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PubMed1986

The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli. A mutagenesis study.

Methods of in vitro mutagenesis were employed to determine the importance of individual nucleotides within the ribosomal RNAs for the structure and function of E. coli ribosomes. A series of defined nucleotides in the genes for the 5 S and 16 S RNA were altered by transition and transversion mutations using either oligonucleotide-directed or bisulfite-catalyzed mutation procedures. Plasmids harbouring the mutated rRNA genes were expressed and the ribosomes containing such altered RNAs were investigated for impairments in RNA-protein interaction assembly and mRNA-coded tRNA binding.

Base Sequence↗
PubMed2004

Increased DNA methylation in the HoxA5 promoter region correlates with decreased expression of the gene during tumor promotion.

Promoter-region DNA methylation inhibits transcription. A two-stage SENCAR (sensitive to mouse carcinogenesis) mouse skin carcinogenicity model was used to examine gene-specific changes in methylation during skin tumor promotion. Analysis was performed on 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin promoted with 9, 18, 27, or 36 mg cigarette smoke condensate (CSC) for 9 wk, or 27 mg CSC for 9 wk and sacrificed 6 wk afterwards (recovery group). Additionally, tumors arising following promotion with 27 mg CSC for 29 wk were assessed. Gene array analysis identified differentially expressed genes. Expression of HoxA5, a tumor suppressor gene, was decreased following 9 wk of treatment with 27 mg CSC, and returned to control levels during recovery. HoxA5 promoter methylation was measured with the enzymatic regional methylation assay (ERMA). DNA was bisulfite-modified, PCR-amplified with primers containing dam sites, incubated with [14C-methyl] S-adenosyl-L-methionine (SAM) and dam methyltransferase for DNA quantification, then incubated with [3H-methyl] SAM and SssI methylase to quantify methylation status. Higher 3H/14C ratios indicate increased methylation. The 3H/14C ratios of animals promoted with 27 or 36 mg CSC (48.2 +/- 6.9 and 24.2 +/- 6.1, respectively) were higher than the control or recovery group ratios (12.3 +/- 0.1 and 12.6 +/- 0.3, respectively); sequence analysis supported these findings. Increased methylation of p16 or O6 methylguanine methyltranferase (MGMT) was detected in 4/8 (50%) of the tumor samples from mice promoted with 27 mg CSC. These data suggest that increased DNA methylation contributes to the downregulation of HoxA5, and combined with hypermethylation of p16 or MGMT, this might facilitate the clonal expansion of increasingly aberrant cells during promotion.

Animals↗
PubMed1980

Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.

Base Sequence↗
PubMed2005

Methylation of the DPYD promoter: an alternative mechanism for dihydropyrimidine dehydrogenase deficiency in cancer patients.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. EXPERIMENTAL DESIGN: DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. RESULTS: No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. DISCUSSION: Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.

Adult↗
PubMed1999

Inactivation of CACNA1G, a T-type calcium channel gene, by aberrant methylation of its 5' CpG island in human tumors.

Using a newly developed PCR-based technique called methylated CpG island amplification, we have identified several DNA fragments that are aberrantly methylated in a colon cancer cell line. One of the fragments, termed MINT31, mapped to human chromosome 17q21, where frequent loss of heterozygosity is detected in various human tumors. By characterizing the genomic sequence around this area, we identified a gene encoding a T-type calcium channel, CACNA1G, as a target for hypermethylation in human tumors. By reverse transcriptase-PCR we detected expression of CACNA1G in normal colon and bone marrow, but expression was absent in the five tumor cell lines in which methylation was found. After treatment with the methylation inhibitor 5-deoxyazacytidine, the expression of CACNA1G was restored in all five cell lines. Detailed methylation mapping of the 5' CpG island by bisulfite-PCR revealed that methylation of a region 300-800 bp upstream of the translation initiation site closely correlated with the inactivation of CACNA1G. This region contained the transcription start site, as determined by 5' rapid amplification of cDNA ends analysis. Aberrant methylation of CACNA1G was also examined in various human primary tumors and was detected in 17 of 49 (35%) colorectal cancers, 4 of 16 (25%) gastric cancers, and 3 of 23 (13%) acute myelogenous leukemia cases. Inactivation of CACNA1G may play a role in cancer development by modulating calcium signaling, which potentially affects cell proliferation and apoptosis.

Bone Marrow Cells↗
PubMed2006

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA.

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.

Base Sequence↗
PubMed2001

Site-specific methylation of the promoter alters deoxyribonucleic acid-protein interactions and prevents follicle-stimulating hormone receptor gene transcription.

