Interaction of mRNA of the p53 protein gene with ribosomal RNA]

By blot hybridization we found that DNA fragments of eukaryotic 18 and 28S rRNAs bind specifically with mRNA. In these experiments the in vitro transcribed mRNA of mouse gene p53 was used. In addition we found that both 18 and 28S rRNAs were able to form intermolecular complexes with mRNAs of several genes 18S rRNA-mRNA; 28S rRNA-mRNA, but fail to bind the antisense RNAs of the same genes. The experimental data allow to suppose that 18S and 28S rRNAs carry several fragments that are complementary to some fragments in the mRNA sequences.     

Optimization of a genome-wide disordered lentivector-based short hairpin RNA library

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro ^R, copGFP, or H-2K ^k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.     

Cystathionase: high-performance liquid chromatography. Molecular cloning in lambda gt11. Nonradioactive immunodetection of fusion protein

A method of purification of rat liver cystathionase by high-performance liquid chromatography (HPLC) utilizing non-ideal gel filtration method is proposed. Resolution factors-flow rate, pH values, ionic strength of the mobile phase-were optimized. Antibodies to the enzyme were purified using an immunosorbent synthesized on the basis of epoxylated Toyopearl-65. Radioimmunoassay and immunoblotting demonstrated antibody monospecificity towards cystathionase. These monospecific antibodies were utilized for detecting enzyme amounts (up to 30 pg) using the avidin-biotin system. Rat cDNA expression library in phage lambda gt11 was screened. The cystathionase cDNA clone was isolated, and the structure of the insert was determined.