Лукьянов Сергей Анатольевич

Научные интересы

Основные научные интересы С. А. Лукьянова лежат в области анализа структуры и функции геномов эукариот.

Премии и заслуги

С. А. Лукьянов является лауреатом премии Президиума РАН им. академика Ю. А. Овчинникова за выдающиеся работы в области физико-химической биологии и биотехнологии за 2006 год за работу «Флуоресцентные белки: поиск, исследование и применение в биотехнологии», премий Международной академической издательской компании «Наука» за лучшую публикацию в издаваемых ею журналах за 1996 и г., победителем конкурсов журнала Биоорганическая Химия на лучшую статью года за 1996, 1997 и г., лауреатом программы «Выдающиеся ученые, молодые доктора и кандидаты наук» и конкурса на присуждение Государственных научных стипендий для ученых.

Основные научные результаты

Методы, разработанные на основе открытого С. А. Лукьяновым эффекта селективной супрессии полимеразной цепной реакции, а также методы с использованием дуплекс-специфической нуклеазы, выделенной в лаборатории С. А. Лукьянова в сотрудничестве с лабораторией морской биохимии Тихоокеанского института биоорганической химии (зав. лабораторией д. б. н. Рассказов В. А.), активно используется в настоящее время в российских и зарубежных лабораториях. Работы, посвященные исследованию флуоресцентных белков, получили мировое признание и перевели на качественно новый уровень технологии прижизненного мечения.

Избранные публикации

  1. Britanova O.V., Shugay M., Merzlyak E.M., Staroverov D.B., Putintseva E.V., Turchaninova M.A., Mamedov I.Z., Pogorelyy M.V., Bolotin D.A., Izraelson M., Davydov A.N., Egorov E.S., Kasatskaya S.A., Rebrikov D.V., Lukyanov S., Chudakov D.M. (2016). Dynamics of Individual T Cell Repertoires: From Cord Blood to Centenarians. J. Immunol. 196 (12), 5005–13 [+]

    The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRβ repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.

  2. Shirmanova M.V., Druzhkova I.N., Lukina M.M., Matlashov M.E., Belousov V.V., Snopova L.B., Prodanetz N.N., Dudenkova V.V., Lukyanov S.A., Zagaynova E.V. (2015). Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2. Biochim. Biophys. Acta 1850 (9), 1905–11 [+]

    Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2.

  3. Matlashov M.E., Bogdanova Y.A., Ermakova G.V., Mishina N.M., Ermakova Y.G., Nikitin E.S., Balaban P.M., Okabe S., Lukyanov S., Enikolopov G., Zaraisky A.G., Belousov V.V. (2015). Fluorescent ratiometric pH indicator SypHer2: applications in neuroscience and regenerative biology. Biochim. Biophys. Acta 1850 (11), 2318–2328 [+]


    SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use.



    Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer.



    Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.



    SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.



    The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.


  4. Yuzhakova D.V., Shirmanova M.V., Serebrovskaya E.O., Lukyanov K.A., Druzhkova I.N., Shakhov B.E., Lukyanov S.A., Zagaynova E.V. (2015). CT26 murine colon carcinoma expressing the red fluorescent protein KillerRed as a highly immunogenic tumor model. J Biomed Opt 20 (8), 88002 [+]

    The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.

  5. Purtov K.V., Petushkov V.N., Baranov M.S., Mineev K.S., Rodionova N.S., Kaskova Z.M., Tsarkova A.S., Petunin A.I., Bondar V.S., Rodicheva E.K., Medvedeva S.E., Oba Y., Arseniev A.S., Lukyanov S., Gitelson J.I., Yampolsky I.V. (2015). The Chemical Basis of Fungal Bioluminescence. Angew. Chem. Int. Ed. 127 (28), 8242–8246 [+]

    Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

  6. Mishina N.M., Mishin A.S., Belyaev Y., Bogdanova E.A., Lukyanov S., Schultz C., Belousov V.V. (2015). Live-Cell STED Microscopy with Genetically Encoded Biosensor. Nano Lett. 15 (5), 2928–2932 [+]

    Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.

