[Immunoprophylaxis of acute respiratory diseases with bacterial multicomponent vaccine VP-4 in children at preschool institutions]. / Immunoprofilaktika bakterial'noi polikomponentnoi vaktsinoi (VP-4) u detei v doshkol'nykh uchrezhdeniiakh.

The prophylactic action of polycomponent vaccine B[symbol: see text]-4, prepared from the antigens of opportunistic bacteria, on morbidity rate in acute respiratory diseases (ARD) of bacterial and mixed (bacterial and viral) etiology in 121 children aged 2-5 years, attending pre-school institutions was evaluated. For comparison, a group of 118 children of the same age from the same institutions was formed. The vaccine was introduced after the schedule consisting of 3 intranasal and 6-9 oral administrations made at intervals of 3-4 days. The duration of the course of immunization was 26 +/- 4 days. The prophylactic effect of B[symbol: see text]-4 on ARD morbidity was evaluated by the number of ARD cases and their duration per child. The prophylactic effect of B[symbol: see text]-4 on ARD morbidity lasted 14 months (the term of observation) after immunization and was manifested by a decrease in the number and duration of ARD cases after administration of the preparation, also in a group of highly susceptible children.

[The reactogenicity and safety of a multicomponent vaccine (VP-4) in the prevention of acute respiratory diseases in preschoolers]. / Reaktogennost' i bezopasnost' polikomponentnoi vaktsiny (VP-4) pri profilaktike ostrykh respiratornykh zabolevanii u doshkol'nikov.

The reactogenicity and safety of poly-component vaccine (VP-4), prepared from the antigens of opportunistic bacteria, in the prophylaxis of acute respiratory diseases (ARD) in children aged 2.6-6 years. The vaccine was administered intranasally in 3 administrations and orally in 6-8 administrations at intervals of 3-4 days for a period of 24 +/- 4 days. The prophylaxis of ARD with the use of VP-4 was carried out in 168 children in 4 children's preschool institutions. The control group was made up of 120 children, attending the same institutions. The study revealed that VP-4 had low reactogenicity and induced short-time systemic and local reactions (common cold, cough). The administration of VP-4 at a period of the epidemic rise on influenza and ARD morbidity did not lead to an increase in the frequency and duration of ARD in the vaccinees, as well as to the exacerbation of chronic infection and the allergization of the body.

Insertions of hybrid P elements in the yellow gene of Drosophila cause a large variety of mutant phenotypes.

A series of yellow mutations associated with a great variety of tissue-specific phenotypes were obtained from several highly unstable Drosophila melanogaster strains carrying the gypsy-induced y2 allele. These mutations are caused by insertion of additional DNA sequences of variable size 69 bp upstream of the yellow transcription start site. These sequences are flanked by identical copies of a deleted 1.2-kb P element arranged in the same or inverted orientation. The central part of the inserted element consists of genomic sequences originating from different regions of the X chromosome. The mutant phenotype caused by these chimeric elements depends on the nature of the sequences present either in the P element or in the central part of the insertion, suggesting that these sequences are able to affect expression of the yellow gene. In addition, sequences present in the central region of the insertions strongly modify the effects of the gypsy-bound suppressor of Hairy-wing [su(Hw)] and modifier of mdg4 [mod(mdg4)] proteins on yellow transcription. Analyses of these mutations give new insights into the mechanisms by which su (Hw) and mod(mdg4) affect enhancer function.

P element-flanked inserts at the yellow locus of Drosophila melanogaster strains.

Large inserts have been found to appear within the yellow (y) locus of several unstable mutant lines derived from a parental strain (y2 sc+ w(aG)) of Drosophila melanogaster. The inserts of the individual mutant lines correspond roughly to a series of multimers (4.1, 7.1, 14.3 and 28.9 kbp, respectively) and are always located at the same position, somewhat upstream of the y gene transcription start site. Each of them begins and ends with a 1-kbp sequence which shares restriction sites with P elements. One of these inserts (14.3 kbp) has been partially sequenced (1270 bp from the 5' end). The first 1185 bp match exactly the sequence of an internally deleted P element. The remaining part of the sequence, on the other hand, is unrelated to P elements. It is assumed that large DNA sequences may be captured by the flanking P-element fragments and inserted into new chromosomal sites.

[Tissue-specific blocking of the EcoRI site adjacent to the pseudogene for mouse oncoprotein p53]. / Tkanespetsificheskoe blokirovanie EcoRI-saita vblizi psevdogena onkobelka p53 myshi.

