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PubMed2006

Deficient expression of the DPD gene is caused by epigenetic modification in biliary tract cancer cells, and induces high sensitivity to 5-FU treatment.

5-FU is the drug most frequently used to treat biliary tract cancer, while dihydropyrimidine dehydrogenase (DPD) is known to be a principal factor in 5-FU drug resistance. However, whether DPD activity and mRNA levels correlate with response to 5-FU is unknown for biliary tract cancers. The precise mechanism of DPD regulation also remains to be elucidated. In the present study, we quantitatively analyzed DPD mRNA in 8 biliary tract cancer cell lines using real-time RT-PCR, and assessed whether DPD mRNA levels correlate with DPD activity or the sensitivity to 5-FU. Finally, we examined the epigenetic gene silencing of DPD using one of the 8 lines, a gallbladder cancer cell line with deficient DPD expression, KMG-C. Strong correlation was found between DPD activity and DPD mRNA expression in the 8 cancer cell lines (R=0.797, P=0.0148). DPD mRNA expression and DPD activity exhibited positive correlation with the IC50 for 5-FU (R=0.658, R=0.644, respectively), although these relationships were not statistically significant. In the KMGC cells with deficient DPD mRNA levels, restoration of DPD expression was observed by 5-Aza-2' deoxycytidine (5-aza-C) treatment in a dose-dependent manner, suggesting gene suppression by promoter hypermethylation. Combined bisulfite restriction analysis was performed to analyze the methylation on CpG islands around the 5'-flanking region and intron 1 of the DPD gene, however, no methylated CpG sites were identified in these regions. In addition, the restored DPD expression level was more strongly induced by the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), than 5-aza-C treatment. These findings suggest that other mechanisms, including histone modification, may be important for DPD suppression. In conclusion, these results may aid the selection of 5-FU chemotherapy following determination of DPD expression in biliary tract cancers. Furthermore, epigenetic gene silencing appears to be an important mechanism of DPD suppression in biliary tract cancer.

Antimetabolites, Antineoplastic↗
PubMed1992

Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid.

The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas. Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction. Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation. Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45. The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity. In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability. Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.

Alleles↗
PubMed2000

Positive display of methylated sites: a novel method for the detection of promoter methylation.

Promoter methylation represents an important mechanism for silencing gene expression in higher eukaryotes. To study methylation of the promoter of the tumor suppressor p16INK4a, a fast and simple method was developed that, in contrast to previous studies, relies on the positive display of methylated sites (PDM). The method is based on bisulfite treatment of DNA, polymerase chain reaction (PCR)-amplification of the modified DNA and restriction digest of de novo created restriction sites to positively display DNA methylation in a background of unmethylated DNA. Since methylated as well as unmethylated DNA is amplified, information on the proportion of both is provided. Using this approach, 33 ductal invasive carcinomas, 4 normal mammary tissues, and 4 cell lines were analyzed for methylation. Methylation in the p16INK4a promoter was detected in 1 of 33 carcinomas (3%) and in 0 of 4 normal tissues. The conclusion is that PDM provides a useful tool in determining the degree and pattern of promoter methylation and is suitable to screen large series of tissue samples.

Base Pairing↗
PubMed1986

A novel general approach to eucaryotic mutagenesis functionally identifies conserved regions within the adenovirus 13S E1A polypeptide.

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.

Adenovirus Early Proteins↗
PubMed2003

Methylation profile of P. lividus sea urchin genes during development.

Methylation pattern has been studied in two genes of sea urchin Paracentrotus lividus using sodium bisulfite method to understand the possible role of DNA methylation during invertebrate development. Three regions of the gene for the hatching enzyme have been analyzed and all of them resulted unmethylated in embryos at different stages of development. Four CpG rich regions have been studied in the gene for DNA methyltransferase: upstream, upstream-exon1, intron 1 and exon 20. The upstream-exon 1 region is always unmethylated, while intron 1 and exon 20 are heavy methylated. Only the upstream fragment changed its pattern of methylation during development. For none of the studied regions the reported data show a general direct correlation between gene expression and methylation process during development.

Animals↗
PubMed2006

Aberrant methylation in pediatric myelodysplastic syndrome.

BACKGROUND: Aberrant methylation of gene promoter region is responsible for inappropriate gene silencing, and it has been associated to initiation and progression of cancer. Aberrant promoter methylation is frequently observed in adult patients with myelodysplastic syndrome (MDS), but in pediatric patients it has been poorly investigated. METHODS: We examined the promoter methylation status of 13 genes in bone marrow cells collected at diagnosis of 21 pediatric patients with MDS (subtype RAEB or RAEB-t). For this analysis, we performed sodium bisulfite treatment of genomic DNA, followed by methylation specific PCR (MSP). RESULTS: In pediatric MDS samples, we observed two genes frequently methylated: CALCA was methylated in 85.7% (18/21) of the analyzed samples and CDKN2B in 50% (6/12). CONCLUSIONS: Our findings indicate that CALCA and CDKN2B are frequently methylated in pediatric MDS. It suggests that aberrant methylation in pediatric MDS seems to be similar to adult MDS, thus pediatric patients could be also benefited with treatment using demethylating agents.

