In vitro suppression as a tool for the investigation of translation initiation.

An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase. Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.

[The origin of resistance to chemicals of naturally occurring isolates of influenza A virus]. / Proiskhozhdenie rezistentnosti k khimiopreparatam u prirodnykh izoliatov virusa grippa A.

The mechanisms responsible for the formation of resistance of influenza A virus isolates during the natural circulation of the influenza viruses in the environment were studied. The influenza viruses H1N1 and H3N2 resistant to remantadine, adapromine, and deitiforine have been isolated in the USSR and Mongolia since 1982. The majority of natural resistant isolates appeared to be atypical both in antigenic properties and genomic structure as compared to the isolates prevalent in the common epidemic process. The nucleotide sequences of the M2 gene of some resistant strains and virus A/PR8/34 used in our country as an attenuation donor for preparation of killed recombinant vaccines. The electrophoretic mobility of genomic RNA of two resistant isolates is similar to that of the vaccine strain X-54 based on the virus A/PR/8/34. In this connection, the appearance of resistant strains in the environment may be due not only to spontaneous mutagenesis or selective drug actions, but also to the involvement into the circulation of vaccinal strains.

Protein-binding DNA regions inside and in the vicinity of the cytochrome P450bm-3 gene from Bacillus megaterium.

Protein-binding DNA-regions inside and in the vicinity of the phenobarbital-inducible P450bm-3 gene from Bacillus megaterium were characterized by gel-retardation technique and foot-printing analysis. Regions with induction-dependent protein binding capacity were shown to be situated in -279bp/-215bp, -215bp/+83bp and +236bp/+425bp fragments. Precise localization of DNA-protein contacts was established by foot-printing analysis for region -215bp/+83bp. The data obtained are discussed in line with previously reported information on the regulatory regions of various phenobarbital-regulated genes.

[The molecular mechanism of the action of antiviral preparations in the adamantane series]. / Molekuliarnyi mekhanizm deistviia antivirusnykh preparatov adamantanovogo riada.

Regions of possible interaction between remantadine and transmembrane M2 protein are revealed by analysis of amino acid substitutions in remantadine- and deutiforin-resistant influenza viruses. The major region includes 5-6 amino acid residues at position 25-31, partially involving the premembrane region and the first position of a hydrophobic membrane-associated domain. The proposed model action of remantadine and its derivatives suggests that remantadine is included into the cell membrane lipid bimolecular layer by its adamantane share and its positively charged NH2-group is exposed to the cell surface. This allows remantadine and its analog to be regarded as molecular "hindrances" for viral particle decapsidation and budding.

Nucleotide sequence of cDNA coding for mink proopiomelanocortin (POMC) and its comparative analysis with POMC mRNA primary structures from pituitaries of other animal species and man.

cDNA for proopiomelanocortin (POMC) of mink has been cloned and sequenced. A comparative analysis of the primary structures of mRNAs coding for proopiomelanocortins of eight animal species and man has been performed. The analysis has revealed conserved and variable POMC mRNA regions. High variability of some of the regions is suggested to be due to the peculiarities of their structural organization. A putative mechanism responsible for the mutations in variable regions is proposed. A tree of the evolutionary relations of POMC has been developed.

[The use of a molecular probe containing a specific cDNA consisting of bacteriophage M13 for detecting the hepatitis A virus in clinical specimens]. / Ispol'zovanie molekuliarnogo zonda, soderzhashchego spetsificheskuiu kDNK v sostave bakteriofaga M13, dlia detektsii virusa gepatita A v klinicheskikh obraztsakh.

A probe was constructed containing a fragment of DNA replica of hepatitis A virus (HAV) RNA within bacteriophage M13 single-stranded DNA which allowed 10(-12) g of viral RNA to be tested. Hybridization of 32P-labeled probe with total RNA from 559 samples of blood, saliva, and urine from patients with viral hepatitis A revealed the presence of HAV RNA in 14% of the samples. In the 1st week of the jaundice period HAV RNA was detected in 40% (15 positive samples out of the 39 tested), in the 2nd week 26% (14 out of 54). HAV RNA was demonstrated in 20 out of 236 blood samples from subjects in a focus of HA outbreak. Six out of 9 subjects subsequently developing the disease were detected 7-10 days before the onset of the clinical symptoms. The proposed method may be useful for detection of HAV carriers in the latent period for their isolation in order to prevent further development of epidemic.