  7. Билан Д.С., Шохина А.Г., Лукьянов С.А., Белоусов В.В. (2015). Основные редокс-пары клетки. Биоорг. хим. 41 (4), 385–402 [+]

    Большая часть живых клеток поддерживает непрерывный поток электронов, обеспечивая себя энергией. Многие соединения представлены в клетке одновременно в окисленном и восстановленном состояниях, формируя таким образом активные редокс-пары. Некоторые из редокс-пар, например, NAD+/NADH, NADP+/NADPH, окисленный/восстановленный глутатион (GSSG/GSH) – универсальны, поскольку они одновременно участвуют в регуляции многих клеточных реакций. Соотношения окисленной и восстановленной форм этих соединений являются важными клеточными редокс показателями. Современные подходы исследований позволяют устанавливать новые функции основных редокс-пар в сложной организации клеточных процессов. В обзоре представлена информация об основных редокспарах клетки, рассмотрено их участие в различных биологических процессах.

  8. 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.

  9. Pletneva N.V., Pletnev V.Z., Souslova E., Chudakov D.M., Lukyanov S., Martynov V.I., Arhipova S., Artemyev I., Wlodawer A., Dauter Z., Pletnev S. (2013). Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 6), 1005–12 [+]

    The yellow fluorescent protein phiYFPv (λem(max) ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow-orange range (535-555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The `yellow' chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  10. Bilan D.S., Pase L., Joosen L., Gorokhovatsky A.Y., Ermakova Y.G., Gadella T.W., Grabher C., Schultz C., Lukyanov S., Belousov V.V. (2013). HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem. Biol. 8 (3), 535–42 [+]

    High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.

  11. Mishina N.M., Markvicheva K.N., Fradkov A.F., Zagaynova E.V., Schultz C., Lukyanov S., Belousov V.V. (2013). Imaging H2O2 microdomains in receptor tyrosine kinases signaling. Meth. Enzymol. 526, 175–87 [+]

    HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation.

  12. Mishina N.M., Markvicheva K.N., Bilan D.S., Matlashov M.E., Shirmanova M.V., Liebl D., Schultz C., Lukyanov S., Belousov V.V. (2013). Visualization of intracellular hydrogen peroxide with HyPer, a genetically encoded fluorescent probe. Meth. Enzymol. 526, 45–59 [+]

    The fluorescent sensor HyPer allows monitoring of intracellular H2O2 levels with a high degree of sensitivity and specificity. Here, we provide a detailed protocol of ratiometric imaging of H2O2 produced by cells during phagocytosis, including instructions for experiments on different commercial confocal systems, namely, Leica SP2, Leica SP5, and Carl Zeiss LSM, as well as wide-field Leica 6000 microscope. The general experimental scheme is easily adaptable for imaging H2O2 production by various cell types under a variety of conditions.

  13. Pletnev S., Pletneva N.V., Souslova E.A., Chudakov D.M., Lukyanov S., Wlodawer A., Dauter Z., Pletnev V. (2012). Structural basis for bathochromic shift of fluorescence in far-red fluorescent proteins eqFP650 and eqFP670. Acta Crystallogr. D Biol. Crystallogr. 68 (Pt 9), 1088–97 [+]