In the male gonad, the FSH receptor (FSHR) gene is expressed only in Sertoli cells. To date, the mechanism(s) responsible for Sertoli cell-specific expression of the FSHR gene are unknown. In this study, DNA methylation at specific sites in the promoter are shown to lead to changes in the DNA-protein interactions at those sites and, subsequently, to transcriptional repression of the gene. The extent of methylation of cytosine residues within the core promoter region of genomic DNA isolated from cells/tissues that expressed, or did not express, the FSHR gene was analyzed by the sodium bisulfite conversion technique. All seven cytosine residues in CpG dinucleotides within the core promoter region were found to be unmethylated in primary cultured rat Sertoli cells that were actively expressing FSHR mRNA. In contrast, in tissues not expressing FSHR the same region of the gene was methylated at each of the CpG dinucleotides examined. In addition, DNA-protein interactions in three primary regulatory regions of the promoter were examined by electrophoretic mobility shift assays (EMSA) with synthetic oligonucleotides containing selectively methylated cytosine residues. Methylation of a CpG sequence within a consensus E box element (CACGTG, -124/-119) decreased the binding affinity of USF1/2 transcription factors for this element. Methylation of the CpG sequence in the Inr region (CCGG, -85/-82) allowed the formation of an additional DNA-protein complex. Methylation at both cytosine residues in the E2F element ((m)CG(m)CG) generated a new methylcytosine-specific DNA-protein complex. The core FSHR promoter region of a mouse Sertoli cell line (MSC-1) that does not express FSHR was shown to be methylated at four CpG dinucleotides. The demethylation of these four sites by treatment of the MSC-1 cells with 5-aza-2'-deoxycytidine (5-azaCdR) activated the transcription of the FSHR gene. Taken together, these results suggest that cytosine methylation is a major factor in the repression of the expression of the FSHR gene.

Animals↗
PubMed2003

MBDin, a novel MBD2-interacting protein, relieves MBD2 repression potential and reactivates transcription from methylated promoters.

We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.

3T3 Cells↗
PubMed2006

Influence of methylated p15 and p16 genes on clinicopathological features in colorectal cancer.

BACKGROUND AND AIM: Genetic silencing by promoter methylation has attracted attention in the carcinogenesis of colorectal cancer. Methylation of the p16(INK4a) gene has been found in primary colorectal cancer. Although the p15(INK4b) gene displays high homology to the p16(INK4a) gene in the amino acid sequence, methylation of p15(INK4b) has not been fully studied. We investigated p15(INK4b) methylation status in patients with colorectal cancer to verify the association between the methylation of p15(INK4b) and clinicopathological features compared with p16(INK4a). METHODS: DNA samples from the tissues of primary colorectal cancer and corresponding adjacent normal colon mucosa were obtained from surgical resections of 88 patients (47 males and 41 females, aged 29-83 years). Methylation-specific polymerase chain reaction was used to analyze p15(INK4b) and p16(INK4a) methylation status after bisulfite modification. Cumulative survival rates (mean follow-up period: 53.2 months) were calculated by the Kaplan-Meier analysis. Methylations of p15(INK4b) and p16(INK4a) genes were detected in 23 (26.1%) and 20 (22.7%) colorectal cancers, respectively. RESULTS: Methylation of p15(INK4b) was not associated with any clinicopathological features. Compared with normal mucosa, the methylation of p15(INK4b) was more prominent in tumor tissue (P < 0.001). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that p15(INK4b) methylaton decreased mRNA expression. Kaplan-Meier analysis showed that patients with stage I-II had a significant difference in survival rate between those with and without methylated p15(INK4b) (P = 0.018). CONCLUSIONS: Our results suggest that methylation of the p15(INK4b) gene contributes to the process of carcinogenesis in colorectal cancer as well as p16(INK4a) and is useful as a prognostic factor in the early stage.

Adult↗
PubMed2006

In situ bisulfite modification of membrane-immobilized DNA for multiple methylation analysis.

Increasing interest in DNA methylation has resulted in the recent development of array-based methods. However, these method require complex sample treatment processes, such as bisulfite modification in the liquid state of every sample. Here, we describe a microarray-based technology for interrogating DNA methylation status of multiple DNA samples at the same time, in which the DNA samples are first dotted on membranes and then treated with bisulfite directly. In this assay, plasmid pUC19 DNA is immobilized on nylon membranes and soaked in bisulfite reaction mixtures for 16h to convert unmethylated Cs to Us, and methylated Cs remain unchanged. The probes for detecting the methylation patterns of CpG sites are hybridized with the converted DNA dotted on the membranes, and the signals are revealed by chemiluminescence of DIG-labeled probes. The experiments show that this method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This new method could be used as a convenient tool for detecting the methylation status of multiple genes for a large number of samples in the future.

Clinical Laboratory Techniques↗
PubMed2006

Analysis of DNA methylation profiles in preimplantation embryos using bisulfite mutagenesis.

For developmental competence of mammalian embryos, dynamic epigenetic changes to both the maternally and paternally derived contributions to the genome of the zygote should be brought about in the early cleavage stages. DNA methylation is a typical epigenetic mark modified during pre-implantation development. Here, we describe how to analyze DNA methylation profiles in early-stage embryos.

Animals↗
PubMed1977

Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base.

Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.

Amino Acyl-tRNA Synthetases↗
PubMed1987

A comprehensive collection of point mutations in the internal promoter of the adenoviral VAI gene.

