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| Structure-functional analysis of chromosomes |  |  One project deals with the (TTAGG)n motif of telomeres at the ends of insect chromosomes. The motif is ancestral in insects but was lost in various taxons. We map its occurrence in arthropods by Southern hybridisation and FISH (fluorescence in situ hybridisation) with the aim to compile the phylogeny of this motif and find out its origin. Structural organisation of telomeres, their role in karyotype evolution and resistance to irradiation are also examined. Our second project deals with the WZ/ZZ sex chromosome system in Lepidoptera. We study differentiation of W-Z by comparative genomic hybridisation (CGH). Molecular composition of the W chromosome is probed by FISH mapping of DNA sequences derived from the W chromosome. The aims are to understand the role of W in sex determination and develop new techniques for genetic sexing. PI: F. Marec | | | | Roles of a co-activator MBF1 in development and oxidative stress | |  During gene activation, co-activators mediate the effect of DNA-binding transcription factors to RNA polymerase. Although co-activators are important for transcription, their in vivo roles are in most cases poorly understood. MBF1 (multiprotein bridging factor 1) is a well-conserved helix-turn-helix protein that appears to stimulate transcription by forming a bridge between certain transcription factors and the TATA-box binding protein (TBP). In the budding yeast, MBF1 cooperates with GCN4, a basic-region leucine zipper (bZIP) regulator of amino acid synthesis, and therefore is important for the yeast growth under histidine starvation.
To address MBF1 role in a multicellular organism, we use the Drosophila model. We have identified new MBF1 partners among Drosophila bZIP proteins, leading to previously unknown MBF1 roles in development and in antioxidant defense. First, MBF1 cooperates with a bZIP protein Tdf (Tracheae defective) during embryogenesis. A recessive tdf allele becomes haplo-insufficient in MBF1-deficient embryos, causing severe lesions in the tracheal and central nervous systems; transcription of a Tdf-dependent reporter gene is reduced in these mutant embryos (Liu et al. 2003, Development).
Second, MBF1 binds to the basic region of D-Jun, a Drosophila ortholog of the proto-oncoprotein c-Jun and the closest relative of yeast GCN4 (see Figure, top). MBF1 protects a redox-sensitive cysteine within the D-Jun basic region from an oxidative modification and thus preserves the DNA-binding activity of the D-Jun/D-Fos dimer (AP-1). An AP-1-dependent developmental process of epithelial tissue closure becomes sensitive to oxidative stress in MBF1-deficient mutants, which also live shorter than controls in the presence of hydrogen peroxide (see Figure, bottom) (Jindra et al. 2004, EMBO J).
Despite its high conservation from yeast to humans, the MBF1 protein is not essential for life under laboratory conditions, even though it has no structural paralogs in either the yeast or Drosophila genomes. This is probably true for many other conserved genes that have not been identified with lethal mutations. Yet, our work shows that such genes may provide an evolutionary advantage as auxiliary factors under conditions of nutritional or chemical stress, encountered in the real world. | | | | Use of Sindbis virus for genetic studies of insect development | |  With the powerful Drosophila model at hand, research on other insects might seem redundant. However, genetic studies on additional species are necessary for us to understand the general developmental mechanisms and how they evolved. One of our goals is to expand reverse genetic techniques so that causal evidence for a gene function may be obtained in any relevant invertebrate.
Specifically, we are interested in genes that govern insect metamorphosis in response to the steroid hormone ecdysone. In Drosophila, ecdysone triggers metamorphosis by acting on its nuclear receptor, which in turn activates a cascade of transcription factors. Among these, Broad-Complex (BR-C) is specifically required for metamorphic events including pupation, extension of adult appendages, differentiation of eyes, and death of obsolete larval organs. Only loss-of-function studies in non-drosophilid species can answer the question whether the role of BR-C (or any other protein) is common to diverse insect orders.
