[Intron 2 of human beta-globin in 3'-untranslated region enhances expression of chimeric genes].

Possibility to enhance heterologous gene expression in mammalian cells by introduction of an intron in 3' untranslated region (UTR) was investigated. To this end, a fragment of human beta-globin gene with intron 2 and flanked exon regions was introduced into vector encoding green fluorescent protein TagGFP2 after the TagGFP2 stop-codon (Int+). The distance between the stop-codon and the exonjunction was 35 nucleotides. It ensured that Int+ mRNA was resistant to degradation by nonsense mediated decay (NMD) machinery. A control vector Int- contained corresponding intronless sequence of the beta-globin mRNA. On the same plasmid, the second gene encoded far-red fluorescent protein Katushka was used to normalize fluorescence for transfection efficiency and expression level in individual cells. Transiently transfected HEK293T cells were analysed by flow cytometry. It was shown that cells transfected with plasmid carrying the Int+ gene possess 1.8 ± 0.2 fold higher green fluorescence compared to Int- cells. The observed effect was used to enhance expression of destabilized variants of yellow fluorescent protein TurboYFP-dest with high degradation rate in mammalian cells. We believe that introduction of beta-globin intron in the 3'-UTR of the chimeric gene can be used to enhance its expression and may be advantageous in some cases when usage of 5'-UTR intron is inappropriate.

[Preparation of prokaryotic cDNA for high-throughput transcriptome analysis].

High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease. The method efficacy was demonstrated on a model experiments.

[Spectral diversity among the members of the family of Green Fluorescent Protein in hydroid jellyfish (Cnidaria, Hydrozoa)].

The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.

[A natural fluorescent protein that changes its fluorescence during maturation]. / Prirodnyi fluorestsentnyi belok, izmeniaiushchii tsvet fluorestsentsii v khode sozrevaniia.

The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.

[Key amino acid residues responsible for the color of green and yellow fluorescent proteins from the coral polyp Zoanthus]. / Vyiavlenie kliuchevykh aminokislotnykh ostatkov, opredeliaiushchikh tsvet zelenogo i zheltogo fluorestsentnykh belkov iz korallovogo polipa Zoanthus.

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Humoral autoimmunity markers in autoimmune endocrine diseases]. / Markery gumoralnogo autoimmuniteta pri autoimmunnykh éndokrinnykh zabolevaniiakh.

To clarify the value of autoantibodies as risk factors of complications in various endocrine abnormalities, the incidence of autoantibodies to thyroid microsomal antigen (ATMA), thyroglobulin, and the surface antigens of the rat islet, adrenal cortex, adenohypophyseal cells and human skin fibroblasts was studied in patients with insulin-dependent mellitus (IDDM), at the onset of the disease and during one-year insulin therapy, non-insulin-dependent diabetes mellitus (NIDDM), Hashimoto thyroiditis, Graves' disease, diabetes associated with thyroidal dysfunction, euthyroid polynodular goiter, Schmidt and polyglandular syndromes and in the population. The antibodies were determined by ELISA. Polyclonal activation of the immune system was found in all abnormalities, except in polyglandular in children. The proportion of patients with more than one type of antibodies was minimal (26.4%) in IDDM and maximal (62.0%) in Graves' disease. Among IDDM patients, polyclonal activation of the immune system was observed more often in women than in men (48.5 vs 8.5%). The persistence of antibodies to fibroblasts in IDDM patients was associated with the development of vascular complications. The latter were observed in 4 of 7 patients who had these antibodies during a year and in none of negative patients. Thus, fibroblast antibodies may have a predicative significance for the development of late diabetic complications. The highest prevalence of these antibodies was discovered in Graves' disease (37.9%) wherein the antibodies may be involved in the development of exophthalmus and pretibial mixedema. Thyroidal dysfunction developed in all IDDM patients with ATMA preserved during a year and in none ATMA-negative patients.(ABSTRACT TRUNCATED AT 250 WORDS)