Biochemistry (Moscow) (v.72, #11)

Noncoding RNAs by J. A. Makarova; D. A. Kramerov (1161-1178).
There has been substantial progress in studies on non-protein-coding RNAs (ncRNAs) during the last decade. Due to the development of new experimental approaches, a wide variety of such molecules have been found. This review considers the major groups of eukaryotic ncRNAs successfully studied in recent years. In particular, snoRNAs and scaRNAs involved in RNA modification and also miRNAs, siRNAs, tasiRNAs, and piRNAs responsible for RNA silencing are discussed. The transcripts of SINEs and SINE-like genes are considered separately.
Keywords: ncRNA; SRA RNA; 7SK RNA; NRSE RNA; snoRNA; scaRNA; miRNA; tasiRNA; piRNA; BC1 RNA; BC200 RNA; 4.5S RNA

Use of short representative sequences for structural and functional genomic studies by I. V. Gainetdinov; T. L. Azhikina; E. D. Sverdlov (1179-1186).
Existing approaches to direct genomic studies are costly and time-consuming. To overcome these problems, a series of tag-based methods utilizing short fragments uniquely representing full-length transcripts/genes from which they originate has been developed. This review summarizes basic principles underlying these methods and their numerous modifications designed for studying transcriptome profiles, searching for unidentified expressed loci, characterization of promoter regions, and high-throughput mapping of various genomic sites, such as hypo-and hypermethylated CpGs, and chromatin-binding and DNase I cleavage sites.
Keywords: functional genomics; transcriptome; tag; SAGE

Bioinformatical and experimental approaches to investigation of transcription factor binding sites in vertebrate genes by T. I. Merkulova; D. Yu. Oshchepkov; E. V. Ignatieva; E. A. Ananko; V. G. Levitsky; G. V. Vasiliev; N. V. Klimova; V. M. Merkulov; N. A. Kolchanov (1187-1193).
The development of computer-assisted methods for transcription factor binding sites (TFBS) recognition is necessary for study the DNA regulatory transcription code. There are a great number of experimental methods that enable TFBS identification in genome sequences. The experimental data can be used to elaborate multiple computer approaches to recognition of TFBS, each of which has its own advantages and limitations. A short review of the characteristics of computer methods of TFBS prediction based on various principles is presented. Methods used for experimental monitoring of predicted sites are analyzed. Data concerning DNA regulatory potential and its realization at the chromatin level, obtained using these methods, are discussed along with approaches to recognition of target genes of certain transcription factors in the genome sequences.
Keywords: genome annotation; recognition of transcription factor binding sites; computer and experimental methods; databases; training samples

Gene expression for VEGF-A, VEGF-C, and their receptors in murine lymphocytes and macrophages by O. I. Stepanova; A. V. Krylov; V. I. Lioudyno; E. P. Kisseleva (1194-1198).
Vascular endothelial growth factors VEGF-A and VEGF-C are the main angiogenic factors that control growth of new blood and lymphatic vessels in the organism, and they also possess several immunoregulatory activities. Expression of VEGF-A and VEGF-C mRNA as well as mRNA for VEGF receptors in lymphocytes and macrophages of naive mice was investigated. Using reverse transcription and subsequent polymerase chain reaction, we found that peritoneal macrophages, thymocytes, and lymph node cells constitutively expressed VEGF-A and VEGF-C mRNA. In addition, macrophages were positive for VEGFR-1, VEGFR-2, VEGFR-3, NRP-1, and NRP-2 mRNA, whereas thymocytes and lymph node cells expressed mRNA of the same receptors except VEGFR-1. These data expand our knowledge concerning gene distribution of VEGF receptors in the organism, in particular, among the cells of the immune system. This suggests that, along with their major angiogenic properties, VEGF family members additionally might also perform important mediatory functions within the immune system.
Keywords: VEGF-A; VEGF-C; receptors of VEGF; angiogenesis; NRP-1; NRP-2

AOP-1 interacts with cardiac-specific protein kinase TNNI3K and down-regulates its kinase activity by Yan Feng; Dong-Qing Liu; Zhen Wang; Zhao Liu; Hui-Qing Cao; Lai-Yuan Wang; Na Shi; Xian-Min Meng (1199-1204).
In the present study, a yeast two-hybrid screening system was used to identify the interaction partners of cardiac troponin I-interacting kinase (TNNI3K) that might serve as regulators or targets, and thus in turn to gain some insights on the roles of TNNI3K. After screening the adult heart cDNA library with a bait construct encoding the ANK motif of TNNI3K, antioxidant protein 1 (AOP-1) was isolated. The interaction between TNNI3K and AOP-1 was confirmed by the in vitro binding assay and coexpression experiments in vivo. The colocalization of TNNI3K and AOP-1 was clarified by confocal immunofluorescence. Moreover, coexpression of AOP-1 inhibited TNNI3K kinase activity in the in vitro kinase assay.
Keywords: AOP-1; protein kinase; protein-protein interaction; TNNI3K; yeast two-hybrid system

The F0 fluorescence yield in intact photosystem II (PSII), Ca-depleted PSII (PSII(-Ca/NaCl)), and Mn-depleted PSII membranes was measured before and after dim light treatment (1–2 min), using flash-probe fluorescence and fluorescence induction kinetic measurements. The value of F0 after the light treatment (F’0) was larger than F0 in dark-adapted PSII membranes and depended on the appearance of the slowly relaxing, reduced plastoquinone pool (t 1/2 = 4 min) formed during preillumination, which was not totally reoxidized before the F’0 measurement. In PSII(-Ca/NaCl) such a pool also appeared, but the F’0 yield was even higher than in intact PSII membranes. In Mn-depleted PSII membranes, the pool did not form. Interestingly, the yield of F’0 in Ca-depleted PSII membranes prepared using chelators (EGTA and citrate) or containing 5 mM EGTA was significantly lower than in PSII(-Ca/NaCl) samples prepared without chelators. These data indicate that chelators inhibit the reduction of QA and QB and formation of the slowly relaxing plastoquinone pool, or alternatively they increase the rate of its oxidation. Such an effect can be explained by coordination of the chelator molecule to the Mn cluster in PSII(-Ca/NaCl) membranes, rather than different amounts of residual Ca2+ in the membranes (with or without the chelator), since the remaining oxygen-evolving activity (∼15%) in PSII(-Ca/NaCl) samples did not depend on the presence of the chelator. Thus, chelators of calcium cations not only have an effect on the EPR properties of the S2 state in PSII(-Ca/NaCl) samples, but can also influence the PSII properties determining the rate of plastoquinone pool reduction and/or oxidation. The effect of some toxic metal cations (Cd, Cu, Hg) on the formation of the slowly relaxing pool in PSII membranes was also studied.
Keywords: photosystem II; oxygen-evolving complex; calcium; plastoquinone QA ; plastoquinone QB ; fluorescence; fluorescence induction kinetics; F0

Profiling of the CD4 receptor complex proteins by G. I. Krotov; M. P. Krutikova; V. G. Zgoda; A. V. Filatov (1216-1224).
Intermolecular complexes produced by the CD4 molecule were studied. To preserve the integrity of weak protein-protein interactions of the CD4 antigen, cells were lysed in a mild nonionic detergent Brij97. Protein constituents of the complex were identified by our previously proposed fluorescence immunoprecipitation assay with subsequent mass spectrometry. In total, 26 proteins associated with CD4 were identified on CEM cells. The CD4 complex included the following major components: tyrosine phosphatase CD45, transferrin receptor CD71, tyrosine kinase Lck, and a lymphocyte phosphatase-associated phosphoprotein LPAP. The CD4 complex also contained some components of cytoskeleton and heat shock proteins. The association between CD4, CD71, and CD45 molecules was confirmed by immunoblotting. The CD4 complexes were not detected on the U937 myeloid cells lacking Lck and LPAP. We attempted to quantitatively characterize the CD4 complex composition.
Keywords: protein complexes; nonionic detergents; immunoprecipitation; mass spectrometry; CD4

As a result of limited tryptic proteolysis of S1 ribosomal protein (molecular mass 60 kD) from Thermus thermophilus, 25 N-terminal amino acid residues and 71 C-terminal amino acid residues are split off and a stable high-molecular-weight fragment with molecular mass of 49 kD is formed that retains RNA-binding properties and is capable of interacting with 30S ribosomal subunit. Earlier, application of a similar procedure for the formation of a fragment of S1 protein from Escherichia coli resulted in splitting of 171 N-terminal amino acid residues with the formation of a 41.3 kD fragment that possesses RNA-binding properties only. Thus, in spite of high homology between E. coli and T. thermophilus proteins, the proteolysis leads to the formation of two different fragments, which points, in our opinion, to the fact of significant differences between their structures.
Keywords: ribosome; S1 ribosomal protein; limited proteolysis; Thermus thermophilus

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1. Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome.
Keywords: dynein; dynactin; microtubules; centrosome; p150Glued; microinjections; melanophores

Molecular cloning and expression analysis of a novel CONSTANS-like gene from potato by J. L. Guo; Q. Yang; F. Liang; Y. J. Xing; Z. Wang (1241-1246).
A full-length cDNA of a StCONSTANS-like (StCOL) gene was cloned from potato (Solanum tuberosum L.) by RTPCR and RACE. The predicted amino acid sequence of this cDNA has a high degree of identity with other homologous members of the CO or COL family. Analysis of mRNA levels for StCOL shows that it is highly expressed in leaves and becomes weaker during tuberization; moreover, is independent of gibberellin A3 and sucrose.
Keywords: StCONSTANS-like; cloning; expression; potato

