Образование

Избранные публикации

1. Gurskaya N.G., Staroverov D.B., Lukyanov K.A. (2016). Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells. Meth. Enzymol. 572, 255–68 [+]

   Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines.

   ID:1527
2. Gurskaya N.G., Pereverzev A.P., Staroverov D.B., Markina N.M., Lukyanov K.A. (2016). Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins. Meth. Enzymol. 572, 291–314 [+]

   Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mechanism of specific degradation of transcripts with a premature stop codon. NMD eliminates aberrant mRNAs arising from mutations, alternative splicing, and other events in cells. In addition, many normal transcripts undergo NMD. Recent studies demonstrated that NMD activity is specifically regulated and that NMD can play a role of global regulator of gene expression. Recently, we developed dual-color fluorescent protein-based reporters for quantification of NMD activity using fluorescence microscopy and flow cytometry (Pereverzev, Gurskaya, et al., 2015). Due to ratiometric fluorescence response, these reporters make it possible to assess NMD activity in live cells at the single-cell level and to reveal otherwise hidden heterogeneity of cells in respect of NMD activity. Here we provide a detailed description of applications of the NMD reporters in mammalian cell lines.

   ID:1528
3. Pereverzev A.P., Matlashov M.E., Staroverov D.B., Lukyanov K.A., Gurskaya N.G. (2015). Differences of Nonsense-Mediated mRNA Degradation Activity in Mammalian Cell Lines Revealed by a Fluorescence Reporter. Bioorg. Khim. 41 (5), 587–91 [+]

   Activity of nonsense-mediated mRNA degradation (NMD) was studied in several mammalian cell cultures using recently developed genetically encoded fluorescence sensor [Pereverzev et al., Sci. Rep., 2015, vol. 5, p. 7729]. This NMD reporter enables measurement of NMD activity in single live cells using ratio of green and red fluorescent proteins signals. The following cell lines were analyzed: mouse colon carcinoma CT26, mouse Lewis lung carcinoma LLC, human T-cell leukemia Jurkat, and spontaneously immortalized human keratinocytes HaCaT. These cell lines demonstrated very different NMD activities. In CT26, NMD activity was low, whereas in LLC it was high (8.5-fold higher than in CT26). Jurkat and HaCaT cells possessed strong heterogeneity and consisted of two cell subpopulations with high and low NMD activities. In addition, we detected high NMD activity in primary culture of mouse embryonic hippocampal neurons.

   ID:1416
4. Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G., Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5, 7729 [+]

   Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.

   ID:1247
5. Переверзев А.П., Маркина Н.М., Янушевич Ю.Г., Городничева Т.В., Минасян Б.Э., Лукьянов К.А., Гурская Н.Г. (2013). УСИЛЕНИЕ ЭКСПРЕССИИ ХИМЕРНЫХ ГЕНОВ ВКЛЮЧЕНИЕМ В ИХ 3’-НЕТРАНСЛИРУЕМУЮ ОБЛАСТЬ ИНТРОНА 2 ГЕНА БЕТА-ГЛОБИНА ЧЕЛОВЕКА. Биоорг. хим. 40 (3), 293–296 [+]

   Исследована возможность увеличения гетерологичной экспрессии в клетках млекопитающих при включении интронов в 3'нетранслируемую область cоответствующего гена. Для этого в экспресси онный вектор, содержащий ген флуоресцентного белка TagGFP2, вводили интрон 2 гена βглобина человека. Данная последовательность встраивалась после стопкодона TagGFP2 на расстоянии 35 нт. от стопкодона до сайта сплайсинга. Это позволило избежать процесса нонсенсопосредован ной деградации мРНК. Использование второй рамки считывания, кодирующей красный флуорес центный белок Katushka, позволило нормировать уровень трансфекции и экспрессии химерных конструкций. Проточная цитофлуориметрия культуры клеток HEK293T, временно трансфициро ванных генетическими конструкциями, содержащими и не содержащими интрон, позволила вы явить увеличение интенсивности флуоресценции в зеленом канале для конструкции с интроном в 1.8 ± 0.2 раза относительно контрольной безинтронной конструкции. Аналогичное усиление уровня экспрессии в 1.7 ± 0.2 раза при наличии интрона 2 βглобина 3'нетранслируемой области было показа но также для гена, кодирующего дестабилизированный вариант флуоресцентного белка TurboYFP. Эффект увеличения уровня экспрессии химерных конструкций в клетках млекопитающих введени ем интрона в 3'нетранслируемую область целевого гена можно использовать в различных модель ных системах, где введение 5'концевого интрона в последовательность является неприемлeмым.