    The crystal structures of the far-red fluorescent proteins (FPs) eqFP650 (λ(ex)(max)/λ(em)(max) 592/650 nm) and eqFP670 (λ(ex)(max)/λ(em)(max) 605/670 nm), the successors of the far-red FP Katushka (λ(ex)(max)/λ(em)(max) 588/635 nm), have been determined at 1.8 and 1.6 Å resolution, respectively. An examination of the structures demonstrated that there are two groups of changes responsible for the bathochromic shift of excitation/emission bands of these proteins relative to their predecessor. The first group of changes resulted in an increase of hydrophilicity at the acylimine site of the chromophore due to the presence of one and three water molecules in eqFP650 and eqFP670, respectively. These water molecules provide connection of the chromophore with the protein scaffold via hydrogen bonds causing an ∼15 nm bathochromic shift of the eqFP650 and eqFP670 emission bands. The second group of changes observed in eqFP670 arises from substitution of both Ser143 and Ser158 by asparagines. Asn143 and Asn158 of eqFP670 are hydrogen bonded with each other, as well as with the protein scaffold and with the p-hydroxyphenyl group of the chromophore, resulting in an additional ∼20 nm bathochromic shift of the eqFP670 emission band as compared to eqFP650. The role of the observed structural changes was verified by mutagenesis.

  14. Mishina N.M., Bogeski I., Bolotin D.A., Hoth M., Niemeyer B.A., Schultz C., Zagaynova E.V., Lukyanov S., Belousov V.V. (2012). Can we see PIP(3) and hydrogen peroxide with a single probe? Antioxid. Redox Signal. 17 (3), 505–12 [+]

    A genetically encoded sensor for parallel measurements of phosphatidylinositol 3-kinase activity and hydrogen peroxide (H(2)O(2)) levels (termed PIP-SHOW) was developed. Upon elevation of local phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) concentration, the sensor translocates from the cytosol to the plasma membrane, while a ratiometric excitation change rapidly and simultaneously reports changes in the concentration of H(2)O(2). The dynamics of PIP(3) and H(2)O(2) generation were monitored in platelet-derived growth factor-stimulated fibroblasts and in T-lymphocytes after formation of an immunological synapse. We suggest that PIP-SHOW can serve as a prototype for many fluorescent sensors with combined readouts.

  15. Bolotin D.A., Mamedov I.Z., Britanova O.V., Zvyagin I.V., Shagin D., Ustyugova S.V., Turchaninova M.A., Lukyanov S., Lebedev Y.B., Chudakov D.M. (2012). Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms. European journal of immunology , [+]

    The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of NGS applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

  16. Britanova O.V., Bochkova A.G., Staroverov D.B., Fedorenko D.A., Bolotin D.A., Mamedov I.Z., Turchaninova M.A., Putintseva E.V., Kotlobay A.A., Lukyanov S., Novik A.A., Lebedev Y.B., Chudakov D.M. (2012). First autologous hematopoietic SCT for ankylosing spondylitis: a case report and clues to understanding the therapy. Bone marrow transplantation , ID:732
  17. Shemiakina I.I., Ermakova G.V., Cranfill P.J., Baird M.A., Evans R.A., Souslova E.A., Staroverov D.B., Gorokhovatsky A.Y., Putintseva E.V., Gorodnicheva T.V., Chepurnykh T.V., Strukova L., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M., Shcherbo D. (2012). A monomeric red fluorescent protein with low cytotoxicity. Nat Commun 3, 1204 [+]

    Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.

  18. Sarkisyan K.S., Yampolsky I.V., Solntsev K.M., Lukyanov S.A., Lukyanov K.A., Mishin A.S. (2012). Tryptophan-based chromophore in fluorescent proteins can be anionic. Sci Rep 2, 608 [+]

    Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with an anionic chromophore and key substitution Val61Lys was obtained. This is the first evidence strongly suggesting that tryptophan-based chromophores in fluorescent proteins can exist in an anionic charged state. Switching between protonated and deprotonated Trp66 in fluorescent proteins represents a new unexplored way to control their spectral properties.

  19. Ivashkin P.E., Lukyanov K.A., Lukyanov S., Yampolsky I.V. (2011). A synthetic GFP-like chromophore undergoes base-catalyzed autoxidation into acylimine red form. J. Org. Chem. 76 (8), 2782–91 [+]

    Fluorescent proteins are widely used in modern experimental biology, but much controversy exists regarding details of maturation of different types of their chromophores. Here we studied possible mechanisms of DsRed-type red chromophore formation using synthetic biomimetic GFP-like chromophores, bearing an acylamino substituent, corresponding to an amino acid residue at position 65. We have shown these model compounds to readily react with molecular oxygen to produce a highly unstable DsRed-like acylimine, isolated in the form of stable derivatives. Under the same aerobic conditions an unusual red-shifted imide chromophore--a product of 4-electron oxidation of Gly65 residue--is formed. Our data showed that GFP chromophore is prone to autoxidation at position 65 Cα by its chemical nature with basic conditions being the only key factor required.