EcoRI fragments of DNA isolated from the different mouse organs were hybridized to radioactivity labelled probe specific for the gene of oncoprotein p53. The analysis of the blot-hybridization points to the existence of the specific blockage of an EcoRI site flanking a 3.3 kb fragment of DNA including the pseudogene p53, isolated from the skin tissue. The existence of a polymorphous EcoRI site localized distally to the pseudogene p53 has been demonstrated in the DNA of mice of different lines.

A variation in the structure of the protein-coding region of the human p53 gene.

An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed BglII site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional BglII site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine----proline substitution in the allele with the additional BglII site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.

[Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene]. / Strukturnaia organizatsiia gena p53 cheloveka. Soobshchenie I. Molekuliarnoe klonirovanie gena p53 cheloveka.

Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.

[Nuclear and cytoplasmic RNA showing hybridization with repetitive sequences B1 and B2 of the mouse genome]. / Iadernye i tsitoplazmaticheskie RNK, gibridiziruiushchiesia s povtoriaiushchimisia posledovatel'nostiami B1 i B2 genoma myshi.

Heavy nuclear RNA contains high amounts of transcripts from repetitive sequences B1 and B2. Cytoplasmic poly(A+)RNA and in particular, polysomal poly(A)+RNA (mRNA) also contain these sequences though their content is much lower. An abundant 2 kb mRNA of mouse liver was found to contain B2 sequence. A novel class of RNA is described namely as low molecular weight heterogeneous (200-400 nucleotide long) poly(A)+RNA. These RNAs contain B1 and B2 sequences and are located mostly in the cytoplasm but not in polysomes. Tumor cells contain high amounts of small B2+RNAs. The content of B1+ small cytoplasmic RNA is much lower. The hybridization tests confirm the existence of extended homology between B1 sequence and 4.5 S small nuclear RNA which has been earlier deduced from comparison of their base sequences. Homology of B1 and small cytoplasmic 7S RNA was also detected. No hybridization of B2 to small nuclear or small cytoplasmic RNAs could be detected.

The sequences homologous to major interspersed repeats B1 and B2 of mouse genome are present in mRNA and small cytoplasmic poly(A) + RNA.

Heavy nuclear RNA contains high amounts of transcripts from repetitive sequences B1 and B2. Cytoplasmic poly(A)+RNA and, particularly, polysomal poly(A)+RNA (mRNA) also include these sequences but in smaller amounts. The abundant 2 kb mRNA of mouse liver are found to have a B2 sequence. These sequences are also found in a new class of low-molecular-weight heterogeneous (200-400 nucleotide long) poly(A)+RNAs. These RNAs are located mostly in cytoplasm rather than in polysomes. The amount of small B2+ RNAs is noticeable larger than that of small B1+ RNAs. Tumour cells seem to contain more small B2 RNA than normal cells. The hybridization tests show that extended homology exists between the B1 sequence and 4.5S small nuclear RNA, which is predicted from comparison of their base sequences. Also, we have found homology between B1 and small cytoplasmic 7S RNA. Hybridization of B2 to sn or sc RNAs has not been observed, although the sequencing reveals partial homology between B2 and 4.5S sn RNAI /1/.

Structural investigation of nuclear RNP particles containing pre-mRNA by different fluorescence techniques.

Ethidium bromide (EB) adsorption isotherms on 30S nuclear RNP particles isolated from liver nuclei has revealed 6% of double-stranded regions in pre-mRNA (dsRNA). It has been established by measurements of the EB fluorescence polarization that the bulk of dsRNA regions in RNP is rigidly attached to RNP. They are longer than 45 degree A. The increase of NaCl concentration from 0.1 up to 0.4 M causes a significant loosening of dsRNA-protein bonds. As a result the dsRNA segments become more flexible. Measurements of energy transfer from fluorescamine (covalently bound to the protein) to EB (adsorbed on dsRNA) have yielded information about dsRNA location. The fact that absorbtion of exciting light by fluorescamine causes pronounced increase of EB fluorescence is consistent with the idea that helical regions of RNA are located outside the RNP particles.

[Nuclear RNP containing pre-mRNA. 16. Cross-linked informofers]. / Iadernye RNP, Soderszhashcie pre-mRNK. XVI. "Zashitye" informofery.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethylsuberimidate and dimethyl-3,3'dithiobispropionimi-date). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers, being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linking reagent has been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or polyparticles) were reconstituted upon mixing or cross-linked informofers with pre-mRNA and removal of 2 M NaCl. The reconstituted particles were indistinguishable from the original ones by several tests.

