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PubMed2005

Frequent methylation of eyes absent 4 gene in Barrett's esophagus and esophageal adenocarcinoma.

Most esophageal adenocarcinomas arise within Barrett's esophagus but the cause of this increasingly prevalent condition remains unknown. Early detection improves survival and discriminant screening markers for Barrett's esophagus and cancer are needed. This study was designed to explore the natural history of eyes absent 4 (EYA4) gene methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated EYA4 as a candidate marker. Aberrant promoter methylation of EYA4 was studied by methylation-specific PCR using bisulfite-treated DNA from esophageal adenocarcinomas, Barrett's esophagus, and normal epithelia, and then confirmed by sequencing. Eight cancer cell lines were treated with the demethylation agent 5-aza-2'-deoxycytidine, and EYA4 mRNA expression with and without treatment was quantified by real-time reverse-transcription PCR. EYA4 hypermethylation was detected in 83% (33 of 40) of esophageal adenocarcinomas and 77% (27 of 35) of Barrett's tissues, but only in 3% (2 of 58) of normal esophageal and gastric mucosa samples (P < 0.001). The unmethylated cancer cell lines had much higher EYA4 mRNA expression than the methylated cancer cell lines. Demethylation caused by 5-aza-2'-deoxycytidine increased the mRNA expression level by a median of 3.2-fold in methylated cells, but its effect on unmethylated cells was negligible. Results indicate that aberrant promoter methylation of EYA4 is very common during tumorigenesis in Barrett's esophagus, occurs in early metaplasia, seems to be an important mechanism of down-regulating EYA4 expression, and represents an intriguing candidate marker for Barrett's metaplasia and esophageal cancer.

Adenocarcinoma↗
PubMed2001

Androgen receptor expression in androgen-independent prostate cancer cell lines.

BACKGROUND: Almost all attempts at establishing prostate carcinoma cell lines have resulted in generation of cells that are androgen-independent, including commonly used LNCaP which expresses androgen receptor (AR) and AR-negative Du145 and PC-3. We attempted to clarify the mechanism(s) responsible for the failure to respond to androgen. METHODS: Cell lines LNCaP, CWR22R, PC-3, Du145, and CA7T2CL were used to examine the AR promoter function with a reporter gene assay and its methylation status by Southern blot, PCR of bisulfite-converted DNA, and 5-aza-2'-deoxycytidine treatment. Structural abnormalities of the AR were identified by sequencing of reverse-transcribed mRNA. RESULTS: All tested AR-positive prostate carcinoma cells were capable of AR transcription at a significantly higher level than PC-3 and Du145, thus suggesting relative deficiency of the transcription factors in the AR-negative cells, further associated with methylation. Examination of CWR22R cells, which express the AR but are androgen-independent, identified an in-frame duplication of exon 3, which resulted in insertion of 39 amino acids in the DNA-binding domain. CONCLUSIONS: Relative deficiency of transcription factors associated with methylation is responsible for the lack of AR promoter function in most of AR-negative cell lines. Mutations in the AR gene are present in the cells that express the AR but are androgen-independent.

Adenocarcinoma↗
PubMed2004

Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure.

Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

Alleles↗
PubMed1979

An improved rapid enzymatic method of RNA sequencing using chemical modification.

A version of rapid gel sequencing procedure based on the analysis of partial endonuclease hydrolizates of chemically modified 5'-32P-labelled RNA is suggested. Complete and selective modification of cytidilic residues by a methoxyamine-bisulfite mixture leads to the unfolding of the RNA secondary structure and, due to this effect, to the generation of a more uniform set of fragments after partial RNAase hydrolysis. The position of cytidines in an RNA sequence can be determined by restricting the hydrolysis of phosphodiester bonds between the modified CMP residues and their 3'-neighbours with T2 and A RNAases. The method was verified with tRNATrp (yeast) and 5S RNA (rat liver and yeast).

Animals↗
PubMed1987

Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants.

The nucleotide sequence of the region containing the promoter-operator for the aroP gene was determined. The start site of aroP transcription was identified by using S1 nuclease mapping and primer extension techniques. Examination of the nucleotide sequence revealed the presence of two "TYR R" boxes which are similar to those identified in the regulatory regions of other genes in the tyrR regulon. Bisulfite-induced aroP operator-constitutive mutants were analyzed, and the base-pair changes responsible for alterations in aroP regulation were located within these boxes.

Acetyltransferases↗
PubMed2002

Desulfovibrio sp. genes involved in the respiration of sulfate during metabolism of hydrogen and lactate.

