Detection of DNA methylation in eucaryotic cells

The methods of molecular biology allow for analyzing the methylation pattern in the whole genome and in particular genes. We differentiate methylated sequences from unmethylated ones by means of cutting the genomic template with methylation-sensitive restriction enzymes or by sodium bisulfite DNA modification. Chemical modification precedes most quantitative and qualitative PCR techniques: MS-PCR, MS-nested PCR, Real-Time PCR, QAMA, HeavyMethyl, MSHRM. Restriction enzymes, on the other hand, may be used together with PCR or hybridisation methods (Southern blot and microarrays). PCRs are conducted with primers specific for methylated and unmethylated sequences and sometimes, similarly to hybridisation techniques, with specifically labeled probes or dyes intercalating to double-stranded nucleic acids. The most advanced methylation detection techniques (MALDI-TOF MS and HPLC) significantly reduce the amount of biological material used for tests, but they require specialist equipment.     

Free and integrated plasmid DNA in spiroplasmas

Spiroplasma citri was found to carry an 8.0 kb plasmid that differed from previously describedS. citri plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid inS. citri (R8A2, Maroc) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome. Significant quantities of integrated plasmid sequences were also observed in the corn stunt spiroplasma,S. kunkelii, while small quantities of free and/or integrated plasmid DNA could be detected in some spiroplasmas serologically and genotypically remote fromS. citri. Integrated plasmid sequences were cloned into theEscherichia coli plasmid pUC13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of fragments, rather than entire plasmid DNA, inserted into specific sites in the spiroplasma chromosome. Although the biological role of the pRA1 plasmid remains unclear, theS. citri subclones containing large quantities of free plasmid exhibited slower growth rates and a tendency to lyse.     

Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.     

DNA probes in detection of spiroplasmas and mycoplasma-like organisms in plants and insects

Abstract DNA probes were applied to detect spiroplasmas and uncultivable mycoplasma-like organisms (MLOs) in infected plants and insects. The probes consisted of pMC5, a plasmid carrying the RNA genes of Mycoplasma capricolum and pRA1, a plasmid recovered from Spiroplasma citri . Southern blot hybridization of pMC5 with digested DNAs of periwinkle plants infected with S. citri , or with various MLOs, yielded 2 heavy and several weaker bands. The heavy hybridization bands were shown to represent rRNA genes of the plant chloroplasts, indicating significant nucleotide sequence homology between the mycoplasmal rRNA genes and those of plant chloroplasts. Some of the weaker hybridization bands, not revealed in DNA of healthy plants, appeared to represent rRNA gene sequences of the infectious agent. Use of the spiroplasma plasmid as a probe enabled the detection of S. citri in infected plant material and in hemolymph of infected leafhoppers at a high sensitivity level.     

Unique secondary and tertiary structural features of the eucaryotic selenocysteine tRNA(Sec).

Cotranslational insertion of selenocysteine into selenoenzymes is mediated by a specialized transfer RNA, the tRNA(Sec). We have carried out the determination of the solution structure of the eucaryotic tRNA(Sec). Based on the enzymatic and chemical probing approach, we show that the secondary structure bears a few unprecedented features like a 9 bp aminoacid-, a 4 bp thymine- and a 6 bp dihydrouridine-stems. Surprisingly, the eighth nucleotide, although being a uridine, is base-paired and cannot therefore correspond to the single-stranded invariant U8 found in all tRNAs. Rather, experimental evidence led us to propose that the role of the invariant U8 is actually played by the tenth nucleotide which is an A, numbered A8 to indicate this fact. The experimental data therefore demonstrate that the cloverleaf structure we derived experimentally resembles the hand-folded model proposed by Böck et al (ref. 3). Using the solution data and computer modelling, we derived a three-dimensional structure model which shows some unique aspects. Basically, A8, A14, U21 form a novel type of tertiary interaction in which A8 interacts with the Hoogsteen sites of A14 which itself forms a Watson-Crick pair with U21. No coherent model containing the canonical 15-48 interaction could be derived. Thus, the number of tertiary interactions appear to be limited, leading to an uncoupling of the variable stem from the rest of the molecule.     