[Structure of nuclear pre-mRNA. XIII. Hybridization properties of triphosphorylated 5'-end fragments]. / Struktura iadernoi pre-mRNK. XII. Gibridizatsionnye svoistva trifosforilirovannykh 5'-kontsevykh fragmentov.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method. These fragments as well as fragments of total pre-mRNA were polyadenylated by ATP:RNA adenyl-transferase and used as templates for the synthesis of [32P]cDNA by reverse transcriptase in the presence of an oligo(dT)-primer, cDNA transcribed from total fragments of pre-mRNA and from 5'-end fragments (5'-cDNA) were hybridized with excess of nuclear pre-mRNA. The extent of hybridization was 65-70 and 80-85% in different experiments. 18% of total cDNA and 35% of 5'-cDNA hybridized with mRNA from polysomes. A high homology between mRNA and triphosphorylated 5'-ends of pre-mRNA may be explained in the terms of splicing. The sequences adjacent to the triphosphorylated 5'-ends of pre-mRNA represent a specific class with complexity about 2.10(5) nucleotides. Less than 30% of 5'-cDNA hybridized with intermediately repetitive DNA, while the main portion hybridized with unique DNA sequences. About 15% of 5'-cDNA contain oligo (dA) sequences, originated from oligo (U) in the pre-mRNA.

Cross-linked informofers.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl.

Hybridization properties of sequences adjacent to triphosphorylated 5'-ends of nuclear pre-mRNA from mouse Ehrlich carcinoma.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method /1/. These fragments as well as fragments of total pre-mRNA of the same size were polyadenylated in vitro by ATP:RNA adenyltransferase and used as templates for the synthesis of [32P] cDNA by reverse transcriptase in the presence of an oligo(dT) primer. The use of cDNA transcribed from the triphosphorylated 5'-end fragments of pre-mRNA (5'-cDNA) and from the total pre-mRNA fragments allows one to calculate the complexity of the 5'-end fraction pre-mRNA and to detect these sequences in polysomal mRNA. Sequences adjacent to 5'-phosphorylated ends of pre-mRNA represent a specific class of sequences with a complexity of about 200 kb. It was also found that about 25% of total pre-mRNA and about a half of sequences adjacent to triphosphorylated 5'-ends are present in polysomal mRNA. A high homology between triphosphorylated 5'-end fragments of pre-mRNA and mRNA sequences may be explained in terms of splicing. Less than 30% of 5'-cDNA hybridized to moderately repetitive DNA while most of them are represented by unique DNA sequences. About 15% of 5'-cDNA contained oligo(dA) sequences originated from oligo(U) in pre-mRNA from which it was transcribed.

[Nuclear ribonucleoproteins containing pro-mRNA. XIV. Structural study using ethidium and fluorescamine]. / Iadernye ribonukleoproteidy, soderzhashchie pro-mRNK. XIV. Issledovanie struktury s ispol'zovaniem étidiia i fluoreskamina.

Nuclear 30S RNP particles were studied by means of fluorescence techniques. It's shown that fluorescamin interacts with NH2-groups of protein molecule. As a result, covalent fluorescent label is formed. Quantum yield (rho), fluorescence spectra, lifetime of excited state (tau) and polarization of fluorescamin complexes with 30S particles were studied. Excitation spectra have their maximum at 395 nm, and fluorescence spectrum at 480 nm. These figures correspond to spectra of fluorescamin complexes with NH2-groups of lysine. Mean quantum yield (rho = 0.27) and lifetime of excited state of fluorescence (tau = 7.8 nsec) were measured. It's shown that fluorescamin forms two types of fluorescent complexes in 30S particles. These complexes differ only by their rho(rho1 = 0.11, rho2 = 0.30) and rho(rho1 = 3.6 nsec, rho2 = 10.0 nsec) by 2.7 times. Migration radius between fluorescamin bound to protein and ethydium bromide adsorbed on double-stranded regions of pre-mRNA in RNP-particles was measured. It's equal to 32 A. Adsorbtion isotherms of ethydium bromide were measured by fluorescence in 0.1 and 0.4 M NaCl. Data obtained showed that 6% of pre-mRNA in 30S particles bound the dye as a strong complex, i. e. this part of pre-mRNA is double-stranded. RNase treatment of RNP had no effect on this value. But the increase of NaCl concentration up to 0.4 M caused the dissociation of protein subunits to some extent followed by appearance of up to 40% free NH2-groups interacting with fluorescamin. Measuring of energy migration from fluorescamin to ethydium bromide showed that double-stranded pre-mRNA regions strictly bound to protein sticked out from RNP particle at a distance of about 27 A. The increase of NaCl concentration up to 0.4 M leads to disruption of this strict bond of double-stranded regions with protein. As a result, these regions of pre-mRNA become labile and move away from the RNP particle at more than 30 A. According to theoretical calculations, there is about 1--2 pre-mRNA hairpins (18--9 base pairs respectively) per one 30S particle.

