Effect of Ionizing Radiation on the Transcript Variants Expression of p21 Gene.

PURPOSE: CDK1A is one of the most important genes that have different key roles in cell lines. This gene has several transcript variants. Investigating of expression of each one actually can be so important because any one of them may have a separate unknown role in cancer cells so can be used to increase therapeutic efficacy. METHODS: A549, MDA-MB-231 and Hek-AD cell lines were used in this study. Firstly, three primers for variants of p21 gene were designed by Snapgene and BLAST software. Secondly, the variants expression was checked for each cell lines by RT-qPCR technique, separately. Then the variants that expressed in the cells were selected for more investigation. Finally 2 Gy irradiation was used to evaluate the effect of that on variants expression. RESULTS: The results show that for all cell lines, primer num1 and 3 expressed before any stimuli. After irradiation, for MDA-MB-231 and A549, the expression of primer num3 was decreased, while for Hek-AD no change was observed. The primer num1 expression after the irradiation was different for the cells, V1 expression was decreased in A549 by fold of 0.03 while expression of this for MDA-MB-231 cells was not changed after 2Gy irradiation. CONCLUSION: It is very necessary to pay attention to the function of each splice variant as well as the response to external stimuli. Understanding the role of each variant in a gene is critical and researchers can use that to improve radiotherapy as well.

Single particle ICP-MS-based absolute and relative quantification of E. coli O157 16S rRNA using sandwich hybridization capture.

Herein, a novel 16S rRNA detection platform was achieved by combining a sandwich hybridization reaction, a single-molecule magnetic capture, and single particle-inductively coupled plasma mass spectrometry amplification. The assay was developed for the direct detection of RNA from dangerous human pathogens and enabled absolute and high-precision quantification of a target with a detection limit of 10 fM.

Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit.

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.

Direct preparation of sticky-ended duplexes within PCR by using caged primers.

Control of the terminal structures of PCR products is crucially important to facilitate molecular biology and biotechnology. Here, we report a new method to prepare PCR products having desired sticky ends directly after thermal cycles. When a pair of caged primers is used, polymerase reaction is site-selectively terminated in front of the caged nucleotide, and the 5'- portion of the primer remains single-stranded throughout the reaction. Successive removal of the photo-cleavable protecting group gives restriction-enzyme free sticky ends on the product. We succeeded in applying this technique to make a recombinant vector bearing GFP gene.

Radiation target analyses of DNA template/primer complexes.

Frozen solutions of low molecular weight DNA template/primer complexes, in the absence and presence of HIV-1 reverse transcriptase, were irradiated with high-energy electrons. Molecules that survived the radiation exposure were quantified and analyzed using radiation target theory. Transfer of radiation-deposited energy was observed by the damage caused. It was found that damage (as a polynucleotide chain break) was observed in one chain when the radiation interaction occurred in the other chain, suggesting a transfer of energy. In contrast, the target sizes of the DNA template/primers were not altered if bound to HIV-1 reverse transcriptase, signifying that the deposited radiation energy is not transferred between protein and nucleic acid.