   ID:1272
6. Gurskaya N.G., Staroverov D.B., Zhang L., Fradkov A.F., Markina N.M., Pereverzev A.P., Lukyanov K.A. (2012). Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins. Nucleic Acids Res. 40 (8), e57 [+]

   Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

   ID:1271
7. Pletneva N.V., Pletnev V.Z., Lukyanov K.A., Gurskaya N.G., Goryacheva E.A., Martynov V.I., Wlodawer A., Dauter Z., Pletnev S. (2010). Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis. J. Biol. Chem. 285 (21), 15978–84 [+]

   The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (lambda(ex) = 480 nm, lambda(em) = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C(alpha) and C(beta) atoms in the sp(3) hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (lambda = 250-300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C(alpha)=C(beta) bond and the conjugated system of pi-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.

   ID:404
8. Pletnev S., Gurskaya N.G., Pletneva N.V., Lukyanov K.A., Chudakov D.M., Martynov V.I., Popov V.O., Kovalchuk M.V., Wlodawer A., Dauter Z., Pletnev V. (2009). Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. J. Biol. Chem. 284 (46), 32028–39 [+]

   KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

   ID:299
9. Zhang L., Gurskaya N.G., Kopantseva Y.E., Mudrik N.N., Vagner L.L., Lukyanov K.A., Chudakov D.M. (2009). [Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP protein]. Bioorg. Khim. 36 (2), 187–92 [+]

   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).

   ID:361
10. Gurskaya N.G., Verkhusha V.V., Shcheglov A.S., Staroverov D.B., Chepurnykh T.V., Fradkov A.F., Lukyanov S., Lukyanov K.A. (2006). Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24 (4), 461–5 [+]

    Разработан новый мономерный флуоресцентный белок Dendra, способный к необратимой фотоконверсии из зеленой флуоресцентной формы в красную. Белок Dendra обладает высокой яркостью флуоресценции и может быть активирован как ультрафиолетовым, так и синим светом.

    ID:81
11. Shkrob M.A., Yanushevich Y.G., Chudakov D.M., Gurskaya N.G., Labas Y.A., Poponov S.Y., Mudrik N.N., Lukyanov S., Lukyanov K.A. (2005). Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J. 392 (Pt 3), 649–54 [+]

    Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

    ID:287
12. Bulina M.E., Lukyanov K.A., Yampolsky I.V., Chudakov D.M., Staroverov D.B., Shcheglov A.S., Gurskaya N.G., Lukyanov S. (2004). New class of blue animal pigments based on Frizzled and Kringle protein domains. J. Biol. Chem. 279 (42), 43367–70 [+]

    The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds.

    ID:290
13. Pakhomov A.A., Martynova N.Y., Gurskaya N.G., Balashova T.A., Martynov V.I. (2004). Photoconversion of the chromophore of a fluorescent protein from Dendronephthya sp. Biochemistry Mosc. 69 (8), 901–8 [+]

    A green fluorescent protein from the coral Dendronephthya sp. (Dend FP) is characterized by an irreversible light-dependent conversion to a red-emitting form. The molecular basis of this phenomenon was studied in the present work. Upon UV-irradiation at 366 nm, the absorption maximum of the protein shifted from 494 nm (the green form) to 557 nm (the red form). Concurrently, in the fluorescence spectra the emission maximum shifted from 508 to 575 nm. The green form of native Dend FP was shown to be a dimer, and the oligomerization state of the protein did not change during its conversion to the red form. By contrast, UV-irradiation caused significant intramolecular changes. Unlike the green form, which migrates in SDS-polyacrylamide gels as a single band corresponding to a full-length 28-kD protein, the red form of Dend FP migrated as two fragments of 18- and 10-kD. To determine the chemical basis of these events, the denatured red form of Dend FP was subjected to proteolysis with trypsin. From the resulting hydrolyzate, a chromophore-containing peptide was isolated by HPLC. The structure of the chromophore from the Dend FP red form was established by methods of ESI, tandem mass spectrometry (ESI/MS/MS), and NMR-spectroscopy. The findings suggest that the light-dependent conversion of Dend FP is caused by generation of an additional double bond in the side chain of His65 and a resulting extension of the conjugated system of the green form chromophore. Thus, classified by the chromophore structure, Dend FP should be referred to the Kaede subfamily of GFP-like proteins.

