OPTIMUM LEVEL OF POSITIVE END-EXPIRATORY PRESSURE IN ACUTE RESPIRATORY DISTRESS SYNDROME CAUSED BY INFLUENZA A(H1NI)PDM09: BALANCE BETWEEN MAXIMAL END-EXPIRATORY VOLUME AND MINIMAL ALVEOLAR OVERDISTENSION.

THE AIM: to determine optimum level ofpositive end-expiratory pressure (PEEP) according to balance between maxi- mal end-expiratory lung volume (EEL V)(more than predicted) and minimal decrease in exhaled carbon dioxide volume (VCO) and then to develop the algorithm of gas exchange correction based on prognostic values of EEL K; alveolar recruitability, PA/FiO2, static compliance (C,,,) and VCO2. MATERIALS AND METHODS: 27 mechanically ventilatedpatients with acute respiratory distress syndrome (ARDS) caused by influenza A (HINJ)pdm09 in Moscow Municipal Clinics ICU's from January to March 2016 were included in the trial. At the beginning of the study patients had the following characteristic: duration offlu symptoms 5 (3-10) days, p.0/FiO2 120 (70-50) mmHg. SOFA 7 (5-9), body mass index 30.1 (26.4-33.8) kg/m², static compliance of respiratory system 35 (30-40) ml/mbar: Under sedation and paralysis we measured EELV, C VCO and end-tidal carbon dioxide concentration (EtCO) (for CO2 measurements we fixed short-term values after 2 min after PEEP level change) at PEEP 8, 11,13,15,18, 20 mbar consequently, and incase of good recruitability, at 22 and 24 mbar. After analyses of obtained data we determined PEEP value in which increase in EELV was maximal (more than predicted) and depression of VCO2 was less than 20%, change in mean blood pressure and heart rate were both less than 20% (measured at PEEP 8 mbar). After that we set thus determined level of PEEP and didn't change it for 5 days. RESULTS: Comparision of predicted and measured EELV revealed two typical points of alveloar recruiment: the first at PEEP 11-15 mbar, the second at PEEP 20-22 mbar. EELV measured at PEEP 18 mbar appeared to be higher than predicted at PEEP 8 mbar by 400 ml (approx.), which was the sign of alveolar recruitment-1536 (1020-1845) ml vs 1955 (1360-2320) ml, p=0,001, Friedman test). we didn't found significant changes of VCO2 when increased PEEP in the range from 8 to 15 mbar (p>0.05, Friedman test). PEEP increase from 15 to 18 mbar and more lead to decrease in VCO2 (from 212 (171-256) ml/min to 200 (153-227) ml/min, p<0,0001, Friedman test, which was the sign of overdistension. Next decrease of VCO2 was observed at PEEP increase from 22 to 24 mbar (from 203 (174-251 ml/min) to 185 (182-257) ml/min, p=0.0025, Friedman test). Adjusted PEEP value according to balance between recruitment and overdistension was higher than the one initially set (16(15-18) mbar vs 12(7-15) mbar, p <0.0001). We observed increase of SpO2 from 93 (87-96) to 97(95-100)% (p<0.0001 followed by decrease in inspiratory oxygen fraction from 60(40-80) to 50(40-60)%(p<0.0001). Low EELV VCO2 and VCO2/EtCO2 at PEEP 8 mbar has low predictive value for death (AUROC 0,547, 0706 and 0.596, respectively).Absolute EELV value at PEEP 18 and 20 mbar were poor predictors of mortality (AUROC 0.61 and 0.65 respectively) Alveolar recruit ability was measured by subtraction of EELV at PEEP 20 and at PEEP II mbar - value below 575 ml was a good predictor of death (sensitivity 75%, specificity 88%, AUROC 0.81). Lowering of VCO2 at PEEP 20 mbar to less than 207 ml/min was a marker of alveolar overdistension and associated with poor prognosis (sensitivity 83%, specificity 88%, AUROC 0,89). C has poor predictive value at PEEP 8 and 20 mbar (AUROC 0,58 and 0,74 respectively. Conclusion: PEEP adjustment in ARDS due to influenza A (H1N1) pdm09 in accordance with balance between recruitment and overdistension (based on EELV and VCO measurements) can improve gas exchange, probably, not leading to right ventricular failure. This value of "balanced" PEEP is in the range between 15 and 18 mbar: Low lung recruitabiilty is associated with poor prognosis. Measurements of EELV and VCO2 at PEEP 8 and 20 mbar can be used to make a decision on whether to keep "high" PEEP level or switch to extracorporeal membrane oxygenation in patient with ARDS due to influenza A (N1H1).

