[Genome-wide lentivector-based pooled shRNA library optimization].

We have optimized lentiviral vector constructs and cassettes for expression of short hairpin RNAs (shRNAs) in order to create genome-wide library capable of inhibition of full variety of human mRNAs. The vector optimization has resulted in 15-20-fold improvement in virus stock titers. We found that in the context of lentiviral vector the most effective structure for the shRNA is simple hairpin with 21 nucleotide stem. The shRNA-expressing lentiviral constructs contain choice of puro(R), copGFP or H-2K(k) selective markers. The efficiency of the optimized library was evaluated in experiments on screening of shRNAs that reactivate oncosuppressor p53 in HeLa cells. The cells contained reporter construct with p53-dependent expression of a fluorescent protein, which allows cytofluorimetric isolation of cell population with reactivated p53.

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.

[A method for obtaining the normalized cDNA libraries based on the effect of suppression of polymerase chain reaction]. / Metod polucheniia normalizovannykh bibliotek kDNK, osnovannyi na éffekte supressii polimeraznoi tsepnoi reaktsii.

A new efficient method for obtaining cDNA libraries with equal representation of all cDNA types (equalized libraries) in a single round of equalization was developed. The method is based on differences in the renaturation kinetics of double-stranded cDNAs of different genes and allow the selection of the equalized single-stranded fraction resulted from the incomplete reassociation of the total cDNA without laborious and inefficient physical separation. The equalized single-stranded fractions are selectively amplified by polymerase chain reaction (PCR). The amplification of other DNA molecules is inhibited due to PCR suppression, i.e. the suppression of amplification of the DNA molecules flanked with long interval terminal repeats in PCR with a primer corresponding to the external moiety of the repeat. The efficiency of the developed method was estimated in obtaining an equalized cDNA library based on mRNA from the activated human T lymphocyte Jurkat cell line.

Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed mRNA.

We have modified recently developed methods of RNA fingerprinting and differential display based on arbitrarily primed PCR which can be used for detection and cloning of differentially expressed mRNAs. Our protocol requires only a single cDNA synthesis for each different RNA sample, in contrast to the multiple cDNA reactions required for differential display method, followed by selective amplification of cDNA sequence fraction by arbitrary and oligo(dT) primers. We have shown that the longer primers (25-29-mers) allow the use of optimal dNTP concentration and higher stringency PCR. Further improvements include using TaqStart antibody for hot start PCR and thermostable enzyme mixes suitable for long-distance PCR. Long-distance PCR enables the method to display bands of up to 2 kb and should result in a higher fidelity of PCR products to the original RNA template. When these improvements are combined the resulting method is highly reproducible, and more than 85% of the differentially expressed bands can be confirmed by Northern blot analysis.

[Analysis of effectiveness of cDNA synthesis, induced using complementary primers and primers containing a noncomplementary base matrix]. / Analiz éffektivnost' sinteza kDNK, initsiirovannogo s komplementarnykh praimerov i praimerov, soderzhashchikh nekomplementarnye matritse osnovaniia.

We have studied the efficiency of DNA synthesis catalyzed by M-MLV reverse transcriptase or Thermus aquaticus DNA polymerase for primers (4-17 nucleotides long) either completely matched or possessing a single mismatched base pair at all possible positions in the primer. It has been shown that DNA synthesis efficiency depends not only on the position of mismatched base pair but on the length and primary structure of the primer. The enzyme, template, and primer concentrations determine the relative level of mismatched DNA synthesis.

[Derivatives of ddUTP, modified at the 5-position of uridine, as substrate terminators of reverse transcriptase. Hydrolysis of oligonucleotides, terminated by these analogs, by phosphodiesterase I]. / Proizvodnye ddUTP, modifitsirovannye v 5-polozhenii uridina, kak substratnye terminatory obratnykh transkriptaz. Gidroliz oligonukleotidov, terminirovannykh étimi analogami, fosfodiésterazoi I.

Synthesis of 2',3'-dideoxyuridine 5'-triphosphate analogues with fluorescent and biotin residues at C5 of uracil base was carried out. The substrate properties of these analogues were studied with AMV, M-MLV, and HIV reverse transcriptase. All 5-derivatives studied were shown to be incorporated into the 3'-terminus of oligonucleotide. The stability of oligodeoxyribonucleotides terminated with ddUTP analogues modified at the 5-position of the pyrimidine residue to the exonuclease action of phosphodiesterase I and Klenow enzyme was more than 1000 times higher than that of nonterminated oligonucleotides.

