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Diethyl pyrocarbonate was used as a probe in mapping early melting stages in supercoiled DNA. It was shown that in the process of early melting of pAO3 DNA two denatured regions (about 15 b.p.) arouse near the left and right boundaries of the cruciform structure. In course of further melting denatured regions appeared within AT-rich stretches and the cruciform structure itself disappeared.
In recent years, triplex-forming oligonucleotides (TFOs) have emerged as powerful tools for site-specific gene modification. Their sequence specificity, binding affinity, and ability to provoke repair and recombination make them promising reagents for altering gene expression. This chapter highlights the binding requirements for triplex formation, identifies a number of chemical modifications that have been used with some success, and discusses studies using TFOs for inhibiting transcription. It also reviews work done using TFOs and related molecules to direct site-specific DNA damage, inducing mutagenesis or sensitizing a site to recombination. TFOs were initially used as positioning devices for nonspecific mutagens but were later discovered to have mutagenic properties of their own in cells with functional nucleotide excision repair (NER) and transcription-coupled repair (TCR) pathways. In subsequent studies triplex formation was able to induce both intramolecular and intermolecular homologous recombination, revealing its potential application for gene therapy. Recent reports demonstrate the ability of these molecules to locate and modify their cognate sites in chromosomal DNA in both cell culture and live animals, laying the foundation for triplex technology in vivo.
The modification status of DNA throughout the life cycle of Bacillus coagulans strain 26 was analysed by restriction analysis with methylation-sensitive enzymes. A significant fraction of the GATC sequences (dam target) in spore DNA contain N6-methyladenine, a modification that is lacking during the vegetative phase. From the modulation of the modification pattern of GATC sites, the existence of a de novo methylase may be inferred. Spore DNA was more sensitive than vegetative cell DNA to BamHI, HpaI, SalI and XhoI, indicating that the sites for these enzymes are modified during the vegetative growth phase.
The possibility of using the 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as the affinity modificator of bacteriophage T7 DNA-dependent RNA polymerase was demonstrated. The UTP derivative used was rather efficient substrate substituting UTP in the transcription reaction performed by the enzyme. The UV treatment of "stopped" reaction complex formed using three of four substrate ribonucleotides, allow to obtain the covalent binding between the enzyme and the reaction product of 9 nucleotides length. The isolation and the analysis of the obtained "nucleotide-peptide" showed that the sequence of modified peptide corresponded to the fragment Tyr802-Lys826, which belonged to the conservative motif C in the enzyme structure. His811 or Asp812 residues belonging to this sequence are most probable targets of the modification.
S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)
I have investigated the thermodynamic and kinetic effects of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation between a 23-bp target duplex and a 15-mer TFO using electrophoretic mobility shift assay, UV melting, isothermal titration calorimetry, and interaction analysis system. The thermodynamic and kinetic analyses have clearly indicated that the PN modification of TFO not only significantly increased the thermal stability of the pyrimidine motif triplex at neutral pH but also increased the binding constant of the pyrimidine motif triplex formation at room temperature and neutral pH by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters has suggested that the more rigidity of the PN TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant of the pyrimidine motif triplex formation at neutral pH. Kinetic data have also demonstrated that the observed PN modification-mediated promotion of pyrimidine motif triplex formation at neutral pH resulted from the considerable decrease in the dissociation rate constant rather than the increase in the association rate constant. This information will present an effective approach for designing chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, which may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
We describe the use of acetoxy-acetyl-aminofluorene-modified DNA probes in several hybridization techniques. Hybrids were detected with the help of a monoclonal antibody raised against AAF-guanosine and a second antibody coupled to an enzyme. The sensitivity achieved with AAF-DNA probes routinely detected 0.25 pg DNA bound to a filter. AAF-DNA probes were highly stable and were prepared by simple chemical modification of DNA. Their use as a possible diagnostic tool is discussed.
Alcohol consumption increases the risk for breast cancer in women by still undefined means. Alcohol metabolism is known to produce reactive oxygen species (ROS), and breast cancer is associated with high levels of hydroxyl radical (*OH) modified DNA, point mutations, single strand nicks, and chromosome rearrangement. Furthermore, ROS modification of DNA can produce the mutations and DNA damage found in breast cancer. Alcohol dehydrogenase (ADH) and xanthine oxidoreductase (XOR) are expressed and regulated in breast tissues and aldehyde oxidase (AOX) may be present as well. Mammary gland XOR is an efficient source of ROS. Recently, hepatic XOR and AOX were found to generate ROS in two ways from alcohol metabolism: by acetaldehyde consumption and by the intrinsic NADH oxidase activity of both XOR and AOX. The data obtained suggests that: (1) expression of ADH and XOR or AOX in breast tissue provides the enzymes that generate ROS; (2) metabolism of alcohol produces acetaldehyde and NADH that can both be substrates for XOR or AOX and thereby result in ROS formation; and (3) ROS generated by XOR or AOX can induce the carcinogenic mutations and DNA damage found in breast cancer. Accumulation of iron coupled with diminished antioxidant defenses in breast tissue with advancing age provide additional support for this hypothesis because both result in elevated ROS damage that may exacerbate the risk for ROS-induced breast cancer.
Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.
The potentials of nickel(II) and cadmium(II) to interfere with the repair of different types of deoxyribonucleic acid (DNA) lesions was investigated. Concerning the nucleotide excision repair pathway, nickel(II) has been shown to reduce the incision and the ligation frequency after ultraviolet (UV)-irradiation. When applying a gel mobility shift assay and HeLa nuclear cell free extracts, nickel(II) diminishes the specific binding of a protein to UV-damaged DNA, suggesting that nickel(II) interferes with the DNA-protein interactions involved in the damage recognition after UV-irradiation. Similarly, the incision frequency is reduced in the presence of low concentrations of cadmium(II). Concerning the repair of oxidative DNA damage induced by visible light, non-cytotoxic concentrations of nickel(II) caused a complete repair inhibition of DNA base modifications like 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) and of DNA strand breaks. Since the repair of DNA damage is essential for the prevention of cancer, its inhibition may account for the carcinogenic action of the respective metal compounds.
The disruption of genomic DNA methylation patterns was the first epigenetic abnormality to be described in human cancer. This imbalance involves the promoter CpG island hypermethylation of tumor-suppressor genes, causing transcriptional repression, and global genomic hypomethylation, leading to chromosomal instability and reactivation of endoparasitic sequences. The relationship between DNA methylation and histone modifications was initially described in the context of the inactivation of female X chromosomes and of the demonstration of strong interactions between the DNA methylation machinery and chromatin modifiers. The repression of tumor-suppressor genes by promoter hypermethylation was also found to be associated with a specific histone modification index. However, this jigsaw was missing a piece: a global view of how the histone modification landscape was distorted in cancer cells. We have recently discovered this piece of the puzzle, demonstrating that the association between DNA methylation and histone modification aberrations in cancer also occurs at the global level. In human and mouse tumors, histone H4 undergoes a loss of monoacetylated and trimethylated lysines 16 and 20, respectively. Most importantly, these alterations occur within the context of the repetitive DNA sequences that also become hypomethylated in transformed cells. The global alterations of histone acetylation status suggest novel pathways by which histone acetyltransferases (HATs), histone methyltransferases (HMTs), and histone deacetylases (HDACs) may play roles as tumor-suppressor genes or oncogenes. In this regard, we have shown how the generation of particular fusion proteins involving HATs in leukemias is associated with an erasure of the monoacetylated lysine 16-H4 marker, whilst the loss of trimethylation at lysine 20-H4 disrupts heterochromatic domains and may reduce the response to DNA damage of cancer cells.
DNA methylation can be analyzed easily by qualitative or quantitative polymerase chain reaction (PCR)-based methods, including methylation-specific PCR (MSP), bisulfite sequencing, methylation-sensitive restriction enzyme PCR, combined bisulfite restriction analysis (COBRA), methylation-sensitive single nucleotide primer extension (Ms-SNuPE), and quantitative real-time MSP. MSP, which couples the bisulfite modification of DNA and PCR, is fast, highly sensitive, specific, and widely applied for DNA methylation analyses. Bisulfite modification converts unmethylated cytosine to uracil, whereas methylcytosine remains unmodified. Most of these methods require specific PCR primers that are designed to distinguish between methylated and unmethylated DNA sequences. Bisulfite sequencing is comparatively time-consuming. Methylation-sensitive restriction enzyme PCR combines methylation-sensitive restriction enzyme digestion and PCR. After enzyme digestion, PCR products are obtained if the enzyme does not digest the methylated CpG sites within the specified DNA region. COBRA, Ms-SNuPE, and quantitative real-time MSP allow the quantitative analyses of DNA methylation.
Modification of DNA bases in mammalian chromatin upon treatment with hydrogen peroxide in the presence of ferric and cupric ions was studied. Ten DNA base products in mammalian chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring after hydrolysis of chromatin and trimethylsilylation of hydrolysates. This technique permitted the analysis of modified DNA bases in chromatin without the necessity of isolation of DNA from chromatin first. Modified bases identified were typical hydroxyl radical-induced products of DNA, indicating the involvement of hydroxyl radical in their formation. This was also confirmed by inhibition of product formation by typical scavengers of hydroxyl radical. The inhibition of product formation was much more prominent in the presence of chelated ions than unchelated ions, indicating a possible site-specific formation of hydroxyl radical when metal ions are bound to chromatin. Hydrogen peroxide in the presence of cupric ions caused more DNA damage than in the presence of ferric ions. Chelation of cupric ions caused a marked inhibition in product formation. By contrast, DNA was damaged more extensively in the presence of chelated ferric ions than in the presence of unchelated ferric ions. The presence of ascorbic acid generally increased the yields of the products, indicating increased production of hydroxyl radical by reduction of metal ions by ascorbic acid. Superoxide dismutase afforded partial inhibition of product formation only in the case of chelated iron ions. The yields of the modified bases in chromatin were lower than those observed with calf thymus DNA under the same conditions.