[The role of recipient cells in the mechanism of pathological calcification of heart valve and vascular transplants].

It was found using the model of subcutaneous implantation in rats that the calcification of the aorta wall occurs by two mechanisms of which one is dependent on, and the other independent of the migration of recipient cells to the transplant.

[Prooxidant and cytotoxic action of N-acetylcysteine and glutathione combined with vitamin Bl2b].

We studied the prooxidant and cytotoxic action of thiols N-acetylcystein (NAC) and glutathione (GSH) combined with vitamin Bl2b. The synergism of action of the thiols and Bl2b resulted in human carcinoma cell damage was found. It was shown that GSH and NAC in physiological doses combined with Bl2b caused the initiation of apoptosis. It was established that prooxidant action of the thiols combined with vitamin Bl2b, i. e. generation and accumulation of hydrogen peroxide in culture medium, led to intracellular oxidative stress and injury of cell redox system. These effects were completely abolished by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine did not suppress the H2O2 accumulation in culture medium but significantly inhibited the cell death induced by the thiols combined with Bl2b. Therefore, the thiols GSH and NAC widely used as antioxidants, in combination with vitamin Bl2b show prooxidant characteristics and induce, with the participation of intracellular iron, apoptotic HEp-2 cell death.

[A method of calculating the discrete secondary structures of globular proteins]. / Metod rascheta diskretnykh vtorichnykh struktur globuliarnykh belkov.

The model of formation of alpha-helices and beta-structures determined by joint action of the three elements: N-terminal, internal and C-terminal fragments are presented. Algorithm for calculation of their localization in a given amino acid sequence was constructed on the base of this model. The preference of the fragments of the amino acid sequence to a definite type of the secondary structure was estimated on the base of corresponding average values of linear discriminant functions dsk (s = alpha, beta, k = N, in, C). The latter were constructed in the previous paper on the base of the revealed significant characteristics. These integral characteristics are used for calculating the localisation of discrete secondary structures. The total prediction for 3 states (alpha, beta, c) given 71% correctly predicted residues (for 4 states alpha, beta, c, t) 62% for the training set, consisting of 72 proteins. For the control set (15 proteins) the accuracy of prediction is about 65%. The essential advantages of this method are: 1) the possibility to localize the discrete secondary structures; 2) the high accuracy of prediction of long secondary structures (for alpha-helices approximately 90%, for beta-structures approximately 80%), which is important for the determination of the protein folding. The influence of mutation on the secondary structure of proteins was investigated. The anormally high stability of the secondary structures of immunoglobulins to mutations was revealed. This probably results from the selection during evolution of such variants of amino acid sequences, which are able to provide the functional variability of antigenic determinants, but keep invariant the tertially structure of protein.

[Search for functional domain boundaries and localization of functional sites based on oligopeptide vocabularies]. / Poisk granits funktsional'nykh domenov i lokalizatsiia funktsional'nykh saitov na osnove oligopeptidnykh slovarei.

A new method based on the analysis of oligopeptide composition of the amino acid sequences from different protein families is presented. We assume, that any protein family can be characterized by the set of oligopeptides (oligopeptides vocabulary). We demonstrate, that oligopeptides vocabulary comparison can distinguish different families from each other and from random sequences. It should be noted, that this comparison can be successfully performed on the set of only 25 dipeptides and without preliminary alignment. We demonstrate, that characteristic peptides are localized in the regions of functional significance, as shown on the example of GTP-binding domain of translation elongation factors. We suggest how to use this method to localize the boundaries of functional domains in amino sequences. On the example of few functional domains we demonstrate, that the average error of prediction does not exceed 3-4 amino acid residue.