同卵双胞胎在复杂性状DNA甲基化调控机制研究中的应用

同卵双胞胎来源于同一个受精卵,DNA序列基本一致,但在某些重要表型上如复杂疾病,并不完全一样。利用表型不一致的同卵双胞胎进行研究,能在遗传背景、母体效应、年龄性别效应等一致的基础上,深入研究分析复杂性状的表观调控机制。而DNA甲基化是最为稳定的一类表观遗传修饰。在人类中,利用同卵双胞胎对印记异常疾病、精神类疾病、自身免疫病及癌症等疾病的DNA甲基化调控研究已经揭示了多个致病基因,为研究疾病的表观调控以及表观遗传学药物的应用打下了基础。本文着重对同卵双胞胎DNA甲基化状态、DNA甲基化遗传力计算以及复杂性状DNA甲基化调控的研究应用及其进展展开综述,以期为复杂性状表观调控机制研究提供借鉴和参考。     

Frameshift mutagenesis by eucaryotic DNA polymerases in vitro

The frequency and specificity of frameshift errors produced during a single round of in vitro DNA synthesis by DNA polymerases-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) have been determined. DNA polymerase-beta is the least accurate enzyme, producing frameshift errors at an average frequency of one error for each 1,000-3,000 nucleotides polymerized, a frequency similar to its average base substitution accuracy. DNA polymerase-alpha is approximately 10-fold more accurate, producing frameshifts at an average frequency of one error for every 10,000-30,000 nucleotides polymerized, a frequency which is about 2- to 6-fold lower than the average pol-alpha base substitution accuracy. DNA polymerase-gamma is highly accurate, producing on the average less than one frameshift error for every 200,000-400,000 nucleotides polymerized. This represents a more than 10-fold higher fidelity than for base substitutions. Among the collection of sequenced frameshifts produced by DNA polymerases-alpha and beta, both common features and distinct specificities are apparent. These specificities suggest a major role for eucaryotic DNA polymerases in modulating frameshift fidelity. Possible mechanisms for production of frameshifts are discussed in relation to the observed biases. One of these models has been experimentally supported using site-directed mutagenesis to change the primary DNA sequence of the template. Alteration of a pol-beta frameshift hotspot sequence TTTT to CTCT reduced the frequency of pol-beta-dependent minus-one-base errors at this site by more than 30-fold, suggesting that more than 97% of the errors at the TTTT run involve a slippage mechanism.     

Cytological and electrophoretic analysis of DNA methylation in the holocentric chromosomes of Megoura viciae (Homoptera, Aphididae).

Chromosomal and purified DNA methylation patterns were determined in the holocentric chromosomes of Megoura viciae by treatment with MspI and HpaII. Both enzymes produced a clear C-like banding pattern but widely digested one telomere of the X chromosome, which appeared as heterochromatic after C-banding treatment and brightly fluorescent after chromomycin A3 staining. Quantitative microfluorometric evaluations of DNA extraction performed on cytological preparations showed that both isoschizomers resulted in the same DNA extraction (about 30%). Contrary to what was found by in situ endonuclease treatment, the electrophoretic patterns of purified and digested DNA showed that digestion with MspI was slightly more extensive than that with HpaII in a zone of fragments ranging from 23 to 9 kb. This result indicates that aphid chromatin is not wholly unmethylated. The discrepancy between electrophoretic and cytological data has been explained by taking into consideration that DNA fragments with high molecular weights could be cleaved in situ by the enzymes but not extracted from the chromatin.     

A third DNA polymerase from Spiroplasma citri and two other spiroplasmas.

Recently, two DNA polymerases (ScA and ScB) were isolated and characterized from Spiroplasma citri. We now have found a third DNA polymerase (ScC) not only in S. citri but also in the serologically related honeybee spiroplasma BC3 and the unrelated flower spiroplasma BNR1. Enzyme ScC is N-ethylmaleimide (NEM) sensitive. The three DNA polymerases from the honeybee spiroplasma seem to be similar to the respective enzymes of S. citri. However, whereas the NEM-resistant enzyme ScA from S. citri and that from the BC3 honeybee spiroplasma are retained on DEAE-cellulose and require 0.09 M KCl for elution, the NEM-resistant enzyme A from the flower spiroplasma BNR1 is not retained.     

A fixed site of DNA replication in eucaryotic cells

We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with ³H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.     