  20. Markvicheva K.N., Bilan D.S., Mishina N.M., Gorokhovatsky A.Y., Vinokurov L.M., Lukyanov S., Belousov V.V. (2011). A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg. Med. Chem. 19 (3), 1079–84 [+]

    Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.

  21. Mishina N.M., TyurinKuzmin P.A., Markvicheva K.N., Vorotnikov A.V., Tkachuk V.A., Laketa V., Schultz C., Lukyanov S., Belousov V.V. (2011). Does cellular hydrogen peroxide diffuse or act locally? Antioxid. Redox Signal. 14 (1), 1–7 [+]

    Understanding of redox signaling requires data on the spatiotemporal distribution of hydrogen peroxide (H(2)O(2)) within the cell. The fluorescent reporter HyPer is a powerful instrument for H(2)O(2) imaging. However, rapid diffusion of HyPer throughout the nucleocytoplasmic compartment does not allow visualization of H(2)O(2) gradients on the micrometer scale. Here we dramatically improved the spatial resolution of H(2)O(2) imaging by applying subcytoplasmic targeting of HyPer. The membrane-attached reporters identified "microdomains" of elevated H(2)O(2) levels within the cytoplasm of the cells exposed to growth factors. We demonstrate that diffusion of H(2)O(2) across the cytoplasm was strongly limited, providing evidence that H(2)O(2) acts locally inside cells.

  22. Shagina I., Bogdanova E., Mamedov I., Lebedev Y., Lukyanov S., Shagin D. (2010). Normalization of genomic DNA using duplex-specific nuclease. BioTechniques 48 (6), 351–355 [+]

    An application of duplex-specific nuclease (DSN) normalization technology to whole-genome shotgun sequencing of genomes with a large proportion of repetitive DNA is described. The method uses a thermostable DSN from the Kamchatka crab that specifically hydrolyzes dsDNA. In model experiments on human genomic DNA, we demonstrated that DSN normalization of double-stranded DNA formed during C0t analysis is effective against abundant repetitive sequences with high sequence identity, while retaining highly divergent repeats and coding regions at baseline levels. Thus, DSN normalization applied to C0t analysis can be used to eliminate evolutionarily young repetitive elements from genomic DNA before sequencing, and should prove invaluable in studies of large eukaryotic genomes, such as those of higher plants.

  23. Bogdanov A.M., Bogdanova E.A., Chudakov D.M., Gorodnicheva T.V., Lukyanov S., Lukyanov K.A. (2009). Cell culture medium affects GFP photostability: a solution. Nat. Methods 6 (12), 859–60 ID:298
  24. Bogdanov A.M., Mishin A.S., Yampolsky I.V., Belousov V.V., Chudakov D.M., Subach F.V., Verkhusha V.V., Lukyanov S., Lukyanov K.A. (2009). Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol.  (5), 459–461 [+]

    Proteins of the green fluorescent protein (GFP) family are well known owing to their unique biochemistry and extensive use as in vivo markers. We discovered that GFPs of diverse origins can act as light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, via green-to-red GFP photoconversion, this process can be observed in living cells without additional treatment.

  25. Shcherbo D., Murphy C.S., Ermakova G.V., Solovieva E.A., Chepurnykh T.V., Shcheglov A.S., Verkhusha V.V., Pletnev V.Z., Hazelwood K.L., Roche P.M., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M. (2009). Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418 (3), 567–74 [+]

    Разработан яркий, мономерный, фотостабильный, pH-стабильный, дальне-красный флуоресцентый белок mKate2. Белок mKate2 хорошо показал себя в качестве метки во фьюзах с рядом белков, как в культуре клеток, так и в трансгенных лягушках Xenopus laevis (совместно с лабораторией Молекулярных основ эмбриогенеза ИБХ РАН).