[Method for calculation of low-energy conformations of alpha-helical pairs of globular proteins]. / Metod rascheta nizkoénergeticheskikh konformatsii par al'fa-spiralei v globuliarnykh belkakh.

The computer model of folding of two alpha-helices of globular proteins was developed. The radicals of amino acids were approximated by spheres with centers located in C beta-atoms. The functional of energy took into account the hydrophobic interactions of alpha-helices, electrostatic contacts of charged and polar side groups of amino acid, Van der Waals' interactions. The conformations with minimum energies of two-helical superstructures G--H from alpha- and beta-chains of horse hemoglobin, sperm whale myoglobin, and erythrocruorin were computed. They have mean deviation 0.7--1.8 A from native conformations of these proteins. Hence, at the self-organization process alpha-helices firstly are "roughly" oriented by hydrophobic interactions, but the choice of stable conformation occurs by Van der Waals' and electrostatic interactions. On this stage the low-energy conformation becomes "frozen" and cannot be significantly rearranged later. The mutual orientation of secondary protein structures are determined mainly by amino acid radical volumes, their hydrophobicity and charge.

[Somatic hypermutagenesis in immunoglobulin genes. II. Properties of somatic mutations and clonal selection]. / Somaticheskii gipermutagenez v immunoglobulinobykh genakh. II. svoistva somaticheskikh mutatsii i klonal'naia selektsiia.

Analysis of the collection of 203 somatic mutations in immunoglobulin genes was carried out. It was shown, that the high frequency of these mutations in CDRs of V-genes may be connected with the high concentration of repeats in these regions. In addition, the observed clusterization of mutations may emerge from simultaneous correction of several pertubations of complementarity in the heteroduplex, formed by the repeat regions. It was revealed, that somatic mutations in FRs are characterized by reliably smaller changes of some important amino acid physical-chemical properties than in CDRs. These data obviously indicate the occurrence of B-lymphocytes clonal selection. Analysis of synonymous substitutions has shown, that stabilizing selection seems to provide the conservatism of FRs (it leads to the conservation of the protein three-dimensional structure) and movement selection may provide the proliferation of B-lymphocytes with considerable changes in CDRs, if these mutations improve antigens binding. Preferential fixation of transitions in comparison with transversions, particularly expressed in FRs, may also be connected with the fact, that transitions lead to smaller changes of amino acid physical-chemical properties and they are rejected by selection to a smaller extent.

[Theoretical analysis of mechanisms of occurrence of DNA deletions in prokaryotic genomes based on direct repeats]. / Teoreticheskii analiz mekhanizmov vozniknoveniia deletsiii DNK v genomakh prokariot na osnove priamykh povtorov.

In the present work a mechanism of deletions emergence on the basis of complementary DNA regions mispairing of direct repeats has been investigated theoretically. A quantitative dependence of the rates of deletions emergence on such parameters of the flanking repeats as the nucleotide composition of repeats, the number of homology damages and the distance between repeated regions has been constructed. It has been proved, that using this relationship one can reliably evaluate the total rates of deletions emergence in the lacI gene sequence of E. coli according to the repeats arrangement in this gene.

[Statistical evidence for the correlation of DNA deletions in prokaryotic genomes with direct repeats]. / Statisticheskoe dokazatel'stvo sviazi deletsii DNK v genomakh prokariot s priamymi povtorami.

In the present work a computer analysis of deletion localization in the sequence of the E. coli lacI gene has been carried out by the statistical weight method. Reliable statistical correlation of the deletions location sites with the arrangement of the most perfect direct repeats revealing the shortest distance between repeated fragments has been shown. At the same time statistical analysis did not reveal reliable connection of deletions localization regions with the expected sites of gyrase recognition, sites and other recombination sites. A conclusion has been drawn, that the mechanism of deletions emergence on the basis of repeats appears to be predominant.