Adolescent↗
PubMed1988

Selective reversible deuteriation of oligodeoxynucleotides: simplification of two-dimensional nuclear Overhauser effect NMR spectral assignment of a non-self-complementary dodecamer duplex.

Oligodeoxynucleotides are reversibly deuteriated at the purine C8 and cytosine C5 positions with deuterioammonium bisulfite at pD 7.8. The exchange reaction is complete after 48 h at 65 degrees C. When an oligomer deuteriated under these conditions is analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy, the purine H8 and cytosine H5 proton signals are selectively removed from the spectrum. A non-self-complementary oligodeoxynucleotide that has been deuteriated in this manner may be annealed with its complement and the resulting heteroduplex analyzed by two-dimensional nuclear Overhauser enhancement (NOESY) spectroscopy. NOE cross-peaks arising from pyrimidine H6-deoxyribose H1' dipolar interactions in both strands are observed, but purine H8-deoxyribose H1' and purine H8-deoxyribose H2',H2" dipolar interactions are only observed for the nondeuteriated strand. The intense cytosine H5-H6 cross-peaks are also removed from the spectrum of the deuteriated strand, which further simplifies interpretation since these strong cross-peaks often interfere with less intense NOE cross-peaks arising from dipolar coupling between purine H8 or pyrimidine H6 and deoxyribose anomeric protons. The resulting spectral simplification allows unambiguous assignments to be made on NOEs that otherwise may be difficult to distinguish. The deuteration procedure is demonstrated with the sequence d(CGTTATAATGCG).d(CGCATTATAACG), which has previously been assigned by traditional NOESY methods [Wemmer, D. E., Chou, S.-H., Hare, D. R., & Reid, B. R. (1984) Biochemistry 23, 2262-2268]. Although the assignment of this dodecadeoxynucleotide may be completed without deuteriation, several NOEs must be assigned indirectly because of degeneracies in the chemical shift of the purine H8 protons.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1996

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

Antisense Elements (Genetics)↗
PubMed1985

Point mutations in the 3' minor domain of 16S rRNA of E.coli.

Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing.

Base Sequence↗
PubMedDate not supplied

Selective modification of cytosines in oligodeoxyribonucleotides.

A single deoxycytidine residing in an oligodeoxyribonucleotide which also contains 5-methyldeoxycytidines can be selectively derivatized with various alkylamines by sodium bisulfite-catalyzed transamination. Selective transamination results because 5-methylcytosine, unlike cytosine, does not form a bisulfite adduct. When the reaction is carried out at pH 7.1, transamination in the oligomer appears to occur to greater than 95% with little or no deamination. This procedure has been used to introduce aminoalkyl or carboxyalkyl side chains at the N4-position of a deoxycytidine in oligonucleotides. These side chains contain potentially reactive amine or carboxy groups which could serve as a sites for further conjugation of the oligomer with a variety functional groups. Oligonucleotides which carry these side chain form duplexes and triplexes with appropriate complementary single-stranded or double-stranded oligodeoxyribonucleotide target molecules. The stabilities of the duplexes are similar to those formed by unmodified oligomers, whereas the stability of the triplexes is approximately 18 degrees C lower than that formed by unmodified oligomers.

Base Sequence↗
PubMed1987

Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

Alcohol Dehydrogenase↗
PubMed1992

Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen.

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type.

Amino Acid Sequence↗
PubMed2003

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications.

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.

CpG Islands↗
PubMed2001

X chromosome inactivation patterns in 45,X/46,XX mosaics.

To investigate X chromosome inactivation (XCI) patterns in 45,X/46.XX mosaics, genomic DNA was extracted from peripheral blood samples of 15 female subjects who showed different proportions of 45,X cell clones. XCI patterns were analyzed using two assays. The first assay was the BstXI restriction endonuclease detection of an X-linked phosphoglycerate kinase (PGK) gene polymorphism following digestion of the DNA with methylation-sensitive HpaII, or with methylation-insensitive AfaI as a control. The second assay was the detection of a CAG triplet repeat polymorphism in the X-linked androgen receptor (AR) gene after sodium bisulfite treatment. Of the 15 subjects, 11 were informative due to heterozygosity for at least one of the polymorphisms (6 were heterozygous for the PGK polymorphism and 9 were heterozygous for the AR polymorphism). Four of the 11 informative subjects (36%) showed extremely skewed XCI for at least one of the polymorphisms, which was a much higher incidence than previously reported for normal females. Moreover, 3 of these 4 women had proportions of 45,X cell clones greater than 20%. Although our results may be due to several possible cytogenetic or molecular mechanisms, the most likely explanation is that cases of 45,X/46,XX that contain relatively high levels of 45,X cell clones probably arose due to structural aberrations of the X chromosome undetectable by conventional karyotyping.

