In situ amplification using universal energy transfer-labeled primers.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used.

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.

Defining a smaller RNA substrate for elongation factor Tu.

A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs. However, the Ala minihelix, where residue A7 is joined directly to A49, binds to EF-Tu less well than the full-sized Ala-tRNA(Ala). Similar data were obtained for Escherichia coli EF-Tu. An in vitro selection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix. After six rounds of enrichment, two groups of RNA were obtained that bound T. thermophilus EF-Tu as well as Ala-tRNA(Ala). Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A, G; D = U, G; Y = U, C) in the randomized region, and Group II molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides. The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA.

Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation.

An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts. The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins. Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites. Most of the mutations did not severely impair the function of these tRNAs in any of the assays. This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA.

In vitro analysis of translational rate and accuracy with an unmodified tRNA.

Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system. To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold. The activity of unmodified Phe-tRNA(Phe) on E. coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons. Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons.

Recognition nucleotides for human phenylalanyl-tRNA synthetase.

The specificity of the interaction between tRNAPhe and phenylalanyl-tRNA synthetase isolated from human placenta was investigated. Using yeast tRNAPhe transcripts with different point mutations it was shown that all the five recognition points for the yeast phenylalanyl-tRNA synthetase (G20, G34, A35, A36 and A73) are also important for the reaction catalyzed by the human enzyme. A set of mutations in nucleotides involved in tertiary interactions of tRNAPhe revealed that mutations which maintained the proper folding of the molecule had almost no influence on the efficiency of aminoacylation. The most striking difference between the yeast and human phenylalanyl-tRNA synthetases involved a mutation in the lower two base pairs of the anticodon stem. This mutation did not affect aminoacylation with the yeast enzyme, but greatly reduced activity with human phenylalanyl-tRNA synthetase.

[Effect of nucleotide replacement on the effectiveness and specificity of the SP6 promotor]. / Vliianie nukleotidnykh zamen na éffektivnost' i spetsifichnost' SP6-promotora.

The interaction of SP6 RNA-polymerase with its promoter (sequence; see text) where +1 is the initiation site, was investigated. The substitution of C for A at +2 position and T for A at +3 creates the AluI-site (-2-1+1+2). The efficiency of the promoter changed in this way is comparable with that of the natural one. The introduced restriction site permits to insert the gene precisely at the initiation site and the synthesis of the transcripts with the authentic 5'-ends. The influence of the nucleotide substitution within -5-10 region of SP6-promoter on its interaction with SP6 and T7 RNA-polymerases was investigated. A new promoter, able to initiate the transcription with both SP6 and T7 RNA polymerases, was obtained.

[Determination of inhibition sites during hydrolysis of polydeoxyribonucleotides by exonucleases III from Bacillus amyloliquefaciens and Escherichia coli]. / Opredelenie uchastkov tormozheniia pri gidrolize polidezoksiribonukleotidov ékzonukleazami III iz Bacillus amyloliquefaciens i Escherichia coli.

The influence of the primary structure of polydeoxyribonucleotides on the rate of hydrolysis with exonuclease III from Bacillus amyloliquefaciens and Escherichia coli was investigated. The substrates used were synthetic oligodeoxyribonucleotides and pBR 322 DNA fragments labeled with 32P at the 5'-termini of one of the chains. According to the data from polyacrylamide gel electrophoresis performed under denaturing conditions, the hydrolysis of these substrates by unsaturating concentrations of B. amyloliquefaciens and E. coli exonuclease III proceeds with several reproducible "stops". The decrease of the reaction rate was shown to take place just before the pyrimidine blocks in the digested DNA chain.

[Exonuclease III from Bacillus amyloliquefaciens]. / Ekzonukleaza III iz Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens cells were found to contain an exonuclease which catalyzes the sequential hydrolysis of mononucleotides from the 3'-termini of duplex DNA. The enzyme was purified to homogeneity and its molecular weight (as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) is 29,000. The exonuclease possesses an additional catalytic activity, i.e., 3'-5' exonuclease specific for the RNA strand in an RNA--DNA hybrid duplex (RNase H activity). In terms of physical and catalytic properties the exonuclease of B. amyloliquefaciens is similar to exonucleases III from E. coli and Haemophilus influenzae and can thus be related to the same class of hydrolases, i.e., 3.1.11.2. However, in comparison with exo III from E. coli, the enzyme from B. amyloliquefaciens exhibits a more strict specificity for the structure of the substrate 3'-end.

[Cleavage of synthetic RNA-DNA hybrids with restriction endonucleases BamH1 and Sau3A]. / Rasshcheplenie sinteticheskikh RNK-DNK-gibridov éndonukleazami restriktsii BamH1 i Sau3A.