[The context analysis of polynucleotide sequences. II. Inverted repeats and complementary palindromes in RNA-polymerase genes]. / Kontekstnyi analiz polinukleotidnykh posledovatel'nostei. II. Invertirovannye povtory i komplementarnye palindromy v genakh RNK-polimeraz.

A new approach to the reconstruction of the RNA secondary structure is suggested on the basis of the method of contextual analysis of polynucleotide sequences. The coding gene regions of beta-, beta'-, sigma-subunits of E. coli RNA polymerase and of phage T7 RNA polymerase were analysed. The clusters of non-random inverted repeats were found in all these genes. The mRNA coded by them can be folded into compact secondary structures. The latter are formed by quite long helices with a few cases of mispairing.

[New elements in the structure of binding sites of glucocorticoid-receptor complex in hormone-regulated genes]. / Novye élementy v strukture saitov sviazyvaniia glikokortikoid-retseptornogo kompleksa v sostave gormonreguliruemykh genov.

An analysis of the structure of DNA sites responsible for binding to glucocorticoid-receptor complex (GlRC) was carried out. The use of the frequency matrices and of a variant of the perception method made it possible to establish that in the GlRC binding site on both sides of the known conservative nucleotide sequence (nucleus) there were additional conservative elements which seemed to be able to modulate the efficiency of GlRC binding. A criterion is worked out for detecting the potential GlRC binding sites in given sequences. It is based on the simultaneous use of several perceptron matrices. The efficiency of detection of GlRC binding sites by means of the proposed criterion is by an order higher than that performed according to the GlRC binding site consensus (Beato et al. [2]).

[Theoretical analysis of the DNA duplication mechanisms in the prokaryotic genomes on the basis of repeats]. / Teoreticheskii analiz mekhanizmov duplikatsii DNK v genomakh prokariot na osnove povtorov.

In the present work a theoretical analysis of the molecular mechanisms on duplications emergence in the genomes of prokaryotes on the basis of direct repeats has been carried out. The correlations obtained have shown, that the duplication rate depends on such parameters as the distance between repeated regions, repeats nucleotide composition and the number of homology damages in them. It has been revealed that the rate of duplications decreases more readily than the deletion rate upon the growth of the distance between the repeats. Such prevalence of deletions over duplications must lead to the elimination of various types of direct repeats from the prokaryotic genomes in the course of their evolution.

[Somatic hypermutagenesis in immunoglobulin genes. I. Connection of somatic mutations with repeats. A statistical weighting method]. / Somaticheskii gipermutagenez v immunoglovulinovykh genakh. I. Sviaz' somaticheskikh mutatsii s povtorami. Metod statvesov.

Based on the analysis of a number of immunoglobulin genes' nucleotide sequences, it has been suggested, that somatic mutations emerge by means of imperfect duplexes correction, formed by mispairing of complementary regions of direct and inverted repeats. In the present work provides new data, confirming this mechanism of somatic hypermutagenesis. It has been shown that the presented sample of V- and J-segments of immunoglobulin genes is abundant in nonrandom imperfect direct repeats and complementary palindromes. To prove the connection of somatic mutations with the correction of imperfect duplexes, made up by the regions of these repeats, we have developed the method of statistical weights, permitting us to analyse the samples of mutations and repeats and to reveal the reliability of the connection between them. Using this method we have investigated the collection of 203 nucleotide substitutions in V- and J-segments and have shown a statistically reliable (P less than 10(-4) connection of these mutation positions with imperfect repeats.

[Context analysis of polynucleotide sequences. Methods of detecting non-random repeats. I. Direct repeats in genes of beta-, beta'-, sigma subunits of Escherichia coli RNA-polymerase]. / Kontekstnyi analiz polinukleotidnykh posledovatel'nostei. Metody vyiavleniia nesluchainykh povtorov. I. Priamye povtory v genakh beta-, beta'-, sigma-sub''edinits RNK-polimerazy iz Escherichia coli.

