack of primer dimers. Changes in the Ct values (ΔCt) of the digested templates are expressed relatively to the undigested samples. Taking into account that about a 2-fold increase in the amount of product results from each successive round of PCR amplification, a ΔCt of 1, 2 and 3 refers to 50%, 75% and 87.5% of template cleavage, respectively. The relationship between the ΔCt and the percentage of methylation can then be described as %Methylation = 100(e−0.7(ΔCt)).

Bottom Line: In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak.Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea.These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

View Article: PubMed Central - PubMed

Affiliation: Centre for Botany Applied to Agriculture (CBAA), Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal.

ABSTRACT
The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

Show MeSH