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A gene encoding superoxide dismutase was revealed in the genome of the thermoacidophilic crenarchaeon Acidilobus saccharovorans. A recombinant expression vector was constructed and transformed into E. coli cells. The novel recombinant superoxide dismutase was purified and characterized. The enzyme was shown to be an iron-dependent superoxide dismutase able to bind various bivalent metals in the active site. According to differential scanning calorimetric data, the denaturation temperature of the enzyme is 107.3°C. The maximal activity of the Fe(II) reconstituted enzyme defined by xanthine oxidase assay is 1700 U/mg protein. Study of the thermal stability of the superoxide dismutase samples with various metal contents by tryptophan fluorescence indicated that the thermal stability and activity of the enzyme directly depend on the nature of the reconstituted metal and the degree of saturation of binding sites.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5′-phosphate and 3′-­hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 A shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

Strain 1633, a novel member of the genus Thermogladius , isolated from a freshwater hot spring, is an anaerobic hyperthermophilic crenarchaeon capable of fermenting proteinaceous and cellulose substrates. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof pathway for glucose metabolism, cytoplasmic NADP-dependent hydrogenase, and several energy-coupling membrane-bound oxidoreductases.

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.

Strain 1860, a novel member of the genus Pyrobaculum , is a hyperthermophilic organotrophic crenarchaeon growing anaerobically with various electron acceptors. The complete genome sequence reveals genes for several membrane-bound oxidoreductases, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the glyoxylate cycle, and the dicarboxylate/4-hydroxybutyrate cycle.

Using Agrobacterium-mediated transformation procedure, a total of 204 primary transformants of the Elizaveta, Lugovskoi and Nevski potato varieties carrying the cry IIIa gene were obtained. A number of biomolecular tests, including Southern hybridization, insert copy number screening, immunofluorescence analysis for assessment of the target gene expression levels, and PCR control for testing of the insert integrity, was carried out for these transformants, as well as the biosafety field trials for the transgenic lines selected during the biomolecular analysis. As a result, 3 transgenic lines (E2, N1, and L5) carrying one insert copy per genome with target gene expression level over 10 ppm were selected.

ISSN 00124966, Doklady Biological Sciences, 2012, Vol. 444, pp. 202–205. © Pleiades Publishing, Ltd., 2012.Original Russian Text © M.V. Dzyuba, A.V. Mardanov, A.V. Beletskii, T.V. Kolganova, M.V. Sukhacheva, A.A. Shelenkov, V.M. Gorlenko, B.B. Kuznetsov, K.G. Skryabin, 2012,published in Doklady Akademii Nauk, 2012, Vol. 444, No. 3, pp. 335–338.

We determined the complete nucleotide sequence of the 41 719 bp mitochondrial genome of the methylotrophic yeast Hansenula polymorpha strain DL-1. It contains genes for three subunits of cytochrome oxidase (cox1, cox2 and cox3), three subunits of ATP synthase (atp6, atp8 and atp9), seven subunits of NADH dehydrogenase (nad1-6 and nad4L), apocytochrome b (cob), four endonuclease/maturase homologs, a ribosomal protein (rps3), large and small rRNAs and a complete set of tRNAs. The structural genes are organized in two major transcriptional units. Phylogenetic, gene content and gene order analyses revealed the close phylogenetic relationship between H. polymorpha and Brettanomyces custersianus, and support the assignment of strain DL-1 to a separate genus rather than including it in the polyphyletic genus Pichia.