A new method of contextual analysis of polynucleotide sequences in developed. The method finds nonrandom repeats in the sequences of N bases in length with given nucleotide frequencies. The coding regions of genes specifying beta-, beta'-, sigma-subunits of E. coli RNA polymerase were analyzed. The high content of short repeats was found to correspond to the secondary structure of globular proteins coded by the genes. The possible evolutionary role of the nonrandom direct repeats in coding regions of genes is discussed.

[Features of the structure and evolution of complex tandemly organized Bsp-repeats in the fox genome. II. Tissue-specific and recombinant BamHI-dimer sites]. / Osobennosti struktury i évoliutsii slozhnykh tandemno organizovannykh Bsp-povtorob genoma lisitsy. II. Tkanespetsificheskie i rekombinatsionnye saity BamHI-dimera.

The complex structure of the clustered Bsp-repeats in fox genome seems to have evolved throughout a long period of time as a result of multiplication, recombination and divergence events. The sequence of the subrepeat (SR) approximately 245 b.p long is the basic substructure for the hierarchically arranged Bam HI-repeat 1468 b.p. long. The monomer consists of 3 SRs with a 43-59% homology. A dimer is composed of 2 monomers with a 93% homology. Amplification of the Bsp-repeats during evolution seems to have occurred at least twice: first--on the SR ancestral form level, second--on the monomer level. Despite profound divergence, there are still conservative regions in SRs with sequences homologous to known functional sites in eukaryotes. However qualitative and quantitative composition of most functional motifs is stringently individual in every SR. The performed analysis revealed that throughout evolution SRs acquired significant amount of motifs homologous to promoter and enhancer regions in tissue-specific genes and virus regulatory regions. Functional motifs in separate SRs are being differently grouped. Most inducible motifs are located in the III and II subrepeats, putative promoters--in the II one; elements participating both in transcriptional and replicational processes--mainly in the I subrepeat. A few ensembles of functional motifs remotely resemble extended regulatory regions of some tissue-specific genes. The monomers are potentially capable of ensuring diverse aspects of transcriptional regulation. As a whole, motifs of the 3 SRs are potentially capable of regulating the RNA synthesis periodicity with respect to the cellular cycle, activation and repression of genetical material in response to signals from the environment (AP-1, AP-2, AP-4, T-antigen, etc) and temporal ("octamers") etc. Apart from the BamHI-dimer, a few homologues fragments were isolated from fox genome and sequenced. Some of them were rearranged with respect to the BamHI-dimer. Inversion locally alters the composition of motifs and the sequence acquires new functional potential. Thus, the analysis of the emergence and development of Bsp-repeat structural variations allows us to consider repetitive DNA sequences as an ideal material in constructing multiprofile regulatory sequences.

[Modular evolution of Bsp-repeats: formation of subrepeats and monomers preceded the divergence of four species of Canidae]. / Blochnaia évoliutsiia Bsp-povtorov: formirovanie subpovtorov i monomerov predshestvovalo raskhozhdeniiu chetyrekh vidov Canidae.

Determination of two nucleotide sequences of satellite-like DNA (Bsp-repeats) segments from the raccoon-like dog genome and analysis of these and three other homologous sequences from distantly related genomes of Canidae (silver fox, domestic dog, and gray fox) show that all these DNA fragments consist of five types of subrepeats (A, B, C, D, and E) and two types of monomers-ACB and ACED. Raccoon-like dog repeated segment of 1620 bp is composed of both type of monomers: ACBACED. Different monomers of the same type could be distinguished by subtypes of subrepeats. Building of a cladogram of 18 subrepeats and analysis of the structures of monomers and higher-order repeated units allowed us to reveal probable evolution events which have determined the hierarchical order in Bsp-repeat organization. Approximate periods of time for these evolution events were calculated from the number of substitutions per branch. Five main types of subrepeats and two types of monomers have been generated and propagated through several steps before divergence of the main phylogenetic trees of Canidae. Combinatorial variability appears to play a fundamental role in forming the hierarchical layers in Bsp-repeats structure.

["DIROM"--an interactive system for planning experiments on directed mutagenesis and design of artificial genes]. / "DIROM"--interaktivnaia sistema dlia planirovaniia éksperimentov po napravlennomu mutagenezu i konstruirovaniiu iskusstvennykh genov.

