DNA amplification using PCR with abutting primers

Molecular Biology - DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is...     

Functions of E3 Ubiquitin Ligase Hyd in Drosophila Tissues

Molecular Biology - The ubiquitin–proteasome system (UPS) is an important regulator of the main cellular processes. The components of the UPS are involved in the regulation of the cell cycle,...     

The influence of CpG (5′-d(CpG)-3′ dinucleotides) methylation on ultrasonic DNA fragmentation

Abstract

DNA methylation is an important way of gene regulation. The variety of methods for DNA methylation analysis based on chemical modification or enzyme digestion has been proposed. However, DNA is able to undergo transformations under physical power. Here, we report that the cytosine methylation in CpG dinucleotides determines the difference in fragmentation rate of methylated and unmethylated DNA under sonication. We found that at the beginning of sonication, methylated DNAs are degraded faster than unmethylated one, and the difference in fragmentation degree can be evaluated with high reliability by quantitative polymerase chain reaction (qPCR). The optimal parameters that provide the greatest difference in amount of amplifiable DNA targets corresponding to fragmentation degree are the following: moderate amplicon size (about 150–250?bp), medium CpG sparseness (one CpG dinucleotide per ～12–14 nucleotides of the chain), and short sonication time (less than 5?min). Along with CpG, the CpA and CpT contents of amplified regions should be taken into account for proper DNA fragmentation by ultrasound as well. The obtained data could be used for elaboration of a method for comparative methylation testing, when there is no need to detect methylation of certain CpG dinucleotides. This method will be simple (can be used by any technician familiar with PCR), low cost (no need to use an expensive reagents), and fast (only brief DNA sonication and conventional qPCR are carried out).

Communicated by Ramaswamy H. Sarma     

Molecular genetic analysis of the interleukin 6 and tumor necrosis factor alpha gene polymorphisms in multiple myeloma

The-174G alpha-->C polymorphism of the interleukin 6 (IL-6) gene promoter and the-308G alpha-->A polymorphism of the tumor necrosis factor alpha (TNF alpha) gene promoter were tested for association with multiple myeloma (MM) varying in severity. Of 69 patients, 19 had aggressive MM, 26 had benign MM, and 24 had unidentified MM. The control group (N = 102) matched the test group in age and sex composition. The two groups did not significantly differ in allele and genotype frequencies of the IL-6 and TNF alpha genes. Genotype CC, which determines low-level expression of IL-6, occurred at a frequency of 0.35 in patients with low-progressing MM and was absent from patients with aggressive MM. The TNF alpha gene showed no association with predisposition to MM or clinical variant of the disease. On this evidence, the polymorphic variants of the cytokine genes were assumed to have no effect on predisposition to MM. As for MM progression, genotype CC of the IL-6 gene was associated with milder clinical signs in patients from Bashkortostan.     

Induction of cyclosporine-sensitive mitochondrial permeability transition pore by substrates forming acetyl-CoA under normal conditions and in type 2 diabetes

Oxidation of pyruvate and palmitoylcarnitine in mitochondria is accompanied by the formation of acetyl-CoA, with its possible participation in the acetylation of various proteins and enzymes that may lead to the inhibition of their functions. This paper studies the effect of the excess of these substrates on respiration and induction of mitochondrial permeability transition pore (MPTP) in mitochondria and liver homogenates of healthy, obese, and type 2 diabetic (T2D) rats and mice. Both substrates produced a reversible inhibition of respiration and induced the opening of MPTP sensitive to cyclosporin A. Induction of MPTP in mitochondria was further activated by calcium ions and inhibited by the NO donor SNAP and NAD–a coenzyme and activator of deacetylation reactions. In obese and T2D animals, the opening of MPTP was stimulated by lower concentrations of L-palmitoylcarnitine than in healthy animals. In these pathologies, an activation effect on the MPTP induction was produced by ammonium ions, in the presence of which the concentration of L-palmitoylcarnitine required for the pore opening was reduced more than twofold. In liver homogenates, an added arginine reduced the probability of the MPTP formation. Analysis of mathematical models has shown that, due to the inhibition of pyruvate dehydrogenase kinase (PDK) by pyruvate, phosphorylation of pyruvate dehydrogenase (PDH) is strongly reduced, and this makes it possible to produce acetyl CoA in a wide range of pyruvate concentrations. The data obtained show that excess substrates that produce acetyl-CoA increase the probability of the MPTP opening, especially in pathologies associated with obesity and T2D. The ability of NO and NAD to inhibit MPTP indicates the participation of phosphorylation and acetylation/deacetylation reactions in this process.     

A simple and effective method for assessing chromatin diminution values in copepods using qPCR

The value of chromatin diminution (CD) in different species of freshwater cyclopoid copepods can differ significantly. The biological and evolutionary roles of these differences remain unclear. To expand the knowledge on CD distribution and magnitude in this group of copepods, a quick method for its evaluation was required. This study proposes a simple approach for CD assessment in copepods using quantitative realtime PCR (qPCR). The magnitude of changes in the genome size was assessed by comparing fluorescence curves of qPCR fragments of target genes for pre- and post-diminution materials. The method was tested on four cyclopoid copepods species. In Cyclops kolensis, CD was assessed as 95.3 ± 1.2; in Acanthocyclops vernalis it was assessed at 94.6 ± 0.8%; at C. insignis, it was 82.3 ± 5.2%; and for the first time, CD was found in Megacyclops viridis at 91.1 ± 2.6%. The advantages of our approach are its rapidity, simplicity and minimal requirements of materials studied.     

A kinetic lattice model of the chain-melting phase transition in lipid membrane: its experimental calibration and physiological implications

A kinetic lattice model of chain-melting phase transition has been developed. Its time scale has been calibrated with the acoustic relaxation spectroscopic data. The model presents a tool for the analysis and predictions of kinetic features of those physiological processes in biological membranes whose mechanism is based on the phase transition in the lipid component. It has been shown that the phase-transitional mechanism can provide a high rate of synaptic exocytosis known for the fastest synapses in the central nervous system. It was also demonstrated that this mechanism can serve as an essential factor of synaptic plasticity.