Contains fulltext : 97937.pdf (Publisher’s version ) (Closed access) We conducted a two-stage genome-wide association study of renal cell carcinoma (RCC) in 3,772 affected individuals (cases) and 8,505 controls of European background from 11 studies and followed up 6 SNPs in 3 replication studies of 2,198 cases and 4,918 controls. Two loci on the regions of 2p21 and 11q13.3 were associated with RCC susceptibility below genome-wide significance. Two correlated variants (r(2) = 0.99 in controls), rs11894252 (P = 1.8 x 10) and rs7579899 (P = 2.3 x 10), map to EPAS1 on 2p21, which encodes hypoxia-inducible-factor-2 alpha, a transcription factor previously implicated in RCC. The second locus, rs7105934, at 11q13.3, contains no characterized genes (P = 7.8 x 10(1)). In addition, we observed a promising association on 12q24.31 for rs4765623, which maps to SCARB1, the scavenger receptor class B, member 1 gene (P = 2.6 x 10). Our study reports previously unidentified genomic regions associated with RCC risk that may lead to new etiological insights.

Complete nucleotide sequence of the 27,266 bp mitochondrial genome of the сephalosporin C producing fungus Acremonium chrysogenum have been determined using whole genome shotgun sequencing approach. The circular mapping molecule encodes a usual set of mitochondrial proteins and RNA genes, including large and small ribosomal RNAs, 19 proteins and 26 tRNA genes and contains 2 introns. All structural genes are located on one strand and are apparently transcribed in one direction. Comparative analysis of this and previously sequenced Pezizomycotina mtDNAs revealed more extensive similarity between A. chrysogenum genome and those of Fusarium clade and specific synthenic patterns characteristic of Hypocrealean mitogenomes. Phylogenetic analysis based on catenated mitochondrial protein sequences confirmed current taxonomic position of A. chrysogenum within Hyprocreales and related taxa.

A somatic cell genome was recently resequenced for a patient with renal cancer. The data were submitted to the NCBI Sequence Read Archive under the accession number SRA012240. Here, we have performed SNP calling for the genome and compared it with several published genomes. We have found 2, 921, 724 SNPs, including 1, 472, 679 newly described ones. Among them, 63, 462 SNPs have been mapped to the Y chromosome and, based on 18 markers, the genome has been ascribed to the R1a1a haplogroup predominant in Russian males. The mitochondrial haplogroup has been determined as U5a, which is also common in the European part of Russia. Short reads unmapped to the hu- man genome were used for the de novo assembly of DNA sequences. This resulted in genome-specific contigs (more than 100 bp in length) with an overall length of 154 kbp (for GAII) and 4.7 kbp (for SOLiD).

The conventional vaccines currently being used to deal with influenza are based on a virus obtained in chicken embryos or its components. The high variability of the major immunogenic surface proteins - hemagglutinin and neuraminidase-require the development of strain-specific vaccines that match the antigenic specificity of a newly emerging virus. Recombinant vaccines based on single viral proteins that could be easily produced in standard expression systems are attractive alternatives to traditional influenza vaccines. We constructed recombinant nanosized virus-like particles based on a nuclear antigen of the hepatitis B virus. These particles expose on the surface the extracellular domain of the M2 protein of the highly pathogenic A(H1N1) 2009 influenza virus. The methods of production of these virus-like particles in Escherichia coli and their purification were developed. Experiments on animals show that M2sHBc particles are highly immunogenic in mice and provide complete protection against the lethal influenza challenge.

Alcohol dehydrogenases belong to the oxidoreductase family and play an important role in a broad range of physiological processes. They catalyze the cofactor-dependent reversible oxidation of alcohols to the corresponding aldehydes or ketones. The NADP-dependent short-chain alcohol dehydrogenase TsAdh319 from the thermophilic archaeon Thermococcus sibiricus was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method using 25%(w/v) polyethylene glycol 3350 pH 7.5 as precipitant. The crystals diffracted to 1.68 A resolution and belonged to space group I222, with unit-cell parameters a = 55.63, b = 83.25, c = 120.75 A.