Point mutations in the adenoviral VAI gene promoter were generated by in vitro mutagenesis with sodium bisulfite and were identified by three phenotypes: the disappearance of certain restriction endonuclease cleavage sites, altered rates of transcription in cell-free extracts, and altered mobility of the RNA product on denaturing acrylamide gels. 25 different point mutations were assayed for their effect on VAI transcription in HeLa S100 extracts. Mutations which change the transcriptional ability of the gene were localized in the internal control regions which are highly conserved among genes transcribed by RNA polymerase III. The magnitude of the change in transcription correlates with the degree of conservation in the consensus sequence at the site of the mutation. Most of the mutations either do not change the transcription rate or reduce transcription less than 6-fold. Two mutations severely reduce the amount of RNA produced, whereas three mutations increase the production of VAI RNA. A second site revertant that partially restores transcription to a mutant gene that produces no detectable VAI has also been isolated.

Adenoviridae↗
PubMed1982

Local mutagenesis within deletion loops of DNA heteroduplexes.

An efficient method has been developed to generate base substitution mutations within deletion loops of DNA heteroduplexes. This method utilizes a heteroduplex formed between a deletion mutant cloned in a plasmid vector and its wild-type counterpart from which two restriction sites had been removed from the vector. The heteroduplex is exposed to sodium bisulfite to deaminate cytosine residues in the single-stranded loop, and the mutagenized plasmid DNA is used to transform a strain of bacteria lacking the enzyme uracil N-glycosylase. Pooled progeny DNA is digested with the two restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA. Point mutants with C . G-to-T . A transitions are obtained at high frequency after a second transformation. To test the feasibility of the approach, the tetracycline resistance gene of pBR322 was chosen as the target sequence. It was found that the proportion of tetracycline-sensitive transformants increased as both the size of the heteroduplex loop and the time of incubation with the mutagen increased and this varied from 20% up to 70%. Nucleotide sequence analysis of several tetracycline-sensitive mutants confirmed that C-to-T transitions had been produced in the segment of DNA corresponding to the deletion loop.

Base Sequence↗
PubMed1993

Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase.

Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly138-SerValGlyAlaSerGlyAsp145) contained an unidentified modification of mass 184 Da located on Ser143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser143 represents the binding site for an electrophilic cofactor required for histidase activity.

Amino Acid Sequence↗
PubMed1984

Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction.

Bisulfite catalyzes transamination of cytidine at the N4 position; the suitability of this reaction for attaching reporter groups to selected cytidine residues in RNA molecules has been investigated. Poly(C) is nearly quantitatively converted to the poly (N4 aminoethyl-C) derivative after 3 hrs at 42 degrees C with ethylene diamine (pK1 = 7.6) and bisulfite. This derivative reacts quantitatively with N-hydroxysuccinimide esters; the linkage of a fluorescent dye, nitrobenzofurazan, to cytidine by this reaction is demonstrated. To direct the bisulfite reaction to selected cytidines within a large RNA molecule, the RNA is hybridized to complementary DNA containing a deletion. Only the cytidines in the single strand RNA loop (corresponding to the DNA deletion) are reactive. Two cytidines in the middle of a 340 base RNA fragment from 16S ribosomal RNA have been modified by this technique.

Amines↗
PubMed2006

MLH3 mutation in endometrial cancer.

MLH3 is a recently described member of the DNA mismatch repair gene family. Based on its interaction with the MutL homologue MLH1, it was postulated that MLH3 might play a role in tumorigenesis. Germ line and somatic mutations in MLH3 have been identified in a small fraction of colorectal cancers, but the role of MLH3 in colorectal cancer tumorigenesis remains controversial. We investigated MLH3's role in endometrial tumorigenesis through analysis of tumor and germ line DNA from 57 endometrial cancer patients who were at increased risk for having inherited cancer susceptibility. Patients with known MSH2 or MSH6 mutations were excluded as well as those who had MLH1-methylated tumors. Sixteen different variants were identified by single-strand conformational variant analysis. Of the 12 missense changes identified, three were somatic mutations. One patient had a germ line missense variant and loss of heterozygosity (LOH) in her tumor specimen. There was no evidence of MLH3 promoter methylation based on combined bisulfite restriction analysis. The identification of inherited missense variants, somatic missense mutations (present in 3 of 57 tumors), and LOH in the tumor from a patient with a germ line missense change suggest a role for MLH3 in endometrial tumorigenesis.

Base Sequence↗
PubMed1993

Deoxycytidine methylation and the origin of spontaneous transition mutations in mammalian cells.

Previously we described a recurrent, site-specific G4784 --> A transition mutation affecting exon V of the Chinese hamster ovary cell RPS14 gene. Because the mutation is located within a CpG dinucleotide, we considered the possibility that deoxycytidine methylation might be responsible for the transition's unusually high frequency and site specificity. Therefore, we used a procedure based on the PCR amplification of bisulfite-modified genomic DNA to analyze the pattern of DNA cytosine methylation in exon V of the CHO cell RPS14 locus. Our data indicate that the CpG dinucleotide targeted by the transition mutation is stably methylated in CHO cell chromosomes. This finding supports the notion that deoxycytidine methylation promotes "spontaneous", site-specific transition mutations in mammalian cells.

Amino Acid Sequence↗