  26. Yampolsky I.V., Kislukhin A.A., Amatov T.T., Shcherbo D., Potapov V.K., Lukyanov S., Lukyanov K.A. (2008). Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs. Bioorg. Chem. 36 (2), 96–104 [+]

    Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

  27. Markvicheva K.N., Bogdanova E.A., Staroverov D.B., Lukyanov S., Belousov V.V. (2008). Imaging of intracellular hydrogen peroxide production with HyPer upon stimulation of HeLa cells with epidermal growth factor. Methods Mol. Biol. 476, 79–86 [+]

    Reactive oxygen species (ROS) regulate both normal cell functions by activating a number of enzymatic cascades and pathological processes in many diseases by inducing oxidative stress. For many years since the discovery of ROS in biological systems, there were no adequate methods of detection and quantification of these molecules inside the living cells. We developed the first genetically encoded fluorescent indicator for the intracellular detection of hydrogen peroxide, HyPer, that can be used for imaging of H2O2 production by cells under various physiological and pathological conditions. Unlike most known ROS indicators, HyPer allows the generation of a real-time image series that give precise information about the time course and intensity of H2O2 changes in any compartment of interest. In this chapter, we describe the method of confocal imaging of hydrogen peroxide production in HeLa cells upon stimulation with epidermal growth factor. The technique described may be accepted with minimal variations for the use in other cell lines upon various conditions leading to H2O2, production.

  28. Shcherbo D., Merzlyak E.M., Chepurnykh T.V., Fradkov A.F., Ermakova G.V., Solovieva E.A., Lukyanov K.A., Bogdanova E.A., Zaraisky A.G., Lukyanov S., Chudakov D.M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4 (9), 741–6 [+]

    Разработан новый флуоресцентный белок Katushka, обладающий флуоресценцией в дальне-красной области спектра, которая является предпочтительной для анализа сигнала внутри тканей животных. Katushka в десять раз ярче, чем созданные ранее дальне-красные флуоресцентные белки и характеризуется высокой скоростью созревания, высокой рН-стабильностью и фотостабильностью. Это делает новый белок идеальным инструментом для прижизненного мечения клеток внутри целых организмов. Создан мономерный вариант белка Katushka, названный mKate, для исследования внутриклеточной локализации белков.

  29. Merzlyak E.M., Goedhart J., Shcherbo D., Bulina M.E., Shcheglov A.S., Fradkov A.F., Gaintzeva A., Lukyanov K.A., Lukyanov S., Gadella T.W., Chudakov D.M. (2007). Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4 (7), 555–7 [+]

    Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.

  30. Chudakov D.M., Lukyanov S., Lukyanov K.A. (2007). Using photoactivatable fluorescent protein Dendra2 to track protein movement. BioTechniques 42 (5), 553, 555, 557 passim [+]

    Photoactivatable fluorescent proteins are capable of dramatic changes in fluorescent properties in response to specific light irradiation. For example, they can be converted from cyan to green, or from green to red, or from nonfluorescent to a brightly fluorescent state. Several types of such proteins were developed recently, and some of them are already becoming popular tools to study protein mobility. Here we provide detailed recommendations on application of the monomeric green-to-red photoconvertible fluorescent protein Dendra2 for protein tracking in living cultured cells.

  31. Chudakov D.M., Chepurnykh T.V., Belousov V.V., Lukyanov S., Lukyanov K.A. (2006). Fast and precise protein tracking using repeated reversible photoactivation. Traffic 7 (10), 1304–10 [+]

    Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we report 'protein rivers tracking' technique based on repeated identical rounds of photoactivation and subsequent images averaging, which results in dramatic increase of imaging resolution for fast protein movement events.