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis . The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS 607 and IS 200 /IS 605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis . Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

A synthetic gene of the B-subunit of Escherichia coli heat-labile toxin, optimized for expression in plants, was designed and synthesized. The recombinant viral vector was constructed on the basis of potato virus X containing the LTB gene instead of the removed triple block of transport genes and the coat protein gene, which provides for LTB expression in plants. The vector is introduced into the plant cells during cell infiltration by agrobacteria incorporating a binary vector, the T-DNA region of which contains a cDNA copy of the recombinant viral genome. Under conditions of posttranscriptional gene silencing inhibition, the LTB yield in Nicotiana benthamiana plants is 1-2% of total soluble protein; in this case, LTB synthesized in plants forms pentameric complexes analogous to those found in the native toxin. The designed viral system of LTB transient expression can be used to obtain in plants a vaccine against enteropathogenic Escherichia coli.

The complete nucleotide sequence of the genome of a new potato virus X (PVX) strain Tula isolated by us has been determined. Based on comparison of the PVX Tula nucleotide sequence with the sequences of 12 other PVX strains, this strain was assigned to the European cluster of PVX strains. Phylogenetic analysis revealed the same phylogeny for both full genome sequences and nucleotide sequences of polymerase and coat protein genes, suggesting that the PVX evolution did not involve recombination between different strains. The full-size cDNA copy of the PVX Tula genome was cloned and the accumulation of the viral coat protein in infected Nicotiana benthamiana was shown to be about twofold higher than for the PVX strain UK3. Based on the PVX Tula genome, a new vector which contained the target gene instead of the removed triple transport gene block and the coat protein gene has been constructed for expression of target proteins in plants. The productivity of the new vector was about 1.5–2-fold higher than the productivity of the vector of the same structure based on the standard PVX strain genome. The new viral vector can be used for superproduction of recombinant proteins in plants.

A rapid and simple technique has been developed to enhance the sensitivity of virus detection in dot-blot hybridization assay by up to 1000 fold. The procedure generally follows that of Baulcombeet al. (1984) but includes moderate heating of the nitrocellulose filter in 10XSSC, 0.5% SDS solution at 55°C after sample application. Using this procedure, four potato viruses (PVX, PVS, PVM and PVY) were detected with cloned virus-specific 32P-cDNA probes in 2 μl spots containing 0.2–2 pg of purufied virus (0.1–1 ng/ml). The procedure was also successfully applied for the detection of PVX, PVS, PVM and PVY in crude potato tuber extracts. ZusammenfassungEin verbesserter dot-blot-Hybridisationsassay, der fur die Ermittlung von Kartoffelviren in ungeremigten Kartoffelknollenextrakten im grosen Masstab geeignet ist Eine schnelle und einfachc Methode wurde entwickelt, um die Empfindlichkeit der Virusermittlutig in einetn dot-blot-Hybridisaiionsassay um bis auf das 1000fache zu erhohen. Das Verfahren ist dem von BAULCOMBE et al. (1984) beschriebenen Verfahren sehr ahnlich, cs wird aber durch eine gemasigte Erhitzung des Nitroceiluiosefilters in 10XSSC, 0.5 %ige SDS-Losung bei 55 °C nach der Probeauftragunj; erganzt. Nach dicser Meihode konnten vier Kartoffelviren (PVX, PVS, PVM und PVY) mit klonicrien virusspezifischen 32P-DNS-Proben in 2 μl Spots, die 0,2–2 pg gereinlgtes Virus (0,1–1 ng/ml) enthielten, ermittelt werden. Die Methode wurde auch erfolgreich bei der Ermittlung von PVX, PVS, PVM und PVY in ungercJnigten Kartoffelknollenexirakten eingestizt.

The amino acid sequences of the large proteins of potexviruses and tymoviruses were aligned. It was shown that three domains of these proteins display significant similarity between the two virus groups. In contrast to central (putative NTPase-helicase) and C-terminal (putative polymerase) domains, the conservative N-terminal domain of the potexvirus/tymovirus large protein displayed no obvious similarity to the respective regions of the large proteins of other Sindbis-like viruses.