Application of poly(A)+RNA patterns method for searching of differentially expressed genes.

Poly(A)+RNA composition differences for normal, fetal and cirrhotic human liver before and after retinoic acid-induced differentiation of the F9 embryonal carcinoma cell line were analyzed by a novel poly(A)+RNA patterns method. The method is based on the polyacrylamide gel electrophoretic analysis of short cDNA termination products, synthesized by reverse transcriptase using poly(A)+RNA as a template, a set of short 5'-end labeled primers, three natural and one terminator deoxyribonucleotide. A number of known differentially expressed genes and some unknown ones were then identified by direct sequencing of the differentially represented bands excised from a gel and searching a complementary mRNA target sites in Genbank database.

Analysis of poly(A)+RNA patterns in human tissues.

A novel method for analysing and comparing the relative amounts of the most abundant (higher than 0.1% abundance) individual mRNAs present in different poly(A)+RNA preparations has been developed. This method is based on the synthesis of short (10-30 nucleotide) cDNA termination products, by reverse transcription of poly(A)+RNA primed with a 5'-labeled oligonucleotide. A set of 30 different oligonucleotides are used as primers in separate reactions, their length and sequences having been chosen to provide more than a 90% probability of initiating synthesis from any individual RNA present in the poly(A)+RNA. Each primer produces about 10-60 bands per track, following polyacrylamide gel electrophoresis under denaturing conditions. Data presented reveals poly(A)+RNA pattern differences for a number of human tissues and identifies changes in RNA patterns between normal tissues and neoplastic human tumors (myoma of the uterus) from several individuals.

[Quantitative analysis of individual RNA in preparations of poly(A)+-RNA mammalian cells]. / Kolichestvennyi analiz individual'nykh RNK v preparatakh poli(A)+-RNK kletok mlekopitaiushchikh.

A new method of simultaneous analysis of the relative abundance of the most abundant individual mRNA's in poly(A)(+)-RNA preparations is described. The method is based on the synthesis of short (10-20 nucleotides) cDNA products by reverse transcription of poly(A)(+)-RNA primed with 5'-labeled oligonucleotides of 9 nucleotide lengths. Three natural nucleotides and one terminator nucleotide are used as substrates for reverse transcriptase. The numbers, lengths and sequence of the oligonucleotides used as primers were chosen to provide more than a 90% probability that synthesis would be initiated from any individual RNA present in the poly(A)(+)-RNA, thus assuring comprehensive analysis of RNA with abundance higher than 0.01%. Each primer produces about 20-60 bands per track following polyacrylamide electrophoresis under denaturing conditions. A full set of 30 oligonucleotides used to analyze a poly(A)(+)-RNA preparation produces an electrophoretic pattern with information capacity similar to that obtained from high resolution 2-dimensional electrophoresis of protein. Using this method we show that the patterns of poly(A)(+)-RNA differ from tissue to tissue, from normal tissues to neoplastic tissue (human myoma of uterus) and during differentiation of a F9 embryonic carcinoma cell line.

Localization of apolipoprotein E in normal and atherosclerotic human aorta.

To elucidate the role of apolipoprotein E (apo E) in atherogenesis, we have investigated the localization of apo E in normal and atherosclerotic aortas as well as in other tissues of 32 post-mortem individuals. Using double immunofluorescence it has been found that normal intima of individuals older than 20 years and some adolescents contained immunoreactive material that reacted with poly- and monoclonal antibodies to apo E. A staining pattern of apo E differed from that of apolipoprotein B, the latter being seen in normal intima of each child older than 7 years. Apo E was present extracellularly in lipid streaks and atheromatous plaques, where its staining was particularly intensive around the necrotic zone of plaques. Some macrophages in the plaques of 4 aortas exhibited apo E-positive staining, while aortic endothelial and smooth muscle cells never contained apo E. Apo E-positive staining was not found in the majority of vessel cells, it was always, however, observed in other types of cells including hepatocytes. Kupffer cells, spleen macrophages and cerebral astrocytes. Our findings indicate that only some macrophages in human aorta may be responsible for the production of apo E that can participate in reverse cholesterol transport. At the same time, apo E accumulation in the aortic wall may promote the development of atherosclerosis.

Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters.

Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P0) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.

[Contacts of Escherichia coli RNA polymerase subunits with nucleotides of lacUV5 promoter]. / Kontakty mezhdu sub"edinitsami RNK-polimerazy Escherichia coli i nukleotidami promotora lacUV5.

We have localized contacts between DNA and subunits of E. coli RNA polymerase (of both holo and core enzymes) along lacUV5 promoter. In the complex with the holo enzyme the anti-sense strand of DNA makes contacts with beta' subunit at -47, -46, -30, -12, +1, +5, +7, +9; beta subunit at -30, -25, -23, -12, +11, +30 and delta subunit at -17, -5, -3 nucleotides, while the sense-strand of DNA makes contacts with beta' subunit at -22, -21, -9, -6, -4, +3, +4, +6, +17; beta subunit at +17, +25, +27, +34; and delta subunit at -35, -18 nucleotides. In the complex with the core enzyme the anti-sense strand of DNA makes contacts with beta'subunit at -47, -23, -5, -3, +5, +7, +9; beta subunit at -23, -16, +11, +30 nucleotides while the sense-strand of DNA makes contacts with beta' subunit at -20, -19, +3, +4, +6, +17; beta subunit at -36, -35, -34, -31, -29, +17, +25, +27, +34 nucleotides alpha subunits of the holo as well as the core enzyme show no contact with DNA in the conditions providing specific complex formation.

[Pyrophosphate analogs in the pyrophosphorolysis reaction catalyzed by Escherichia coli RNA polymerase]. / Analogi pirofosfata v reaktsii pirofosforoliza, kataliziruemoi RNK-polimerazoi Escherichia coli.

Processive pyrophosphorolysis of RNA from ternary RNA polymerase-nascent RNA-delta D111 T7 DNA complex has been followed in the absence of nucleoside triphosphates. Series of inorganic pyrophosphate analogs were investigated for their ability to sustain the reaction and to compete with inorganic pyrophosphate for the reaction. Methylenediphosphonic, imidodiphosphonic, phosphonacetic acids, inorganic triphosphate, methylenediphosphonic and phosphate were found to be capable of substituting the inorganic pyrophosphate in RNA degradation reaction with tantamount efficiency. They give rise to nucleoside monophosphates for phosphonoacetic acid, nucleoside triphosphates for inorganic pyrophosphate and inorganic triphosphate, nucleoside triphosphates analogs for methylenediphosphonic, imidodiphosphonic acids and methylenediphosphonic acid phosphate as the low molecular weight product of the reaction. The problem of specific interaction of RNA polymerase with nucleoside triphosphates and inorganic pyrophosphate is discussed in the terms of structural requirements for the compounds to be a potent substrate for RNA polymerase.

[Reaction of pyrophosphorolysis catalyzed by Escherichia coli RNA polymerase]. / Reaktsiia pirofosforoliza, kataliziruemaia RNK-polimerazoi Escherichia coli.

E. coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+. Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis. The reaction was realized in the absence of added nucleoside triphosphates. Nucleoside triphosphates are low molecular mass products of the reaction. The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration. The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis. Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture. RNA as short as 70-8 nucleotides long can be produced at the conditions used. It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA. Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation. The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised.

[Methylation of E. coli RNA polymerase with dimethylsulfate]. / Metilirovanie RNK-polimerazy E. coli dimetilsul'fatom.

DNA dependent RNA polymerase from E. coli was methylated with dimethylsulfate. After the methylation the enzymatic activity was lost. Addition of two methyl groups per enzyme monomer completely inactivated enzyme with respect to RNA synthesis but couldn't prevent enzyme binding to DNA. Methylated enzyme was able to form tight complexes with DNA and to compete with the native enzyme for the formation of rifampicin resistant complex with DNA. The ratio of the binding constants of the native and methylated enzymes to DNA was determined to be equal to 3. Methylated enzyme was not able to form the first phosphodiester bound as revealed from pyrophosphate exchange reaction studies.