Synthetic oligodeoxyribonucleotides, containing one or two ribonucleotides in the recognition sequence, and RNA--DNA hybrids were tested for their activity in cleavage with BamH1 and Sau3A endonucleases. The replacement of dG with G in the first position of BamH1-site (GGATCC) of one of the chains does not affect the rate of the BamH1 hydrolysis. The similar heteroduplex, containing G residue in the second position, displays a decreased rate of the BamH1 hydrolysis of the modified strand and, to a lesser extent, of the unmodified complementary strand. Oligodeoxyribonucleotides in complex with oligoribonucleotides can be cleaved with the excess of BamH1 and Sau3A, oligoribonucleotides remaining intact.

[Surgical treatment of atherosclerotic occlusion of the femoral, popliteal and tibial arteries]. / Khirurgicheskoe lechenie ateroskleroticheskikh okkliuzii bedrennoi, podkolennoi i bertsovykh arterii.

An analysis of results of the treatment of 182 patients with atherosclerotic occlusions of femoral, popliteal and tibial bones has shown the necessity of an individual approach to choosing the method of restorative operation on the vessels. More favourable remote results were obtained after autovenous shunting "in situ" and by the method of vein reversion.

A study on the unprimed poly (dA-dT) synthesis catalyzed by preparations of E. coli DNA polymerase I.

Evidence was obtained indicating that the initiation of poly (dA-dT) de novo synthesis is provided by deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase present in preparations of E. coli DNA polymerase I and capable of catalyzing the unprimed polymerization of dNDP. dNDP-transferase synthesyzes short oligonucleotides which form template-primer complexes repeatedly replicated by DNA polymerase I. This conclusion was based on the following observations: the abolition of the lag period of poly (dA-dT) synthesis by preincubation of DNA-polymerase I preparations with dADP and dTDP; the presence of oligo (dA-dT) among the preincubation products; the suppressive effect of dithiothreitol and N-ethylmaleimide (inhibitors of dNDP-transferase) on the de novo, but not on the primed synthesis of poly (dA-dT), catalyzed by preparations of DNA-polymerase I.

[Unprimed synthesis of poly (d(A-T)), catalyzed by a preparation of Escherichia coli DNA polymerase I]. / Issledovanie prirody bezmatrichnogo sinteza d(A-T)-sopolimera, katalizirchemogo preparatami DNK-polimerazy I Escherichia coli.

The initial events of the de novo synthesis of poly[d(A-T)], catalyzed by preparations of E. coli DNA-polymerase I, were investigated. The data provide evidence that deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase), the enzyme which is able to catalyze unprimed polymerization of dNDP, participates in the process of initiation. This conclusion is based on the following data: 1) preincubation of E. coli DNA-polymerase I preparation with dADP and dTDT abolishes a lag-period in the poly[d(A-T)] synthesis; 2) dithiothreitol and N-ethylmaleinide, inhibitors of dNDP-transferase, inhibit de novo synthesis of [d(A-T)]-copolymer by preparations of E. coli DNA-polymerase I but do not effect primed synthesis ensured by this enzyme. High concentration of the substrate have similar effect. Using two-dimentional thin-layer chromatography and microcolumn chromatography on TEAE-cellulose we have shown that preliminary incubation of DNA-polymerase I preparations with dADP and dTDP results in the synthesis of short oligonucleotides (from di- to decanucleotides). Hydrolysis of these oligonucleotides with dilute sulfuric acid demonstrates that among the reaction products prevail oligoadenylates and oligothymidylates, but an appreciable amounts of heterooligomers including oligo[d(A-T)] were revealed as well. The model of so called de novo synthesis of regular polynucleotides is proposed, according to which dNDP-transferase, an accompanying enzyme in the preparations of DNA-polymerase I E. coli, is carrying out the synthesis of short oligonucleotides which form template-primer complexes repeatedly replicated by the DNA-polymerase I E. coli.

[Separation of the enzyme catalyzing polymerization of deoxyribonucleoside diphosphates from preparations of E. coli DNA-polymerase I]. / Vydelenie fermenta kataliziruiushchego polimerizachiiu dezoksinykleoziddifatov, iz preparatov DNK-polimerazy I E. coli.

The enzyme which catalyses template independent synthesis of polydeoxynucleotides from deoxynucleoside diphosphates was separated from E. coli DNA polymerase I by DEAE-cellulose chromatography followed by ultrafiltration through the M-50 Amicon filter. The ultrafiltration data indicate that the molecular weight of the enzyme is not higher than 50,000. The enzyme is not able to use deoxynucleoside triphosphates, ribonucleoside di- or triphosphates as substrates for the polymerization. The reaction of template independent polymerization proceeds with a lag period varying from 2 to 20 hours (for different preparations of enzyme) and is activated by Mg2+ (the optimal concentration 1-2 . 10(-3) M). The pH optimum of the reaction is at 8.5. The optimal concentration of deoxyribonucleoside diphosphates is 10(-3) M, and its increase strongly inhibits polymerization. The enzyme was supposed to be called deoxynucleoside diphosphate: olygonucleotide deoxynucleotidyltransferase (catalyzing polymerization without template). The presence of the enzyme in the preparations of E. coli DNA-polymerase I can explain the ability of the latter to catalyze the untemplated synthesis of poly dG : poly dC.