Holliday junctions are formed in concentrated solutions of purified products of DNA amplification

Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.     

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how “stress” might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function¹. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats². Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.     

Oncolytic Viruses in the Therapy of Lymphoproliferative Diseases

Molecular Biology - Cancer is a leading causes of death. Despite significant success in the treatment of lymphatic system tumors, the problems of relapse, drug resistance and effectiveness of...     

Method for the quantitative study of the growth of fibroblastic cells in tissue culture

The method is elaborated for a quantitative evaluation of hypodermic fibroblast element growth in tissue culture of albino rats. The method is based on determination of the growth activity and intensity for each of the explant growth zones (compact, necwork-like zones and zone of migrating elements). The method permits a rapid and an exact evaluation of the influence of different substances added to the culture medium.     

Activity of proteolytic enzymes in the kidney and blood serum of rabbits during implantation of different polyurethanes

It is shown that the activity of neutral proteinase both in homogenate and in blood serum increases by the 14th day the D-1 sample being implanted. In the subsequent periods after implantation the enzyme activity in homogenate is the same. Three and six months after implantation the neutral proteinase activity in blood serum decreases as compared to the norm. The activity of acid proteinase in rabbit kidney homogenates lowers by the 90th day after implantation both for the D-1 and for A-10 samples. For the D-1 sample the enzyme activation in blood serum is observed by the 30th day after implantation, three months later it falls to reach the normal level in 6-12 months and inhibition activity on the 30th day after implantation on the A-10 sample. Such changes in the activity of enzymes in homogenates and blood serum may reflect certain stages of polyurethane biodestruction participation of various enzymic systems of the organism in these processes.     

Inactivation of bacteriophage PM2 during storage

The kinetics of bacteriophage inactivation in the medium that is optimal for its storage has been studied at temperatures from 4 to 55 degrees C. The plot of Arrhenius dependence of the constant of inactivation rate consists of the two linear parts with the energies of activation Ea = 25 kcal/mol for 4-37 degrees C and Ea = 91 kcal/mol for 37-55 degrees C. The DNA of inactivated bacteriophage remained mostly in superspiralized form and completely preserved its biological activity as tested by transfection in spheroplasts. The analysis of inactivation kinetics suggests ageing of virions cultivated at 4 degrees C. The addition of watersoluble antioxidant amoxipin did not change the inactivation kinetics. The addition of antioxidant ionol with twin-80 increased the inactivation that was paralleled by the bacteriophage DNA degradation.     

Results of the examination of patients with viral hepatitis for HBsAg in Kashka-Darya Province, Uzbek SSR

The practical use of the reverse passive hemagglutination test for the examination of viral hepatitis patients revealed the presence of HBsAg, on the average, in 28.4 +/- 0.95% of cases. This antigen occurred most frequently among the patients aged up to 1 year (42.8 +/- 4.18%), as well as in the second quarter of the year (33.3 +/- 2.3%). The proportion of patients with hepatitis B in the total number of patients with viral hepatitis was 20%.     

TRAPPII subunits are required for the specificity switch of a Ypt-Rab GEF

Ypt-Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt-Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport. An interesting possibility is that Ypt-Rabs coordinate separate steps of the transport pathways. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5-7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120-Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.