To develop a better understanding of respiration by sulfate-reducing bacteria, we examined transcriptional control of respiratory genes during growth with lactate or hydrogen as an electron donor. RNA extracts of Desulfovibrio desulfuricans subsp. aestuarii were analyzed by using random arbitrarily primed PCR. RNA was reverse transcribed under low-stringency conditions with a set of random primers, and candidate cDNAs were cloned, sequenced, and characterized by BLAST analysis. Putative differentially expressed transcripts were confirmed by Northern blot analysis. Interestingly, dissimilatory bisulfite reductase was upregulated in the presence of hydrogen. To link these transcriptional changes to the physiology of sulfate-reducing bacteria, sulfide was measured during growth of several strains of Desulfovibrio on hydrogen or lactate, and this revealed that hydrogen-grown cells produced more sulfide per unit of cell mass than lactate-grown cells. Transcription of other redox proteins was characterized by Northern blotting to determine whether or not they were also transcribed to higher levels in hydrogen-grown cells. Growth on lactate produced greater transcription of [NiFe] hydrogenase. H(2)-grown cells transcribed the adenylylsulfate reductase b subunit and HmcA to higher levels. The results we describe here provide new insight into the continuing debate over how Desulfovibrio species utilize redox components to generate membrane potential and to channel electrons to sulfate, the final electron acceptor.

Bacterial Proteins↗
PubMed2005

Targeting of the activation-induced cytosine deaminase is strongly influenced by the sequence and structure of the targeted DNA.

Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitro AID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first approximately 100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.

Ampicillin Resistance↗
PubMed2006

Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: Theoretical and empirical considerations.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. METHODS: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. RESULTS: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. CONCLUSION: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Chromosomes, Human, Pair 18↗
PubMed1980

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Capped ribopolymers lacking a sequence complementary to the common 3' end of the influenza virion RNA segments effectively stimulated transcription of these RNAs by the virion-associated transcriptase. Thus, stimulation of transcription results not from hydrogen bonding between the capped RNA and the 3' end of the virion RNA but presumably from a specific interaction of the capped RNA with protein(s) in the transcriptase complex. Although no specific nucleotide sequence was required for priming activity, capped mRNAs with diminished secondary structure were preferred as primers. Inosine-substituted or bisulfite-modified capped reovirus mRNAs were at least 3- to 5-fold more effective as primers than were the native capped mRNAs. On the other hand, inosine substitution or bisulfite treatment of the uncapped form of reovirus mRNAs converted them from essentially inactive species to potent inhibitors of the transcriptase reaction primed by either ApG or globin mRNA. These effects of reduced secondary structure also most probably reflect an interaction of the exogenous RNAs with transcriptase protein(s). The results obtained from screening a series of native uncapped ribopolymers were consistent with inhibitory activity requiring the absence of most hydrogen bonding in the ribopolymer and also suggested that specific structural feature(s) of the nucleotides in the chain were important.

Base Sequence↗
PubMed1984

Probing the structure of 16 S ribosomal RNA from Bacillus brevis.

A majority (approximately 89%) of the nucleotide sequence of Bacillus brevis 16 S rRNA has been determined by a combination of RNA sequencing methods. Several experimental approaches have been used to probe its structure, including (a) partial RNase digestion of 30 S ribosomal subunits, followed by two-dimensional native/denatured gel electrophoresis, in which base-paired fragments were directly identified; (b) identification of positions susceptible to cleavage by RNase A and RNase T1 in 30 S subunits; (c) sites of attack by cobra venom RNase on naked 16 S rRNA; and (d) nucleotides susceptible to attack by bisulfite in 16 S rRNA. These data are discussed with respect to a secondary structure model for B. brevis 16 S rRNA derived by comparative sequence analysis.

Bacillus↗
PubMed1976

Location of accessible bases in Escherichia coli formylmethionine transfer RNA as determined by chemical modification.

Chemical modification of Escherichia coli tRNAfMet with 1 M chloroacetaldehyde, pH 5.5-6.0 at 25 degrees C, has been found to result in alteration of six cytidine and five adenosine residues in the molecule. The modified cytidine residues are the same as those previously found to be reactive with sodium bisulfite at pH 6.0. The accessible adenosine residues are A36 in the anticodon, A58 in the T psi C loop, and A73, A74, and A77 in the 3; terminal sequence. No modification of adenosine residues in the dihydrouridine or variable loops or of adenosine residues on the 3' side of the anticodon loop could be detected. Treatment of fMet-tRNAfMet with chloracetaldehyde gave the same pattern of midofication as was observed with deacylated tRNAfMet. Chemical modification of E. coli tRNAfMet with 2 sodium bisulfite, pH 7.0 at 25 degrees C, resulted in selective modification of exposed uridine residues in the tRNA. Only three sites were found to be reactive: U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. The overall pattern of chemical modification of tRNAfMet is very similar to that found by others for yeast tRNAPhe, supporting the idea that many of the tertiary interactions in the two tRNAs are the same. The adenosine residue at position 58 in the center of the T psi C loop of the initiator tRNA shows unusual reactivity, however, being modified by chloroacetaldehyde at the same rate as the 3' terminal adenosine residue. This result is in sharp contrast to the uniform resistance of nucleotides in the T psi C loop of yeast tRNAPhe to chemical modification.