Modified chitin-binding domain (ChBD) from Bacillus circulans chitinase A1 with W42F mutation in chitin-binding site was genetically fused to different positions within alpha-subunit of glutaryl-7-aminocephalosporanic acid acylase (GLA) gene. Hybrid proteins were efficiently expressed in E. coli cells as soluble, enzymatically active and correctly processed holoenzymes. ChBD-GLA fusions were easily affinity purified on chitin column by changing the salt concentration of binding and elution buffer. The developed one-step affinity purification procedure is thus a promising approach for scaled-up isolation of GLA variants for preparation of industrial biocatalysts as well as for structure-functional studies.

Latent amino acid repeats seem to be widespread in genetic sequences and to reflect their structure, function, and evolution. We have recently identified latent periodicity in more than 150 protein families including protein kinases and various nucleotide-binding proteins. The latent repeats in these families were correlated to their structure and evolution. However, a majority of known protein families were not identified with our latent periodicity search algorithm. The main presumable reason for this was the inability of our techniques to identify periodicities interspersed with insertions and deletions. We designed the new latent periodicity search algorithm, which is capable of taking into account insertions and deletions. As a result, we identified many novel cases of latent periodicity peculiar to protein families. Possible origins of the periodic structure of these families are discussed. Summarizing, we presume that latent periodicity is present in a substantial portion of known protein families. The latent periodicity matrices and the results of Swiss-Prot scans are available from http://bioinf.narod.ru/del/.

Matrix 2 protein ectodomain (M2e) is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek). Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1) and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1) and A/Chicken/Kurgan/05/05 RG (H5N1) to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1). Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.

We have applied information decomposition to show the presence of many sequences in prokaryotic genomes that possess latent dinucleotide periodicity but have not been found before. More than two hundred DNA sequences having such a latent periodicity were found. These sequences belonged to the 94 different microbial genomes. The classification made has shown that all of the sequences could be assigned to the 45 classes according to the type of latent dinucleotide period. A probable functional and evolutional meaning of the latent dinucleotide periodicity in different prokaryotic genomes is discussed.

The techniques of Information Decomposition, Cyclic Profile Alignment, and Noise Decomposition allowed us to search for latent repeats within protein families of various functions. We have found out 94 protein families with a family-specific periodicity. In each case, the periodic element was found in greater than 70 % of family members. Latent periodicity profiles with specific length and signature was obtained in each case. The possible relationship between the periodic elements thus identified and the evolutionary development of the protein families are discussed with specific reference to the possibility that there is a correlation between the periodic elements and protein function.

Here, we have applied information decomposition, cyclic profile alignment, and noise decomposition techniques to search for latent repeats within protein families of various functions. We have identified 94 protein families with a family-specific periodicity. In each case, the periodic element was found in greater than 70% of family members. Latent periodicity profiles with specific length and signature were obtained in each case. The possible relationship between the periodic elements thus identified and the evolutionary development of the protein families are discussed with specific reference to the possibility that there is a correlation between the periodic elements and protein function.

The expression and assembly of the hepatitis B virus (HBV) nucleocapsid protein (HBcAg) were investigated in plants using viral vectors. Constructs based on either Potato virus X (PVX) or Cowpea mosaic virus (CPMV) containing the sequence of HBcAg were able to infect the appropriate host plants and remained genetically stable during infection. Analysis of HBcAg expression revealed that the protein can self-assemble into core-like particles and that the assembled material could be partially purified by differential centrifugation. Thus, the use of viral vectors can be considered a practical method for rapid production of assembled HBcAg particles in plants. This approach provides a means whereby a variety of chimaeric particles can be assessed quickly and cheaply for various diagnostic and vaccine applications.