  32. Belousov V.V., Fradkov A.F., Lukyanov K.A., Staroverov D.B., Shakhbazov K.S., Terskikh A.V., Lukyanov S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3 (4), 281–6 [+]

    Разработан уникальный флуоресцентный сенсор HyPer для прижизненного мониторинга колебаний концентрации одного из важнейших регуляторов биологических процессов — перекиси водорода. Имеющий белковую природу, HyPer может быть экспрессирован в клетках или направлен в определенный клеточный компартмент. Благодаря высокой специфичности и чувствительности, HyPer может быть использован для отслеживания колебаний концентрации перекиси водорода на уровне единственной клетки или клеточной органеллы.

  33. 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.

  34. Bulina M.E., Lukyanov K.A., Britanova O.V., Onichtchouk D., Lukyanov S., Chudakov D.M. (2006). Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed. Nat Protoc 1 (2), 947–53 [+]

    The phototoxic red fluorescent GFP-like protein KillerRed has recently been described. The phototoxicity of KillerRed exceeds that of EGFP by at least 1,000-fold, making it the first fully genetically encoded photosensitizer. KillerRed opens up new possibilities for precise light-induced cell killing and target protein inactivation. Because KillerRed is encoded by a gene, it can be expressed in a spatially and temporally regulated manner, under a chosen promoter, and fused with the desired protein of interest or localization signal. Here we provide a protocol for target protein inactivation in cell culture using KillerRed. As KillerRed is a new tool, the protocol focuses on aspects that will allow users to maximize the potential of this protein, guiding the design of chimeric constructs, recommended control experiments and preferred illumination parameters. The protocol, which describes target protein visualization and subsequent inactivation, is a 2- or 3-d procedure.

  35. Bulina M.E., Chudakov D.M., Britanova O.V., Yanushevich Y.G., Staroverov D.B., Chepurnykh T.V., Merzlyak E.M., Shkrob M.A., Lukyanov S., Lukyanov K.A. (2006). A genetically encoded photosensitizer. Nat. Biotechnol. 24 (1), 95–9 [+]

    Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to beta-galactosidase and phospholipase Cdelta1 pleckstrin homology domain.

  36. Lukyanov K.A., Chudakov D.M., Fradkov A.F., Labas Y.A., Matz M.V., Lukyanov S. (2006). Discovery and properties of GFP-like proteins from nonbioluminescent anthozoa. Methods Biochem Anal 47, 121–38 ID:284
  37. 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.

  38. Chudakov D.M., Lukyanov S., Lukyanov K.A. (2005). Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23 (12), 605–13 [+]

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.

  39. Lukyanov K.A., Chudakov D.M., Lukyanov S., Verkhusha V.V. (2005). Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6 (11), 885–91 [+]

    The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.

  40. Yampolsky I.V., Remington S.J., Martynov V.I., Potapov V.K., Lukyanov S., Lukyanov K.A. (2005). Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata. Biochemistry 44 (15), 5788–93 [+]

    A model compound for the chromophore within the purple nonfluorescent GFP-like chromoprotein asFP595 was synthesized. The postulated structure of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone, was taken from the high-resolution crystal structure analysis of intact asFP595 [Quillin, M. L., Anstrom, D., Shu, X., O'Leary, S., Kallio, K., Lukyanov, K. A., and Remington, S. J. (2005) Kindling Fluorescent Protein from Anemonia sulcata: Dark-State Structure at 1.38 A Resolution, Biochemistry 44, 5774-5787]. Erlenmeyer lactonization and oxidation of the methylene group attached to the heteroaromatic moiety with selenium dioxide were used at the key stages of the synthesis. The spectral properties of the model chromophore in solution and their dependence on the pH and polarity of the solvent were investigated. In water, the chromophore was found to exist in two forms, neutral and anionic, with a pK(a) of 7.1. In a dimethylformamide solution, the spectral properties of the anionic form closely match those of the native protein, demonstrating that under these conditions, the compound is an excellent model for the chromophore within native asFP595.