[Nuclear RNP, containing pre-mRNA. XIV. Nature of particles, containing 5'- and 3'-end structures]. / Iadernye RNP, soderzhashchie pro-mRNK. XIV. Priroda chastits, soderzhashchikh 5'-i 3'-kontsevye struktury.

Some characteristic peculiarities of the 5'-end and 3'-end structures of pre-mRNA isolated from nuclear RNP particles have been investigated: presence of triphosphorylated nucleotides on the 5'-ends, as a characteristic of the primary product of transcription; presence of modified 5'-ends -- blocked and methylated structures -- caps; presence of poly(A) blocks attached to the 3'-end of pre-mRNA during post-transcriptional transformation of the latter. It was shown that pre-mRNA isolated from nuclear RNP particles contained triphosphorylated nucleotides as well as a "cap" structure at the 5'-ends. On the 3'-ends of pre-mRNA from nuclear RNP particles isolated in the presence of a RNAse inhibitor, the presence of poly(A) blocks have been shown. These poly(A) structures are separated very easily from pre-mRNA during mild RNAase digestion. This is the reason why they are not detected in 30S monoparticles, containing pre-mRNA fragments connected with one informofer. Almost all poly(A) complexes in this condition are combined with proteins and have the sedimentation coefficient of about 14S. It was concluded that formation of nuclear pre-mRNA containing RNP-particles take place just after the onset of RNA synthesis. All processing steps occur with pre-mRNA packed in nuclear RNP particles.

[Structure of nuclear pre-mRNA. XI. Triphosphorylated and blocked 5'-ends in the pre-mRNA]. / Struktura iadernoi pro-mRNK. XI. Trifosforilirovannye i blokirovannye 5'-kontsy v pro-mRNK.

The nature of the 5'-termini in pre-mRNA isolated from Ehrlich carcinoma cells has been investigated. To discriminate between triphosphorylated 5'-ends and capped structures different methods were used including treatment by alkaline phosphatase and several chromatographic methods. It was shown that heavey pre-mRNA contains a significant number of non-blocked triphosphorylated nucleotides at the 5'-end termini. However, phosphatase resistent, blocked 5'-termini were also found. 5'-terminal nucleotides in triphosphorylated pre-mRNA are G in a 3 : 2 ratio. In contrast to nuclear pre-mRNA cytoplasmic poly(A)+mRNA does not contain triphosphorylated 5'-ends but does contain the "cap" structure only. To elucidate the pre-mRNA topography the localization of homopolymeric regions of pre-mRNA, poly(A) and oligo(U), in relation to 5'terminal structures has been investigated. The experiments showed that the distance between 3'-terminal poly(A) sequences and 5'-end triphosphates is longer than 1500--2000 nucleotides. At the same time the distance between the latter and oligo(U) in pre-mRNA is much shorter.

[Nuclear ribonucleoproteins containing messenger RNA. 12. Fluorescence studies of the secondary structure of pre-mRNA in nuclear RNP-particles]. / Iadernye ribonukleoproteidy, soderzhashchie informatsionnuiu RNK. 12. Izuchenie vtorichnoi struktury pro-mRNK v sostave iadernykh RNP-chastits fluorestsentnymi metodami.

Secondary structure of pre-mRNA in nuclear ribonucleoprotein particles (30S-particles) was examined using fluorescent dyes: acridine orange, acriflavine and ethidium bromide. Comparison of ethidium bromide and acriflavine adsorption isotherms for RNP-particles and free RNA and a study of acridine orange dimerization on binding to RNP revealed that 70% of pre-mRNA in 30S-particles is accessible for the dye binding. Dye molecules were adsorbed on double-stranded sequences (11--12% of the total amount of RNA in 30S-particles) and on the single-stranded parts of RNA (58--59% of 30S-particles), the rest part of RNA was unaccessible for the dye binding. A method involving measurements of acriflavine fluorescence quantum yields was used for the determination of nucleotide composition of double-stranded parts of RNA in the 30S-particles. AU-nucleotide content thus obtained was approximately 50%, as was established also for free pre-mRNA. Na+ ions weaken the interaction between the protein and pre-mRNA in 30S particles and increase mobility of double-stranded parts of this nucleic acid.