We identified latent periodicity in catalytic domains of approximately 85% of annotated serine-threonine and tyrosine protein kinases. Similar results were obtained for other 22 protein families and domains. We also designed the method of noise decomposition, which is aimed to distinguish between different periodicity types of the same period length. The method is to be used in conjunction with the method of cyclic profile alignment, and this combination is able to reveal structure-related or function-related patterns of latent periodicity. Possible origins of the periodic structure of protein kinase active sites are discussed. Summarizing, we presume that latent periodicity is the common property of many catalytic protein domains.

tRNAs and their derivatives are known to be involved in various genetic processes from bacteria to primates. Revealing evolutionary ancestors and relatives of the modern tRNAs with further functional analysis could shed light on their role in expansion of interspersed repeats, their promoter activity, and so on. Although tRNA-like sequences were earlier found and described in many gene regions and most known SINE repeats contain tRNA part, there are still a lot of unknown entries. tRNA-like sequences could also help to find out new families of genetic repeats. Presence of tRNA-like sequences was detected in various genetic structures. Their relative distribution pattern over these sequences and preliminary functional analysis are presented.

Transfer RNA (tRNA)-like sequences were searched for in the nine basic taxonomic divisions of GenBank-121 (viruses, phages, bacteria, plants, invertebrates, vertebrates, rodents, mammals, and primates) by an original program package implementing a dynamic profile alignment approach for the genetic texts' analysis, in using 22 profiles of tRNAs of different isotypes. In total, 175,901 previously unknown tRNA-like sequences were revealed. The locations of the tRNA-likes were considered over the regions whose functional meaning is described by standard Feature Keys in GenBank. Many regions containing the tRNA-like sequences were recognized as known repeats. A mode of distribution of the tRNA-like sequences in a genome was proposed as expansion in a content of the various transposable elements. An analysis of the integrity of RNA polymerase III inner promoters in the tRNA-like sequences over the GenBank divisions has shown a high possibility of generating new copies of short interspersed nuclear element (SINE) repeats in all divisions, excepting primates. The numerous tRNA-likes found in the regions of RNA polymerase II promoters have suggested an adaptation of RNA polymerase III promoter to a binding of RNA polymerase II.

Thermoproteus uzoniensis 768-20 is a thermoacidophilic anaerobic crenarchaeon isolated from a solfataric field in Kamchatka, Russia. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, beta-oxidation of fatty acids, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the dicarboxylate/4-hydroxybutyrate cycle, hydrogenase, and sulfur reductase.

Strain 768-28 was isolated from a hot spring in Kamchatka, Russia, and represents a novel member of the Vulcanisaeta genus. The complete genome sequence of this thermoacidophilic anaerobic crenarchaeon reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, beta-oxidation of fatty acids, and sulfate reduction.

Oscillochloris trichoides is a mesophilic, filamentous, photoautotrophic, nonsulfur, diazotrophic bacterium which is capable of carbon dioxide fixation via the reductive pentose phosphate cycle and possesses no assimilative sulfate reduction. Here, we present the draft genome sequence of Oscillochloris trichoides subsp. DG-6, the type strain of the species, which has permitted the prediction of genes for carbon and nitrogen metabolism and for the light-harvesting apparatus.

Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5′-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shuntdependent expression to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shunting proved to be lethal. Finally, by using an artificial stem structure that blocks scanning, we obtained direct evidence that ribosome shunt operates during CaMV infection.

The 5′ untranslated leader of potato virus X (PVX) RNA is shown when contiguous to the coding sequence, to enhance the expression of the neomycin phosphotransferase II gene (NPTII) in Nicotiana tabacunz protoplasts in vivo. The level of transient expression of the NPTII gene in protoplasts provided by a plasmid containing the coding sequence of the NPTII gene under the control of 35S cauliflower mosaic virus (CaMV) promoter and terminator elements served as the baseline control. Insertion of the viral 5′ untranslated leader sequence upstream of the NPTII ATG codnn increased the level of expression 4-fold. An 83 nucleotide (nt) leader sequence (lacking only one nucleotide of the complete PVX leader) and a truncated version with a 28 deletion at the 3′ end both had similar enhancing abilities. The 28 nt CA region of the PVX leader alone had no enhancement properties.