  41. Chudakov D.M., Verkhusha V.V., Staroverov D.B., Souslova E.A., Lukyanov S., Lukyanov K.A. (2004). Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22 (11), 1435–9 [+]

    In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.

  42. 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.

  43. 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.

  44. Chudakov D.M., Feofanov A.V., Mudrik N.N., Lukyanov S., Lukyanov K.A. (2003). Chromophore environment provides clue to "kindling fluorescent protein" riddle. J. Biol. Chem. 278 (9), 7215–9 [+]

    asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

  45. Chudakov D.M., Belousov V.V., Zaraisky A.G., Novoselov V.V., Staroverov D.B., Zorov D.B., Lukyanov S., Lukyanov K.A. (2003). Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21 (2), 191–4 [+]

    Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

  46. Shagin D.A., Rebrikov D.V., Kozhemyako V.B., Altshuler I.M., Shcheglov A.S., Zhulidov P.A., Bogdanova E.A., Staroverov D.B., Rasskazov V.A., Lukyanov S. (2002). A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res. 12 (12), 1935–42 [+]

    Охарактеризован новый фермент — дуплекс-специфическая нуклеаза (ДСН) из гепатопанкреаса камчатского краба. ДСН обладает высокой специфичностью к двухцепочечной ДНК при отсутствии гидролитической активности по отношению к одноцепочечной ДНК и РНК и стабильность в широком диапазоне температур. Благодаря уникальной комбинации свойств ДСН является идеальным инструментом для удаления двухцепочечной ДНК из сложных смесей нуклеиновых кислот.

  47. Terskikh A., Fradkov A., Ermakova G., Zaraisky A., Tan P., Kajava A.V., Zhao X., Lukyanov S., Matz M., Kim S., Weissman I., Siebert P. (2000). "Fluorescent timer": protein that changes color with time. Science 290 (5496), 1585–8 [+]

    We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

  48. Matz M.V., Fradkov A.F., Labas Y.A., Savitsky A.P., Zaraisky A.G., Markelov M.L., Lukyanov S.A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17 (10), 969–73 [+]

    Открыты новые флуоресцентные белки из коралловых полипов класса Anthozoa с разными цветами флуоресценции от сине-зеленого до красного. Выявление флуоресцентных и окрашенных GFP-подобных белков у неспособных к биолюминесценции коралловых полипов опровергло распространенное мнение, что такие белки функционируют только в составе биолюминисцентных систем и прояснило природу разнообразной флуоресцентной окраски коралловых рифов — явления, которое на протяжении многих лет не находило правильного объяснения.

  49. Kazanskaya O.V., Severtzova E.A., Barth K.A., Ermakova G.V., Lukyanov S.A., Benyumov A.O., Pannese M., Boncinelli E., Wilson S.W., Zaraisky A.G. (1997). Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis. Gene 200 (1-2), 25–34 [+]

    Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.

  50. Zaraisky A.G., Ecochard V., Kazanskaya O.V., Lukyanov S.A., Fesenko I.V., Duprat A.M. (1995). The homeobox-containing gene XANF-1 may control development of the Spemann organizer. Development 121 (11), 3839–47 [+]

    At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stage embryo and have observed the following effects. First, microinjections of the mRNA in the animal blastomeres and the blastomeres of the marginal zone elicited massive migration of cells to the interior of the embryo at the early gastrula stage. Second, overexpression of XANF-1 in the ventral marginal zone (VMZ) resulted in the appearance of an additional centre of gastrulation movements and the formation of a secondary axis. In addition we showed that synthetic XANF-1 mRNA was able to cause dorsal-type differentiation in VMZ explants extirpated from the microinjected embryos at the beginning of gastrulation. These results suggest that XANF-1 may control the main functions of cells of the Spemann organizer.

  51. Zaraisky A.G., Lukyanov S.A., Vasiliev O.L., Smirnov Y.V., Belyavsky A.V., Kazanskaya O.V. (1992). A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo. Dev. Biol. 152 (2), 373–82 [+]

    To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.