Disruption of a fur-like gene inhibits magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 by Yijun Huang; Weijia Zhang; Wei Jiang; Chengbo Rong; Ying Li (1247-1253).
In this study, a genomic library of Magnetospirillum gryphiswaldense MSR-1 strain was constructed and a fur-like gene (encoding Fur protein, ferric uptake regulator) was isolated and sequenced. This gene consisted of 420 bp and encoded 139 amino acid residues. To investigate the function of this gene in MSR-1, a fur mutant was generated by double crossover with a kanamycin cassette inserted into its coding region. Iron uptake and magnetosome formation were dramatically inhibited by disruption of fur. Iron content analysis of the fur mutant indicated that it contained approximately 0.037% by dry weight, which was at least 10-fold less than that observed in the wild type. Electron microscopy revealed the absence of a magnetosome in the fur mutant, although it was able to tolerate 1 mM H2O2 at 10-fold higher level than wild-type. These data suggest that Fur protein may possess a novel function in magnetic bacteria.
Keywords: Magnetospirillum gryphiswaldense ; ferric uptake regulator (Fur); magnetosome formation

Cooperative interaction of the high-potential hemes (Ch) in the cytochrome subunit of the photosynthesizing bacterium Ectothiorhodospira shaposhnikovii was studied by comparing redox titration curves of the hemes under the conditions of pulse photoactivation inducing single turnover of electron-transport chain and steady-state photoactivation, as well as by analysis of the kinetics of laser-induced oxidation of cytochromes by reaction center (RC). A mathematical model of the processes of electron transfer in cytochrome-containing RC was considered. Theoretical analysis revealed that the reduction of one heme Ch facilitated the reduction of the other heme, which was equivalent to a 60 mV positive shift of the midpoint potential. In addition, reduction of the second heme Ch caused a three-to four-fold acceleration of the electron transfer from the cytochrome subunit to RC.
Keywords: purple bacteria; reaction center; cytochromes; electron transport; mathematical model; Ectothiorhodospira shaposhnikovii

Aberration of morphogenesis of siliceous frustule elements of the diatom Synedra acus in the presence of germanic acid by T. A. Safonova; V. V. Annenkov; E. P. Chebykin; E. N. Danilovtseva; Ye. V. Likhoshway; M. A. Grachev (1261-1269).
Addition of germanic acid into the culture medium of the diatom Synedra acus subsp. radians (Kutz.) Skabitsch. had nearly no influence on the culture growth at the Ge/Si molar ratio 0.01, but stopped it at ratios 0.05 and higher. It was shown by mass-spectrometry that at the Ge/Si ratio 0.01 germanium was incorporated in both the cytoplasm and siliceous valves, whereas at Ge/Si 0.05 it was incorporated into the cytoplasm but almost failed to accumulate in the valves. At Ge/Si 0.1 germanium was accumulated in the cytoplasm, but its incorporation into the valves terminated. Studies on the cell morphology by light, epifluorescence, and transmission electron microscopy showed that high concentrations of germanic acid induced disorders in morphogenesis of the siliceous frustule and accumulation of large rhodamine-stainable electron-dense inclusions. Model chemical experiments with over-saturated solutions of silicic acid in the presence of polyallylamine revealed that addition of 5% germanic acid considerably accelerated coagulation of silica. Hence, the toxic effect of germanic acid on diatoms could be caused by changes in coagulation of silica.
Keywords: diatoms; silica; germanium; polyallylamine; morphogenesis; epifluorescence and transmission electron microscopy

β-synuclein protein from Xenopus laevis: Overexpression in Escherichia coli of the GST-tagged protein and production of polyclonal antibodies by Zhu Yuan; Xinyu Zhao; Fei Yan; Jian Zhao; Huanyi Liu; Shaoquan Xiong; Jiong Li; Lijuan Chen; Yuquan Wei (1270-1278).
This report presents a procedure to obtain and purify recombinant β-synuclein from Xenopus laevis expressed in Escherichia coli as a glutathione-S-transferase fusion protein. After identification by mass spectrometry, the protein was then used to raise anti-X. laevis β-synuclein polyclonal antibodies, which were suitable to detect the presence of β-synuclein in X. laevis brain by Western blot. This is the first report of a positive identification of β-synuclein in an amphibian at the protein level.
Keywords: Xenopus laevis ; β-synuclein; Escherichia coli ; β-synuclein expression; antibody production