An earlier reported method for revealing latent periodicity of the nucleotide sequences has been considerably modified in a case of small samples, by applying a Monte Carlo method. This improved method has been used to search for the latent periodicity of some nucleotide sequences of the EMBL data bank. The existence of the nucleotide sequences' latent periodicity has been shown for some genes. The results obtained have implied that periodicity of gene structure is projected onto the periodicity of primary amino acid sequences and, further, onto spatial protein conformation. Even though the periodic structure of gene sequences has been eroded, it is still retained in primary and/or spatial structures of corresponding proteins. Furthermore, in a few cases the study of genes' periodicity has suggested their possible evolutionary origin by multifold duplications of some gene's fragments.

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5′-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem–loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5′-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.

Heterologous expression of proteins can be dramatically improved as a result of optimized mRNA translation. 5’-Untranslated mRNA sequences (leaders of translation) are one of the possible targets for such an optimization. “Translational enhancers” for foreign protein expression in plants could be derived from 5’-leaders of genomic RNAs of positive-sense RNA viruses. Genomic RNA of these viruses functions as messenger RNA in infected plant cell and apparently efficiently competes with host mRNAs for the components of translation machinery. Alternatively, subgenomic RNAs for viral coat proteins, featuring their own translation initiation sites should be efficiently translated, since coat proteins are synthesized in a substantial amount during viral reproduction.

In the past 7-8 years considerable effort has been expended to produce transgenic plants that resist virus infection, insects, herbicides and disease development. The crop improvement and the desired phenotype is conferred by transformation, usually with a single foreign gene. Selection of transformants demands the use of selectable markers, which usually encode enzymes which inactivate either a herbicide or an antibiotic. Also we use certain foreign genes known as reporter genes (for review see [7]).

The design of de novo proteins which ensures the desirable three-dimensional (3-D) structures is the best way to test our understanding of the principles of protein structure and protein folding. The first successful engineering of a de novo protein, namely four-helix bundle, has been reported seven years ago (Regan and DeGrado, 1988) and then a number of attempts have been made in this direction during the past years (for a recent review see Sander, 1994). Most of the designed structures mimicked the 3-D structures of some natural proteins. In 1988 four of us have reported a design of a de novo protein albebetin (Figure 1) with a predetermined 3-D fold consisting of a four-stranded antiparallel β-sheet with one side screened by two α-helices (Finkelstein et al., 1988). This fold does not contradict any structural rule but has been never observed up to now in natural proteins. It consists of two identical αββ units, therefore we have called our de novo protein albebetin. We have expressed albebetin in a cell-free translational system and in E.coli and investigated it by various physical and chemical techniques (Dolgikh et al., 1991, 1992; Fedorov et al., 1992; Chemeris et al., 1994). It has been shown that the de novo protein is relatively stable and nearly as compact as natural proteins, it unfolds cooperatively at high urea concentrations and possess structural features of a definite structure consistent with the predesigned one. An eight amino acids biologically active human interferon fragment was attached to albebetin in order to obtain a de novo protein with a grafted biological function (Dolgikh et al., 1993).

The regulatory activity of a 826 bp DNA fragment located upstream of the pTiBo542 TL-DNA gene 6b coding region was analyzed in transgenic tobacco, using beta-glucuronidase (gus) as a reporter gene. The region was shown to drive organ-specific, wound- and auxin-inducible expression of the reporter, the effect being dependent on the type and concentration of auxin.

The problem of searching for functional sites in protein molecules is far from solving. The straight experimental way is to isolate molecules of both receptor and ligand, co-crystallize them, obtain X-ray data of this complex, analyze it and directly find interacting sites [Burt et al. 1991]. Unfortunately in most cases this ideal could not be reached because of known difficulties in cloning, obtaining, purification, crystallization etc. of both components. This method provides full information about structures of recognition sites but not about particular biological functions of amino acids constituting these sites (e.g. binding, or signal transduction etc.). The usual way to obtain functional information is changing the structure of selected protein sites (via site-directed mutagenesis or chemical modification) followed by comparative biological testing. Step-by-step mutagenesis of the whole protein molecule remains difficult and time-consuming task. Selection of proper mutagenesis strategy makes it enormously multi-variant, although some simplistic approaches (e.g. alanine mutagenesis) could be successful. Thus it is preferential to find probable functional sites and start mutagenesis from it.