Base Sequence↗
PubMed1991

Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.

The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Gene expression profiles of human BPH (II): Optimization of laser-capture microdissection and utilization of cDNA microarray.

Laser capture microdissection (LCM) enables the dissection of heterogeneous components of tissues, helping to investigate the molecular properties of these tissues. We have reported gene expression profiles in human benign prostatic hyperplasia (BPH) using LCM and quantitative real-time PCR. In the current work, we extended the previous observations. Firstly, we studied the relationship between the number of dissected acini and amplification by PCR, and found that androgen receptor (AR) and 18S rRNA transcripts were successfully amplified from total RNA obtained from one prostate acinus. Furthermore, LCM-dissected samples were applicable to methylation-specific PCR of E-cadherin promoter gene after bisulfite modification of genomic DNA. Next, we performed cDNA microarray analysis to screen gene expression profiles in the epithelium and stroma. RNA was amplified by T7-RNA polymerase and labeled with Cy3 and Cy5. Epithelium-related or stroma-related genes were identified through cDNA microarray. We confirmed true gene expression levels by quantitative real-time PCR. In epithelium, E-cadherin and serine protease 2, Kunitz-type gene expression levels were significantly elevated, while the connective tissue growth factor gene expression level was significantly elevated in stroma. Thus, LCM-dissected samples were applicable to various molecular examinations including methylation-specific PCR and cDNA microarray, and this will contribute to a precise understanding of the molecular profiles of prostate glands.

DNA↗
PubMed1996

Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region.

The galectin-1 gene is developmentally regulated gene whose activity is strongly modulated during cell differentiation and transformation. We have previously shown that galectin-1 promoter constructs are highly active when transiently transfected in cells both expressing and not expressing the endogenous gene and that the basal activity is determined by a small region encompassing the transcription start site (from positions -50 to +50). We have now investigated the role of DNA methylation in galectin-1 gene expression. Southern blot analysis with HpaII and MspI endonucleases and sodium bisulfite analysis of genomic DNA from expressing and nonexpressing cell lines and cell hybrids showed a close correlation between gene activity and demethylation of the 5' region of the galectin-1 gene. We found that the galectin-1 promoter region is fully methylated, at every CpG site on both strands, in nonexpressing differentiated rat liver (FAO) and thyroid (PC C13) cells and unmethylated in the expressing undifferentiated liver (BRL3A) and thyroid transformed (PC myc/raf) cell lines. In addition, reactivation of the silent FAO alleles in FAO-human osteosarcoma (143tk-) hybrid cells is accompanied by a complete demethylation of the promoter region. Finally, when galectin-1 chloramphenicol acetyltransferase (CAT) promoter constructs were methylated in vitro by SssI methylase at every cytosine residue of the CpG doublets and transfected into mouse fibroblasts, the transcription of the CAT reporter gene was strongly inhibited.

Animals↗
PubMed2006

Isolation of sulfate-reducing bacteria from Tunisian marine sediments and description of Desulfovibrio bizertensis sp. nov.

Several strains of sulfate-reducing bacteria were isolated from marine sediments recovered near Tunis, Korbous and Bizerte, Tunisia. They all showed characteristics consistent with members of the genus Desulfovibrio. One of these strains, designated MB3(T), was characterized further. Cells of strain MB3(T) were slender, curved, vibrio-shaped, motile, Gram-negative, non-spore-forming rods. They were positive for desulfoviridin as bisulfite reductase. Strain MB3(T) grew at temperatures of 15-45 degrees C (optimum 40 degrees C) and at pH 6.0-8.1 (optimum pH 7.0). NaCl was required for growth (optimum 20 g NaCl l(-1)). Strain MB3(T) utilized H(2) in the presence of acetate with sulfate as electron acceptor. It also utilized lactate, ethanol, pyruvate, malate, fumarate, succinate, butanol and propanol as electron donors. Lactate was oxidized incompletely to acetate. Strain MB3(T) fermented pyruvate and fumarate (poorly). Electron acceptors utilized included sulfate, sulfite, thiosulfate, elemental sulfur and fumarate, but not nitrate or nitrite. The G+C content of the genomic DNA was 51 mol%. On the basis of genotypic, phenotypic and phylogenetic characteristics, strain MB3(T) (=DSM 18034(T)=NCIMB 14199(T)) is proposed as the type strain of a novel species, Desulfovibrio bizertensis sp. nov.

DNA, Bacterial↗