Acetaldehyde↗
PubMed2004

Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer.

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.

Breast Neoplasms↗
PubMed1996

Pyrene-labeled DNA probes for homogeneous detection of complementary DNA sequences: poly(C) model system.

DNA and RNA probes are important analytical reagents in molecular biology and in the detection of infectious and genetic diseases. However, the present polynucleotide probe technology is complex and labor-intensive. We have been investigating the possibility of using fluorescent-labeled DNA probes to develop assays which do not require the separation of free from hybridized probe (homogeneous assays). Such assays are possible if the fluorescence efficiency or fluorescence anisotropy of the fluorescent label changes upon hybridization of probe with target DNA. In this article we examine pyrene as a fluorescent label for DNA or RNA probes. Experiments were performed using a model system in which poly(C) and poly(I) are respectively the probe and target sequences. A small fraction of the nucleotide bases of poly(C) was randomly labeled with pyrene using the bisulfite-catalyzed diamine reaction. The results show that the uncorrected emission spectrum of pyrene-poly(C) decreases by a factor of 4 and shifts toward longer wavelengths upon hybridization with poly(I) at saturating concentrations. The average lifetime changes from 10.78 to 4 ns. These fluorescence changes occur in a wide range of chemical environments, including the high salt concentrations normally used to increase the velocity of the hybridization reaction in clinical assays. The pyrene label can thus be used to readily detect the amount of poly(I) in an unknown sample without having to separate free and bound labeled probe. To unravel the mechanism responsible for the observed changes in fluorescence intensity upon hybridization, we have performed polarized fluorescence intensity measurements and analyzed the results by approximate steady-state expressions that allow evaluation of the relative contributions of changes in lifetimes (fluorescence efficiency) and rotational motions to the changes in fluorescence intensity. The results indicate that the latter changes are due chiefly to changes in lifetime or fluorescence efficiency and that these changes seem to be due to the movement of the pyrene label to a more hydrophilic environment upon hybridization.

DNA Probes↗
PubMed2005

MLH1 germline epimutations as a factor in hereditary nonpolyposis colorectal cancer.

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by heterozygous germline sequence mutations of DNA mismatch repair genes, most frequently MLH1 or MSH2. A novel molecular mechanism for HNPCC has recently been suggested by the finding of individuals with soma-wide monoallelic hypermethylation of the MLH1 gene promoter. In this study, we determined the frequency and role of germline epimutations of MLH1 in HNPCC. METHODS: A cohort of 160 probands from HNPCC families who did not harbor germline sequence mutations in the mismatch repair genes were screened for methylation of the MLH1 and EPM2AIP1 promoters by combined bisulfite and restriction analyses. Allelic expression and family transmission of MLH1 were determined using polymorphisms in intron 4 and the 3' untranslated region. RESULTS: One of 160 individuals had monoallelic MLH1 hypermethylation in peripheral blood, hair follicles, and buccal mucosa, indicative of a soma-wide alteration. Monoallelic transcription of the paternal MLH1 allele was shown using a heterozygous expressed polymorphism within the 3' untranslated region. The hypermethylated allele was maternally transmitted, however, the mother and siblings who inherited the same maternal homologue were unmethylated at MLH1, suggesting the epimutation arose as a de novo event. CONCLUSIONS: Germline MLH1 epimutations are functionally equivalent to an inactivating mutation and produce a clinical phenotype that resembles HNPCC. Inheritance of epimutations is weak, so family history is not a useful guide for screening. Germline epimutations should be suspected in younger individuals without a family history who present with a microsatellite unstable tumor showing loss of MLH1 expression.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP.

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

DNA↗
PubMed2003

Expression of ECRG4, a novel esophageal cancer-related gene, downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma.

AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

Carcinoma, Squamous Cell↗
PubMed1991

[Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA].

Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA. Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened. It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism. In contrast, modification of two DNA strands led to induction of mutations. The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9%. Mutations were analysed by restriction mapping and sequencing. All of them were G----A transitions.

DNA Damage↗