Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.     

Molecular dynamics simulation of the substitution of serine for the conserved glycine in the G-loop in the cdc28-srm yeast mutant using the crystal lattice of human CDK2 kinase

Two-nanosecond molecular dynamics simulations of the crystal lattice of an active complex of pT160-CDK2 kinase/cyclin A/ATP-Mg²⁺/substrate were performed. The simulations showed that the structures of the wild-type CDK2 complex and the mutant CDK2 complex involving the substitution G16S-CDK2 corresponding to the yeast substitution G20S-CDC28 differ noticeably and the differences between the structural conformations are most pronounced in the regions that play a key role in the kinase functioning. The results of the computer calculations were used to consider the structural elements that may affect the kinase activity, the regulatory phosphorylation, and the binding of protein kinase with cyclins and substrates.     

Protein trypsin inhibitor from potato tubers

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.     

Tobacco transformants expressing the <Emphasis Type="Italic">Medicago truncatula</Emphasis> ornithine aminotransferase cDNA

The Medicago truncatula ornithine aminotransferase cDNA was cloned under the potent constitutive 35S RNA promoter of the cauliflower mosaic virus and transferred into the genome of tobacco Nicotiana tabacum SR1 plants. Transformed tobacco plants grew better in salinity stress, but did not differ in proline content under normal or stress conditions from control plants. It was assumed that the role of ornithine aminotransferase in the molecular mechanisms of stress resistance is not associated with additional proline synthesis.     

Maternal effect for genes encoding 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in Drosophila melanogaster

We studied the maternal effect for two enzymes of the pentose cycle, 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD), using a genetic system based on the interaction of Pgd^? and Zw^? alleles, which inactivate 6PGD and G6PD, respectively. The presence and formation of the enzymes was investigated in those individuals that had not received the corresponding genes from the mother. We revealed maternal forms of the enzymes, detectable up to the pupal stage. The activities of “maternal” 6PGD and G6PD per individual increased 20-fold to 30-fold from the egg stage to the 3rd larval instar even in the absence of normal Pgd and Zw genes. Immunologic studies have shown that the increase in 6PGD activity is due to an accumulation of the maternal form of the enzyme molecules. We revealed a hybrid isozyme resulting from an aggregation of the subunits of isozymes controlled by the genes of the mother and embryo itself. These results indicate that the maternal effect in the case of 6PGD is due to a long-lived stable mRNA transmitted with the egg cytoplasm and translated during the development of Drosophila melanogaster.