The RUSNANOTECH 2011 International Forum on Nanotechnology was held from 26–28 October 2011, in Moscow, Russia. It was the fourth forum organized by RUSNANO (Russian Corporation of Nanotechnologies) since 2008. In March 2011 RUSNANO was established as an open joint-stock company through the reorganization of the state corporation Russian Corporation of Nanotechnologies. RUSNANO's mission is to develop the Russian nanotechnology industry through co-investment in nanotechnology projects with substantial economic potential or social benefit. Within the framework of the Forum Science and Technology Program, presentations on key trends of nanotechnology development were given by foreign and Russian scientists, R&D officers of leading international companies, universities and scientific centers. The science and technology program of the Forum was divided into four sections as follows (by following hyperlinks you may find each section's program including videos of all oral presentations): Nanoelectronics and Nanophotonics Nanomaterials Nanotechnology and Green Energy Nanotechnology in Healthcare and Pharma (United business and science & technology section on 'RUSNANOTECH 2011') The scientific program of the forum included more than 50 oral presentations by leading scientists from 15 countries. Among them were world-known specialists such as Professor S Bader (Argonne National Laboratory, USA), Professor O Farokzhad (Harvard Medical School, USA), Professor K Chien (Massachusetts General Hospital, USA), Professor L Liz-Marzan (University of Vigo), A Luque (Polytechnic University of Madrid) and many others. The poster session consisted of over 120 presentations, 90 of which were presented in the framework of the young scientists' nanotechnology papers competition. This volume of Journal of Physics: Conference Series includes a selection of 47 submissions. Section editors of the proceedings: Nanoelectronics and nanophotonics Corresponding Member of Russian Academy of Sciences, Professor Anatoly Dvurechenskii (Institute of Semiconductor Physics, RAS). Nanomaterials Member of Russian Academy of Sciences, Professor Mikhail Alfimov (Photochemistry Center, RAS), Professor Igor Suzdalev (Semenov Institute of Chemical Physics, RAS), Member of Russian Academy of Science, Professor Vyacheslav Osiko (Prokhorov General Physics Institute, RAS), Member of Russian Academy of Science, Professor Aleksey Khokhlov (Physical department of Moscow State University). Nanotechnology and green energy Corresponding Member of Russian Academy of Sciences, Professor Eduard Son (Joint Institute for High Temperatures, RAS). Nanotechnology in Healthcare and Pharma Member of Russian Academy of Sciences, Professor Konstantin Skryabin (Bioengineering Center, RAS), Member of Russian Academy of Sciences, Professor Rem Petrov (RAS), Corresponding Member of Russian Academy of Sciences, Professor Sergey Deev (Institute of Bioorganic Chemistry).

We have sequenced 6006 bp DNA of a region from the Bacillus subtilis SHgw chromosome known to contain riboflavin biosynthesis genes (rib gene cluster, 210 degrees on the B. subtilis genetic map). Five of the seven open reading frames found within the sequence are shown to represent the genes ribG, ribB, ribA, ribH and ribTD. The calculated molecular masses for the putative translation products are 39,305, 23,481, 44,121, 16,287 and 14,574 daltons respectively. The five rib genes are transcribed as a polycistronic 4277 nucleotide messenger RNA. The steady-state level of the transcript is negatively regulated by riboflavin. A cis-acting element necessary for regulation was mapped by analysis of constitutive mutations within the 5' untranslated region of the operon. The element is at least 48 bp in length and does not bear obvious similarity to well defined prokaryotic regulatory elements. The molecular mechanism of regulation remains unknown, but the data presented argue against regulation by attenuation.

A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli. Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg.