[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.

[Synthesis of the chromophores of fluorescent proteins and their analogs].

Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4H-imidazol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroaminoacids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroaminoacids, and in the third part we summarize cyclizations of N-acyldehydroaminoacids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.

[Synthesis of biosynthetic precursors of red fluorescent proteins' chromophores].

A method for the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones, the corresponding chromophore of green fluorescent protein (GFP) with acylaminoalkyl substituents at position 2 of imidazole core have been developed. These structures represent biosynthetic precursors of the chromophores of red fluorescent proteins. The method is based on masking of the dehydrotyrosine fragment with beta-hydroxytyrosine moiety The key stages of the synthesis include peptide coupling of beta-hydroxytyrosine with the N-acetylamino acid of choice, unmasking of dehydrotyrosine by O-acylation with subsequent elimination, and cyclization of the obtained derivatives of 3-acylaminocinnamic acid in basic media.

[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.

[Genetically encoded photoimmunosensitizer].

Photosensitizer-antibody conjugates are successfully used for targeted elimination of cancer cells bearing specific membrane markers. This method is known as photoimmunotherapy. However, chemical conjugation of photosensitizer and antibody poses a number of complications such as low reproducibility, aggregation and unconjugated photosensitizer impurities. Here we report a fully genetically encoded photoimmunosensitizer, consisting of an anti-HER2/neu miniantibody 4D5scFv and a phototoxic fluorescent protein KillerRed. Both domains in this photoimmunosensitizer retained their functional qualities - high affinity for HER2/neu antigen and phototoxicity respectively. 4D5scFv-KillerRed fusion protein showed high specificity for HER2/neu-over-expressing cells and effectively lowered their viability upon illumination.

[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).

[Synthesis and properties of chromophores of fluorescent proteins].

We describe the existing approaches to the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones - model chromophores of fluorescent proteins and their nonnatural analogs. We discuss in detail the chemical (acid-base and redox reactions, cis-trans isomery, etc.) and spectral properties of the chromophores and the influence of substitutes and the environment. The study of synthetic chromophores allows for modeling of the photophysical characteristics of fluorescent proteins.

[Chromoproteins of the green fluorescent protein family: properties and applications].

The distribution in nature and the spectral and structural properties of chromoproteins of the green fluorescent protein (GFP) family and their differences from one another and other fluorescent proteins of this family are considered. Discussed in detail are practical applications of the chromoproteins and their mutant variants that have unique characteristics not found among natural proteins of the GFP family, such as far-red or photoconvertible fluorescence, a large Stokes shift, enhanced phototoxicity, etc.

[The ability of green fluorescent proteins for photoconversion under oxygen-free conditions is determined by the chromophore structure rather than its amino acid environment].

Photoconversion of various green and cyan fluorescent proteins to the form fluorescing in the red area of the visible spectrum under the oxygen-free conditions was studied. Such photoconversion has earlier been described for the EGFP green fluorescent protein. Phylogenetically distant fluorescent proteins that have a low homology of their amino acid sequences but contain chemically identical chromophores based on a Tyr residue were shown to be susceptible to this type of photoconversion. At the same time, the ECFP protein, which has 92% homology with EGFP but contains a chromophore based on tryptophan did not undergo the photoconversion. Thus, it is precisely the chromophore structure, rather than the amino acid environment that determines the ability of green fluorescent proteins to display photoconversion to the red fluorescent state under anaerobic conditions.

[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.

[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.

[An analysis of the expression of the genes containing the LeR-1 and VeR-1 sequences in the embryogenesis, regeneration and in the intact tissues of newts]. / Analiz ékspressii genov, soderzhashchikh posledovatel'nosti LeR-1 i VeR-1 v émbriogeneze, pri regeneratsii i v intaktnykh tkaniakh u tritonov.

A method of construction of amplified cDNA libraries was developed on the basis of selective inhibition of cDNA amplification and modified for the studied models for analysis of expression of the genes containing LeR-1 and VeR-1 sequences. Time-related changes in expression of these genes were studied during regeneration of the adult lens and during embryogenesis of newts. The pattern of expression of the LeR-1 and VeR-1 genes proved to be not tissue-specific. A fragment adjoining 5'-area of the LeR-1 gene was obtained using a new approach: rapid amplification of cDNA ends by polymerase chain reaction (5'-RACE-PCR). Analysis of the LeR-1 clone primary structure using Gene Bank did not show essential homology with the known nucleotide sequences. Sequence LeR-1 is characterized by evolutionary conservation of genomic DNA in Pleurodeles waltlii, Xenopus laevis, and Rana temporaria.

[In vitro cloning of DNA fragments by one polymerase chain reaction]. / Klonirovanie fragmentov DNK in vitro za odnu polimeraznuiu tsepnuiu reaktsiiu.

An improved version of the in vitro DNA cloning method described earlier is proposed. The method allows amplification by polymerase chain reaction (PCR) of individual DNA molecules of an unknown primary structure followed by sequencing. The modifications described here provide for in vitro cloning by means of a 40-45-cycle PCR (the original protocol required two consecutive amplifications). In addition, the in vitro cloning is suggested to be carried out in special 96-well plates in the presence of ethidium bromide; upon UV irradiation, the wells containing amplified DNA fluoresce to make the analysis of all 96 wells unnecessary. The improved protocol makes the preparation of individual in vitro clones more straightforward and less expensive.

[A method for obtaining the normalized cDNA libraries based on the effect of suppression of polymerase chain reaction]. / Metod polucheniia normalizovannykh bibliotek kDNK, osnovannyi na éffekte supressii polimeraznoi tsepnoi reaktsii.

A new efficient method for obtaining cDNA libraries with equal representation of all cDNA types (equalized libraries) in a single round of equalization was developed. The method is based on differences in the renaturation kinetics of double-stranded cDNAs of different genes and allow the selection of the equalized single-stranded fraction resulted from the incomplete reassociation of the total cDNA without laborious and inefficient physical separation. The equalized single-stranded fractions are selectively amplified by polymerase chain reaction (PCR). The amplification of other DNA molecules is inhibited due to PCR suppression, i.e. the suppression of amplification of the DNA molecules flanked with long interval terminal repeats in PCR with a primer corresponding to the external moiety of the repeat. The efficiency of the developed method was estimated in obtaining an equalized cDNA library based on mRNA from the activated human T lymphocyte Jurkat cell line.

[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.

[The cloning of fragments of homeobox genes expressed during planarian regeneration]. / Klonirovanie fragmentov gomeobokssoderzhashchikh genov, ékspressiruiushchikhsia v khode regeneratsii planarii.

The polymerase chain reaction with degenerate primers corresponding to the most conservative amino acids 16-21 (ELEKEF) and 49-54 (WFQNRR) of the Antennapedia class homeodomain was used for the amplification of cDNA from regenerating planarians (asexual race of Dugesia tigrina). A total of six new Antennapedia-like homeobox sequences, designated Dutarh-1-Dutarh-6 (Dugesia tigrina asexual race homeobox gene), were obtained. Their comparison with other homeobox genes using a "Genebee" software (the EMBL Data Library) showed that all sequences except Dutarh-6 belong to the Antennapedia class. Dutarh-6 is closely related to a recently described novel homeobox gene subfamily that includes mouse mesodermal homeobox genes Mox-1 and Mox-2 and rat homeobox gene Gax.

[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.