We present a computer system "DIROM" for oligonucleotide-directed mutagenesis and artificial gene design experiments planning and support. "DIROM" allows to search for optimal oligonucleotides according to such parameters as sufficient energy of oligonucleotide-target hybridization, secondary structure of oligonucleotide and target DNA, presence of alternative attachment sites in target DNA, terminal G/C pairs presence. Both single-stranded and double-stranded vector mutagenesis methods are implemented. It can be also used for optimal primer selection for polymerase chain reaction, sequencing etc. "DIROM" can search for both existent and potential carry out vector+target sequence construction. Both amino acid and nucleotide sequences can be operated.

[Genome organization and primary structure of the BamHI fragment of highly repetitive DNA from Hordeum vulgare]. / Genomnaia organizatsiia i pervichnaia struktura BamHI-fragmenta vysokopovtoriaiushcheisia DNK Hordeum vulgare.

The primary structure of three highly repetitive DNA sequences, which are: the most widespread repeats in a number of Hordeum species, represented by 10(5) copies in the genome of Hordeum vulgare, and belong to the BamHI fragment, was studied. The organization of this fragment in the barley genome was described. The computer analysis of the primary structure indicated a great number of inverted repeats (two of them are flanked by short direct repeats) in the BamHI fragment and revealed the short subrepeats of the A, B, C types, five potential RNA-polymerase II sites, one site for RNA polymerase III, open reading frame of 294 b.p. or 210 b.p., sites for polyadenylation and enhancer sequence. The presence of a mobile element (95-568 bp) is very likely in the BamHI fragment, taking into account the location of the mentioned structures along the fragment. This mobile element contains the subrepeats of only A and B types, all sites of transcription initiation, the open reading frame and the inverted repeats on both ends, flanked by the short direct repeats. The transposition mechanisms probably play an essential part in the origin and propagation of the studied BamHI fragment. The presence of potential functional sites in the fragment indicates the possibility of transcription and translation of some highly repetitive Hordeum vulgare DNA.

[Features of the structure and evolution of complex, tandemly organized Bsp-repeats in the fox genome. I. Structure and internal organization of the BamHI-dimer]. / Osobennosti struktury i évoliutsii slozhnykh, tandemno organizovannykh, Bsp-povtorov genoma lisitsy. I. STruktura i vnutrenniaia organizatsiia BamHI-dimera.

A 1468 b.p. DNA BamHI-fragment homologous to the Bsp-repeat was isolated from the fox genome and sequenced. This fragment is an hierarchically arranged dimer. Its 734 b.p.-monomers consist of three subrepeats (SR), each 245 b.p. long, abundant with overlapping imperfect tandem repeats which in turn are rich in short direct related repeats (each 4-7 b.p. in size). The latters are mainly composed of AG, TG dinucleotides and their complements CT, CA. All subrepeats in the BamHI-dimer are flanked by motifs homologous to Jeffreys' sites. At certain points the sites are doubled. The above data allow to assume that the Bsp-repeat complex structure is likely to have developed throughout long multi-step evolution of relatively simple DNA sequences which had emerged de novo. Single substitutions, small inserts and deletions, multiple duplication and recombination events seem to have most contributed to the evolution of the Bsp-repeats. Single substitutions in SRs with respect to the consensus are not equally distributed along their length. A wave-like pattern of this distribution is the evidence for non-random character of mutations accumulation. A correspondence was noted between conservative regions in SR and the presence therein of functional motifs homologous to the binding sites of already known regulatory proteins.

[Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)aminobenzylidene]m derivatives of oligodeoxyribonucleotides. IV. Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA]. / Komplementarno adresovannoe alkilirovanie 16S rRNK Escherichia coli 2',3'-O-[4-methyl-N-(2-khlorétil)aminobenzilidenovymi] proizvodnymi oligodezoksiribonukleotidov. IV. Opredelenie uchastkov sviazyvaniia 16S rRNK s benzilidenovymi proizvodnymi d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA.

By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV] followed by the RNase H treatment a number of 16S rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.