Modification of a Coulter EPICS V orthogonal laser-based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome-bearing spermatozoa from the bull, boar, and ram can be resolved using this system.
The modification of the human placenta DNA polymerase alpha by the imidazolides of dNMP was investigated. The modification was shown to occur only in the simultaneous presence of the template and the primer. This process, however, doesn't depend on the complementary interaction of the nucleotide base with the template. The Kd values of the complexes between the different nucleotides and DNA polymerase alpha were estimated. The affinity of Im-dTMP was determined from the dependence of the Kapp of the enzyme inactivation rate on the reagent concentration. The Kd values for dNMP, dNDP, dNTP were estimated using the protective effect of these nucleotides under the enzyme modification by Im-dTMP. The comparison of the interaction efficiency between the polymerase and dNMP, dNDP, dNTP (complementary or non-complementary to the template) allow to conclude that the nucleotide discrimination occurs on the dNTP level, i. e. dNMP and dNDP upon forming the complex with the enzyme, don't interact complementarily with the template. The additional contacts between the enzyme and the nucleotide terminal phosphate were supposed to form only for the complementary dNTP. The studies allowed to put forward a hypothetical model of the template complementary dNTP binding to the polymerases. The role of the hydrophobic interaction of the nucleotides with the enzyme as well as the possible influence of the nucleotide gamma-phosphate group on the template--dNTP complement formation. The Watson-Crick bound formation of the nucleotide with the template was supposed to be followed by the additional conformational rearrangement of the nucleotide triphosphate chain. The latter process leads to the formation of additional contacts between the enzyme and the nucleotide gamma-phosphate.
It is well recognized that there is a surprising degree of phenotypic variation among genetically identical individuals, even when the environmental influences, in the strict sense of the word, are identical. Genetic textbooks acknowledge this fact and use different terms, such as 'intangible variation' or 'developmental noise', to describe it. We believe that this intangible variation results from the stochastic establishment of epigenetic modifications to the DNA nucleotide sequence. These modifications, which may involve cytosine methylation and chromatin remodelling, result in alterations in gene expression which, in turn, affects the phenotype of the organism. Recent evidence, from our work and that of others in mice, suggests that these epigenetic modifications, which in the past were thought to be cleared and reset on passage through the germline, may sometimes be inherited to the next generation. This is termed epigenetic inheritance, and while this process has been well recognized in plants, the recent findings in mice force us to consider the implications of this type of inheritance in mammals. At this stage we do not know how extensive this phenomenon is in humans, but it may well turn out to be the explanation for some diseases which appear to be sporadic or show only weak genetic linkage.
Knowledge about preimplantation development is important both for basic reproductive biology and for practical applications, including livestock breeding and regenerative medicine. During preimplantation development, epigenetic modifications such as DNA methylation and histone modifications are involved in the regulation of imprinted and non-imprinted genes, in the initiation of X chromosome inactivation, and the adjustment of telomere length. The underlying events are particularly vulnerable to external factors. Characterization of expression profiles in in vivo-derived embryos of different developmental stages and understanding the mechanisms and dynamics underlying the reprogramming process are the first steps towards the analysis of the complex gene regulatory networks. They provide a baseline for the analysis of manipulated embryos of all mammalian species, including humans, to improve embryo technologies and related therapeutic applications.
BACKGROUND: Aberrant methylation of promoter-associated cystosine-guanine (CpG) islands is an epigenetic modification of DNA frequently observed in adult patients with acute lymphocytic leukemia (ALL). This epigenetic modification has been associated with gene silencing, malignant transformation, and aging. It is not known whether there are epigenetic differences between pediatric patients and adult patients with ALL. METHODS: To investigate the methylation characteristics of pediatric patients with ALL and to determine whether DNA methylation can explain prognostic or biologic differences between pediatric and adult patients, the authors analyzed the methylation status of 7 promoter-associated CpG islands in 16 pediatric patients with ALL and compared them with the methylation characteristics of a cohort of adult patients with ALL. The genes analyzed included the estrogen receptor gene (ER), multidrug resistance gene 1 (MDR1), p15, C-ABL, CD10, p16, and p73. RESULTS: The mean methylation densities of ER, MDR1, CD10, p15, and C-ABL were 25.4%, 16.4%, 5.23%, 4.24%, and 4%, respectively. P16 was methylated in 11.7% of patients, and p73 was methylated in 17.6% of patients. One patient (6.2%) had methylation of 0 genes, 15 patients (93.7%) had methylation of >/= 1 gene, and 4 patients (25%) had methylation of 3-4 genes. Methylation of all these genes was < 2% (or methylation specific polymerase chain reaction negative) in nonneoplastic tissues. A significant inverse correlation was observed between methylation of CD10 and CD10 expression. No differences were observed between the methylation characteristics of pediatric patients and adult patients. CONCLUSIONS: The results indicate that DNA methylation is common in pediatric patients with ALL and that methylation of the genes studied does not account for prognostic differences between pediatric patients and adult patients with ALL.