[The study of protein-peptide hormones using genetic engineering methods (review of the literature)]. / Issledovanie belkovo-peptidnykh gormonov metodami geneticheskoi inzhenerii (obzor).

Use of genetic engineering technique in cloning and sequence assay of cDNA and genes coding for mammal protein and peptide hormones is reviewed. Recent data on molecular aspects of gene reorganization in some inherited disorders are considered involving analytical methods for evaluation of gene activity, especially of genes coding for peptide hormones. Sequence assay of mRNA coding for mammal proopiomelanocortins (precursor of adrenocorticotropic hormone), beta-lipotropic hormones of pituitary body, is discussed.

Isolation of bioluminescent functions from Photobacterium leiognathi: analysis of luxA, luxB, luxG and neighboring genes.

Genes encoding luminescence of Photobacterium leiognathi have been cloned in Escherichia coli. The luminescent clones were readily apparent. Among them, a clone containing a recombinant plasmid with a 13.5-kb insertion was identified. This DNA fragment contained all of the luminescence-encoding genes. The luciferase-encoding genes (lux) in this DNA fragment were localized. We have sequenced a part of the cloned lux region and identified the luxA, luxB and luxG genes encoding the alpha and beta subunits of luciferase and a gamma protein with an Mr of 26,180, respectively. The analysis of deduced amino acid sequences and comparison with known luciferase sequences from Vibrio harveyi, indicate the common origin of these proteins.

[The change in functional activity and primary structure of the M2 protein in variants of the influenza virus resistant to remantadine and deitiforin: common and individual differences from the original strain]. / Izmenenie funktsional'noi aktivnosti i pervichnoi struktury belka M2 u rezistentnykh k remantadinu i deitiforinu variantov virusa grippa: obschie i individual'nye otlichiia ot iskhodnogo shtamma.

The two variants of influenza A/Victoria/35/72 (H3N2) virus resistant simultaneously to remantadine, deitiforin, adapromine and amantadine were obtained while passaging the virus in presence of remantadine or deitiforin. Both variants differed from the parental strain in optimal pH for hemolysis, transcriptase activity and in amino acid sequence of M2 protein. Maximal hemolytic activity of the parental strain is registered at pH 5.2, for the variants cultured in the presence of remantadine or deitiforin at pH 5.5 and 5.8, respectively. In contrast to NH4OH, remantadine and deitiforin do not exert inhibition of virus-induced hemolysis. Transcriptase activity of resistant variants is about 50% higher as compared with parental strain (enzyme source--whole virus particles or RNP). The M2 protein of the remantadine variant has 2 amino acid substitutions: 31 (Ser----Asn) and 59 (Met----Leu); the deitiforin variant has 3 substitutions: 14 (Met----Leu), 30 (Ala----Val) and 59 (Met----Leu). The phenotypic resistance of the virus seems to be determined by the mutations in the hydrophobic protein region (30,31); the other substitutions (14,59) may modify conformational structure and functional activity of the viral proteins.

[Chemico-enzymatic synthesis and cloning of human angiogenin gene in M13mp8 phage]. / Khimiko-fermentativnyi sintez i klonirovanie gena angiogenina cheloveka v sostave faga M13mp8.

The nucleotide sequence coding for human angiogenin has been deduced from the published amino acid sequence with the use of codons preferentially utilized in highly expressed E. coli genes. It was divided into forty-three oligonucleotides, which were synthesized by automatic gene assembler and then joined by DNA ligase into three double-stranded blocks, the blocks were consequently cloned and ligated in M13mp8 phage, and the resultant 389-bp DNA sequence coding for human angiogenin was analysed by chain-terminator sequencing technique.

[Construction of probes for hybridization with hepatitis A virus RNA based on phage M13]. / Konstruirovanie zondov dlia gibridizatsii s RNK virusa gepatita A na osnove faga M13.

