[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.

[Coding region of far-red fluorescent protein katushka contains a strong donor splice site].

Computer analysis predicted a strong donor splice site within the 3'-part of the far-red fluorescent protein Katushka coding region. To test the functional activity of this site a model vector has been constructed. This vector encoded Katushka and green fluorescent protein TagGFP2 with a gene fragment of tafazzin in between. Normal splicing of this pre-mRNA should result in a frameshift between Katushka and TagGFP2. Alternatively, after splicing at internal katushka donor splice site appearance of Katushka-TagGFP2 fusion protein was expected. Expression of this construct in a mammalian cell culture led to bright red and green fluorescence. Therefore, katushka-specific donor splice site is functional. Disruption of this splice site by several silent substitutions resulted in red-only fluorescent cells that corresponded to normal splicing. The mutant katushka can be used for visualization of pre-mRNA splicing at single cell level by fluorescence microscopy and flow cytometry.

[Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP protein].

The site-directed mutagenesis of the monomeric red fluorescent protein TagRFP and its variants was performed with the goal of generating reversibly photoactivatable fluorescent proteins. Amino acids at positions 69, 148, 165, 179, and 181 (enumeration according to the green fluorescent protein GFP) were shown to play a key role in the manifestation of the photoactivatable properties. A reversibly photoactivatable red fluorescent protein KFP-HC with excitation and emission maxima at 585 and 615 nm, respectively, was generated. The KFP-HC fluorescent intensity was decreased by 5-10 times under green light (530-560 nm) irradiation (due to the fall of the fluorescence quantum yield) and restored under irradiation with blue light (450-490 nm) or after incubation in the dark (time of half reconstruction of 30 min).

[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 family of genes of multidomain free lectins from a planarian: structure, expression, and use as markers for regeneration monitoring ]. / Semeistvo genov multidomennykh svobodnykh lektinov planarii: struktura, ékspressiia i ispol'zovanie v kachestve markerov dlia nabliudeniia za regeneratsiei.

A family of genes of the agamic race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[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.

[Selective suppression of polymerase chain reaction]. / Selektivnaia supressiia polimeraznoi tsepnoi reaktsii.

The selective suppression of the polymerase chain reaction and methods based upon it (construction of cDNA libraries from low amounts of biological material, subtractive hybridization and differential display of mRNA, fast cloning of full-size cDNA, chromosome walking, cloning in vitro, and others) are reviewed. These methods display a high effectiveness and, taken together, enable intricate DNA analyses to be performed--from the search for nontrivial sequences to the total sequencing of the corresponding genes.

[Cloning cDNA for the ha-SDGF gene from a Syrian hamster cell line with increased metastatic potential using subtractive hybridization]. / Klonirovanie s pomoshch'iu vychitaiushchei gibridizatsii kDNK gena ha-SDGF iz linii kletok siriiskogo khomiachka s povyshennym potentsialom metastazirovaniia.

Using subtractive hybridization, a cDNA library containing over 50% of clones specific for a highly metastatic cell line was obtained from two hamster embryo fibroblast lines with different metastatic potentials. Most of the clones (83%) contained new sequences. One clone contained the ha-SDGF gene cDNA homologous to SDGF cDNA from rodents. The level of ha-SDGF mRNA expression was considerably higher in the highly metastatic cell line.

[Selective amplification of evolutionally conserved expressed sequences]. / Selektivnaia amplifikatsiia évoliutsionno konservativnykh ékspressiruiushchikhsia posledovatel'nostei.

An efficacious method of cloning the sequences common for two cDNAs was proposed. The method was used for constructing a library of expressed sequences evolutionarily conserved for human and hamster.

[Highly-effective subtractive hybridization of cDNA]. / Vysokoéffektivnaia vychitaiushchaia gibridizatsiia kDNK.

A scheme for subtractive hybridization is described allowing for a 500-1000 fold enrichment of low abundant cDNA. The scheme is based on the previously described principle of normalization of an initial mixture of differently represented cDNAs in the single-stranded portion of a tracer after the first round of subtraction and the principle of a trapper excluding the fraction of the double-stranded cDNAs formed during the first round from the subsequent PCR-amplification. The technique is simple and makes unnecessary the separation of the tracer, driver and hybrids formed after the subtraction.