Separation of naturally occurring or induced methylated nucleobases of DNA by reversed-phase high-performance liquid chromatography

Valencia, Richard; Han, N'guyen Công; Bertaux, O

doi:10.1016/s0021-9673(00)96021-7

Cited by 14 publications

(9 citation statements)

References 29 publications

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“…The purified DNA was hydrolyzed in sealed glass tubes with 90% (vol/vol) formic acid (1 ptl/lxg of DNA) at 1750C for 30 min and subjected to analysis of base composition by reversedphase high-performance liquid chromatography (HPLC) (14). A Shimadzu LC-6A HPLC system was employed in combination with a reversed-phase column, uBondapak phenyl (300 x 3.9 mm, Waters Division of Millipore), which was placed after a Guard-Pak precolumn (Waters Division of Millipore).…”

Section: Methodsmentioning

confidence: 99%

DNA methylation as a mechanism of transcriptional regulation in nonphotosynthetic plastids in plant cells.

Ngernprasirtsiri¹,

Kobayashi²,

Akazawa³

1988

Proc. Natl. Acad. Sci. U.S.A.

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Transcription of amyloplast DNA in a heterotrophic line of cultured cells of sycamore (Acer pseudoplatanus L.) appeared to be greatly suppressed. A mutant cell line obtained from the heterotrophic line is green and autotrophic.Heavy modification of amyloplast DNA with a variety of methylated bases was demonstrated by analysis of the acid hydrolysate of DNA by high-performance liquid chromatography, but little modification of chloroplast DNA from the green line was detected. When plastid DNAs from the original and green cell lines were digested with methyl-sensitive restriction enzymes, DNA methylation was detected in regions containing the genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL), subunits of chloroplast coupling factor 1 (atpA, -B, and -E), the apoprotein of P700 (psaA), and ribosomal protein S4 (ips4) but not the genes for 16S rRNA and the 32-kDa QB protein (psbA) in the original line, whereas no methylation was observed in the green line. The genes for which methylation was not detectable were found to be active as templates for in vitro transcription by Escherichia coli RNA polymerase, but the methylated genes were apparently inactive. Methylation of DNA is a likely mechanism for the regulation of expression of amyloplast DNA in sycamore cells.

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Section: Methodsmentioning

confidence: 99%

DNA methylation as a mechanism of transcriptional regulation in nonphotosynthetic plastids in plant cells.

Ngernprasirtsiri¹,

Kobayashi²,

Akazawa³

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…In other studies C was detected [53,60,[93][94][95][96][97], while both bases, C and 5mC ( Table 1) were determined in a smaller number of cases [87][88][89][90][91], where genome-wide methylation was expressed as the ratio of 5mC vs. C.…”

Section: Minor Dna Bases Identified By Liquid Chromatographymentioning

confidence: 97%

“…Thus Deoxyadenosine, deoxyguanosine and thymidine [108] 7-methylguanine [94] Seven minor methylated bases [95] Seven deoxyribonucleoside monophosphates [56] Cytidine , uridine, [109] N N [60,[97][98][99][100][101][102][103][104][105][106][107][108][109][110] and deoxycytidine [60,[106][107][108][109][110] were determined in several studies, either separately or together [106,107]. In most of the above studies, separation was achieved with reversed-phase columns.…”

Section: Minor Dna Bases Identified By Liquid Chromatographymentioning

confidence: 99%

“…Several studies have shown that the four major DNA bases [93][94][95][96][97] can be detected by LC (Table 1). Moreover, the four corresponding deoxynucleosides have been simultaneously detected in some cases [109,110].…”

Section: -Methyldeoxycytidinementioning

confidence: 99%

“…The frequency of N6-methyladenine, which is another minor base, frequently encountered in plant genomes, involved in the regulation of expression, was also investigated using LC. The methylated A base [53,55,58,92,[94][95][96] was determined in a number of studies, while in other studies detection of N6-methyladenine and 5mC was also performed [55,58,92].…”

Section: -Methyldeoxycytidinementioning

confidence: 99%

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Seeking the 5th Base of DNA Using Chromatographic Methods of Analysis

Kouidou¹,

Kovatsi²,

Ioannou

2010

COC

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Expression of genetic information is related to the presence of 5-methylcytosine in DNA. The distribution of 5-methylcytosine varies in different tissues and its frequency is drastically modified according to the developmental stage of the cell as well as under pathological conditions. Among other applications, the presence of "irregular" 5-methylcytosine distribution is currently used as a marker of carcinogenesis as well as of abnormal neurological and other pathological conditions. Analysis of the distribution and mostly the frequency of this modified base, applying an accurate and reproducible method, is a challenge, frequently met by the application of various methods involving the use of analytical techniques.In this review we present the high-throughput analysis of DNA methylation by High Performance Liquid Chromatography, Denaturing High Performance Liquid Chromatography and Gas Chromatography. Moreover, we discuss in detail the principles, as well as the advantages and disadvantages of the above methods, their application for analyzing either global or gene-specific methylation and the biological issues addressed in these studies. In addition, we discuss the detection, by the same methods, of other minor DNA bases, besides 5-methylcytosine, as well as the choice of base derivatives monitored in these studies. Finally, we present information regarding the evaluation of analytical data relative to those obtained by molecular techniques and discuss the particular clinical applications for which the use of analytical techniques can be particularly valuable.

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Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

1990

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During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run‐on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5‐methylcytosine was most abundant. The presence of 5‐methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run‐on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts.

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Separation of naturally occurring or induced methylated nucleobases of DNA by reversed-phase high-performance liquid chromatography

Cited by 14 publications

References 29 publications

DNA methylation as a mechanism of transcriptional regulation in nonphotosynthetic plastids in plant cells.

DNA methylation as a mechanism of transcriptional regulation in nonphotosynthetic plastids in plant cells.

Seeking the 5th Base of DNA Using Chromatographic Methods of Analysis

Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

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