Three fragments of a DNA copy complementary to tre hepatitis A virus RNA have been isolated from the pHAV-VPI-22 plasmid and cloned in M 13 mp 8 vector. The 140, 250 and 390 b. p. fragments corresponded to the viral genome fragment coding for VP1 protein. Single-stranded DNAs of the hybrid phages with 250 b. p. and 390 b. p. inserts have been used for radioactive probe synthesis. The specificity of the probes for viral RNA was confirmed in hybridization with hepatitis A virus RNA purified from the virus-infected cell culture. The hybridization was shown to detect up to 10(-12)-10(-13) g of the virus. A principal possibility to use the probes in diagnostic purposes was demonstrated by the virus detection in blood samples obtained from patients with hepatitis A infection.

Molecular cloning of a bovine immunoglobulin lambda chain cDNA.

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.

The mink proopiomelanocortin gene: characterization of cDNA and chromosomal localization.

A cDNA library from the mink pituitary was screened using as probe a synthetic oligodeoxyribonucleotide, 5'-TTCATGACCTCCGA-3', corresponding to the endorphin region of bovine proopiomelanocortin (POMC) cDNA. As a result, several clones containing inserts complementary to POMC mRNA were identified. The sequence of one of the fragments (585 bp, 65% of the total length of mRNA) was determined. A high degree of homology (over 80%) among the primary structures of sequences from mink, man, and bovine cDNA POMC was established. With the cloned mink cDNA fragment as probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of mink POMC sequences and mink chromosomes in the mink-Chinese hamster panel allowed us to assign the POMC gene to mink chromosome 11.

[Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi]. / Nukleotidnaia posledovatel'nost' genov alpha- i beta-sub''edinits liutsiferazy Photobacterium leiognathi.

Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

[Synthesis, cloning and primary structure of cDNA for proopiomelanocortin from the pituitary gland of the mink (Mustella vison)]. / Sintez, klonirovanie i opredelenie pervichnoi struktury kDNK proopiomelanokortina iz gipofiza norki (Mustella vison).

Total RNA was extracted from Mustella vison pituitary gland, and cDNA for proopiomelanocortin mRNA was synthesised and cloned. A 600 b. p. insert encoding for total ACTH, beta LPH and 3'-nontranslated end of the mRNA was sequenced using the Maxam-Gilbert technique.

[Comparative analysis of primary structure of M-genes in remantadine-resistant and remantadine-sensitive strains of influenza virus A/FPV/Weybridge (H7N7) strains]. / Sravnitel'nyi analiz pervichnykh struktur M-genov remantadinustoichivogo i remantadinchuvstvitel'nogo shtammov virusa grippa A/FPV/Weybridge (H7N7).

Full-length DNA copies of M-gene of remantadine-sensitive and remantadine-resistant variants of the influenza virus strain A/FPV/Weybridge (H7N7) have been synthesised and cloned. Complete nucleotide sequences of both cDNAs were determined by the Maxam-Gilbert method. There are three nucleotide substitutions, two of which lead to amino acid changes in M1 and M2 proteins. The existence of M3 protein, a polypeptide 68 amino acids long, encoded by the negative strand of RNA, is suggested. Amino acid changes in M2 and M3 proteins and their relation to the remantadine resistance are discussed.

[Synthesis, cloning and determination of the primary structure of DNA complementary to mRNA of prolactin from the human pituitary gland]. / Sintez, klonirovanie i opredelenie pervichnoi struktury DNK, komplementarnoi mRNK prolaktina iz gipofiza cheloveka.

The poly(A)-containing mRNA from human pituitary and prolactinoma have been purified and translated in the cell-free system from rabbit reticulocytes. mRNA from prolactinoma was shown to be enriched with specific prolactin mRNA. DNA complementary to the prolactin mRNA from human pituitary was obtained and cloned. Sequencing of the 900 bp insert by the Maxam-Gilbert technique suggested the cDNA cloned to cole for the previously published amino acid sequence, mismatches with mRNA from prolactinoma occurring at the third positions of codons and thus not causing amino acid substitutions.