Inhibition of eucaryotic DNA polymerases by phosphonoacetate and phosphonoformate

Phosphonoacetate was found to be an inhibitor of the DNA polymerase α from three human cells, HeLa, Wi-38, and phytohemagglutinin-stimulated lymphocytes. The inhibition patterns were determined. The apparent inhibition constants (K_ii) were about 30 μm. Thus the DNA polymerase α is 15 to 30 times less sensitive to Phosphonoacetate than the herpesvirus-induced DNA polymerase. The DNA polymerase α from Chinese hamster ovary cells and calf thymus was also inhibited. The DNA polymerases β and γ from the eucaryotic cells were relatively insensitive to phosphonoacetate. The sensitivity of the DNA polymerase α and the relative insensitivity of the DNA polymerase β and γ appeared to be general characteristics of the vertebrate polymerases, DNA polymerases from two other eucaryotic cells, yeast DNA polymerase A and B and tobacco cell DNA polymerase, were inhibited by phosphonoacetate, and to about the same extent as the α-polymerases. Fourteen phosphonate analogs were examined for inhibition of the HeLa DNA polymerase α. Only one, phosphonoformate, was an inhibitor. The mechanism of inhibition for phosphonoformate was analogous to that for phosphonoacetate.     

Silver staining of native and denatured eucaryotic DNA in agarose gels

A modified method of silver staining for native and denatured eucaryotic DNA in 1% agarose gel is described. This method is at least fivefold more sensitive than ethidium bromide staining, with a detection limit of 2.5 ng for total DNA. The calibration curve is linear within the range 5-30 ng of single-stranded and double-stranded DNA. This method is especially advantageous for electrophoretic assessment of DNA molecular weights.     

DNA methylation and obesity traits: An epigenome-wide association study. The REGICOR study

Obesity is associated with increased risk of several diseases and has become epidemic. Obesity is highly heritable but the genetic variants identified by genome-wide association studies explain only limited variability. Epigenetics could contribute to explain the missing variability. The study aim was to discover differential methylation patterns related to obesity. We designed an epigenome-wide association study with a discovery phase in a subsample of 641 REGICOR study participants, validated by analysis of 2,515 participants in the Framingham Offspring Study. Blood DNA methylation was assessed using Illumina HumanMethylation450 BeadChip. Next, we meta-analyzed the data using the fixed effects method and performed a functional and pathway analysis using the Ingenuity Pathway Analysis software. We were able to validate 94 CpGs associated with body mass index (BMI) and 49 CpGs associated with waist circumference, located in 95 loci. In addition, we newly discovered 70 CpGs associated with BMI and 33 CpGs related to waist circumference. These CpGs explained 25.94% and 29.22% of the variability of BMI and waist circumference, respectively, in the REGICOR sample. We also evaluated 65 of the 95 validated loci in the GIANT genome-wide association data; 10 of them had Tag SNPs associated with BMI. The top-ranked diseases and functions identified in the functional and pathway analysis were neurologic, psychological, endocrine, and metabolic.     

Detection and differentiation of cross-link formation in an eucaryotic DNA population

A new method to differentiate an eukaryotic DNA population in terms of the frequency of cross-linking sites in each DNA or each class of DNA is presented. Selective cross-link formation can be introduced to a certain class of DNA or a certain region of DNA sequences by use of DNA cross-linking agents due to the differences in the frequency of cross-linking sites in eukaryotic DNA.     

The role of eucaryotic cell surface glycoconjugates in DNA replication

Multiple forms of ricin have been isolated from castor bean seeds. Two forms, ricin-1 and ricin-2, differ in their isoelectric pI values and toxicity towards IMR-32 cells. Inhibition of IMR-32 DNA polymerase α₂ is more pronounced with ricin-1 (65%) than with ricin-2 (10%). Ricin B chain (pI = 5.2) isolated from ricin-1 binds to IMR-32 cell surfaces as well as inhibits DNA polymerase α₂ activity when studiedin vitro. The presence of galβ-linked glycoconjugates near the active site of IMR-32 DNA polymerase α₂ has been proposed. Replication modulators which bind to the glycose portion of the enzymes involved in the replication system may need a mandatory binding to cell surface glycoconjugates for their activity.