    ID:820
14. Verkhusha V.V., Chudakov D.M., Gurskaya N.G., Lukyanov S., Lukyanov K.A. (2004). Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins. Chem. Biol. 11 (6), 845–54 [+]

    The mechanism of the chromophore maturation in members of the green fluorescent protein (GFP) family such as DsRed and other red fluorescent and chromoproteins was analyzed. The analysis indicates that the red chromophore results from a chemical transformation of the protonated form of the GFP-like chromophore, not from the anionic form, which appears to be a dead-end product. The data suggest a rational strategy to achieve the complete red chromophore maturation utilizing substitutions to favor the formation of the neutral phenol in GFP-like chromophore. Our approach to detect the neutral chromophore form expands the application of fluorescent timer proteins to faster promoter activities and more spectrally distinguishable fluorescent colors. Light sensitivity found in the DsRed neutral form, resulting in its instant transformation to the mature red chromophore, could be exploited to accelerate the fluorescence acquisition.

    ID:291
15. Martynov V.I., Maksimov B.I., Martynova N.Y., Pakhomov A.A., Gurskaya N.G., Lukyanov S.A. (2003). A purple-blue chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins. J. Biol. Chem. 278 (47), 46288–92 [+]

    A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).

    ID:819
16. Gurskaya N.G., Fradkov A.F., Pounkova N.I., Staroverov D.B., Bulina M.E., Yanushevich Y.G., Labas Y.A., Lukyanov S., Lukyanov K.A. (2003). A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem. J. 373 (Pt 2), 403–8 [+]

    We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

    ID:1402
17. Labas Y.A., Gurskaya N.G., Yanushevich Y.G., Fradkov A.F., Lukyanov K.A., Lukyanov S.A., Matz M.V. (2002). Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. U.S.A. 99 (7), 4256–61 [+]

    The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.

    ID:1401
18. Gurskaya N.G., Fradkov A.F., Terskikh A., Matz M.V., Labas Y.A., Martynov V.I., Yanushevich Y.G., Lukyanov K.A., Lukyanov S.A. (2001). GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507 (1), 16–20 [+]

    We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission lambda(max)=615-640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.

    ID:827
19. Gurskaya N.G., Savitsky A.P., Yanushevich Y.G., Lukyanov S.A., Lukyanov K.A. (2001). Color transitions in coral's fluorescent proteins by site-directed mutagenesis. BMC Biochem. 2, 6 [+]

    Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters.

    ID:1400
20. Rebrikov D.V., Britanova O.V., Gurskaya N.G., Lukyanov K.A., Tarabykin V.S., Lukyanov S.A. (2000). Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization. Nucleic Acids Res. 28 (20), E90 [+]

    Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.

    ID:1399
21. Lukyanov K.A., Fradkov A.F., Gurskaya N.G., Matz M.V., Labas Y.A., Savitsky A.P., Markelov M.L., Zaraisky A.G., Zhao X., Fang Y., Tan W., Lukyanov S.A. (2000). Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275 (34), 25879–82 [+]

    It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

    ID:560
22. Gurskaya N.G., Diatchenko L., Chenchik A., Siebert P.D., Khaspekov G.L., Lukyanov K.A., Vagner L.L., Ermolaeva O.D., Lukyanov S.A., Sverdlov E.D. (1996). Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal. Biochem. 240 (1), 90–7 [+]

    The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.

    ID:1403
23. Diatchenko L., Lau Y.F., Campbell A.P., Chenchik A., Moqadam F., Huang B., Lukyanov S., Lukyanov K., Gurskaya N., Sverdlov E.D., Siebert P.D. (1996). Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A. 93 (12), 6025–30 [+]

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

    ID:1404