RNA interference (RNAi) is the most promising approach to loss-of-function genetic analyses in non-model organisms, where targeted mutagenesis is not feasible. The main problem is how to deliver the interfering double-stranded RNA, sufficient for a specific gene knock-down, into the organism cells. We have solved this problem by using a recombinant virus Sindbis, which infects some of insect tissues without killing the host. Once in the host cell, the Sindbis RNA genome (see Figure, top) including an inserted foreign gene is rapidly expressed, producing either protein (such as GFP) or double-stranded RNA that can trigger degradation of a specific endogenous mRNA and thereby silence the gene. We have demonstrated Sindbis-mediated RNAi in vivo using the silkworm Bombyx mori as a pilot system. BR-C RNAi disrupted silkworm pupation as well as the morphogenetic (differentiation of the adult wings, eyes and legs, see Figure) and degenerative (programed death of the larval silk glands) aspects of metamorphosis. These effects correspond to those of BR-C mutations in Drosophila, suggesting that the role of BR-C in metamorphosis is evolutionarily conserved (Uhlirova et al. 2003, PNAS USA). | | | | Evolutionary genetics of insects | | | Research is done in two areas – molecular evolution of gene families of Drosophila and population genetics of Ips typographus. Development-related multigene family Idgf (Imaginal disc growth factors) of D. melanogaster is a unique model of similar gene complexes, which can reveal to what extent is evolution of the individual family members affected by their physical linkage or different cytological location. The bark beetle, I. typographus, is a serious pest of spruce forests, causing enormous damages by its periodical outbreaks. Knowledge of genetic characteristics of its populations (genetic variability, population size, migration, etc.) can significantly contribute toward better management practices. PI: M. Žurovcová | | | | Conserved features of promoter architecture | | Detailed studies of the binding consensus of Drosophila and Bombyx transcription factor Fork head revealed unexpected consistency in the distribution of the putative cognate Fork head sequences in the insect labial gland promoters. To understand this phenomenon we have developed theoretical framework including the statistical software ELSEA for the study of conserved features of promoter architecture. A novel database of expression-connected mammalian promoter sequences has been established in an effort to extend this observation to other systems (PRESTA. PI: V. Mach
| Database | Human | Mouse |
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| Release | 1.0, April 2002 | 1.0, April 2002 |
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| Promoters | 552 | 241 |
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| EST clones | 26116 | 7657 |
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| EST libraries | 661 | 273 |
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more.. | | | | Interaction of the nuclear receptor NHR-25 with β-catenin signaling | |  The gonad of Caenorhabditis elegans is remarkable in that its proximal-distal organization depends on asymmetry of a single cell division, which produces a distal tip cell (DTC), necessary to lead gonad differentiation, and anchor cell (AC) that will induce the vulva. Necessary for this asymmetry is a β-catenin/MAPK pathway that culminates at the nuclear effector TCF (POP-1) and that promotes the distal fate. We have shown that a nuclear receptor NHR-25 counters the effect of the β-catenin/MAPK signaling and that proper specification of both the distal and proximal fates requires a balance between NHR-25 and the β-catenin/MAPK pathway activity. Silencing of NHR-25 via RNAi causes up to two extra DTCs to arise instead of the proximal AC (see figure). In addition, NHR-25 RNAi allows otherwise sterile pop-1 mutants to develop DTCs and gonads. NHR-25 blocks POP-1-dependent transcription by binding to β-catenin SYS-1, while another β-catenin WRM-1 inhibits NHR-25 activity. Since NHR-25 belongs to an ancient nuclear receptor family (including Ftz-F1, SF-1, and LRH-1 proteins) whose members functionally interact with β-catenin in mammals, the cross-regulation between NHR-25 and β-catenin/MAPK signaling, uncovered in the C. elegans gonad, may present a broadly conserved mechanism of cell fate acquisition during differentiation (Asahina et al. 2006, Dev Cell). | | | | The steroid deficiency gene ecdysoneless | |  Insects require the steroid hormone ecdysone for molting, metamorphosis, and reproduction. Drosophila mutants deficient in steroid production are therefore very useful for understanding the hormonal regulation of development and physiology. Among these mutants, ecdysoneless[1] (ecd[1]) has been extensively used since its discovery by Garen and colleagues in 1977, although the gene and its defect have remained unknown. We have identified ecdysoneless and shown that a single amino acid change in the conserved but as yet functionally undescribed Ecd protein is responsible for the steroid deficiency. However, our mosaic analyses with another, ecd-null mutation, have shown that Ecd has a primarily steroid-independent, cell-autonomous function. While ecd(-/-) clones fail to survive in proliferating imaginal discs, mutant clones of follicle cells cause fusions of adjacent egg chambers in the ovary (see Figure, top). When mutant clones are induced in the germ-line, loss of Ecd leads to egg chamber degeneration prior to vitellogenesis (see Figure, bottom left). Interestingly, mosaic egg chambers, in which only half of the 16 cells lack Ecd while half carry the female sterile ovoD1 mutation, mature (see Figure, bottom right). Finally, lethal ecd-null homozygotes could not be rescued by feeding the hormone or by Ecd expression targeted to the ecdysone-producing endocrine gland. These results indicate that Ecd plays an essential cell-autonomous role, which is independent of the free-circulating hormone (Gaziova et al. 2004, Development). | | | | Developmental roles of a nuclear receptor NHR-25 in C. elegans | |  For a mysterious reason, the simple nematode Caenorhabditis elegans possesses 284 nuclear hormone receptor (NHR) genes, which is five times the number in humans. These receptors are all orphan, and only about 15 are well conserved in either Drosophila or mammals. Thus far very few NHRs have been ascribed a biological role in the worm. NHR-25 is a well-conserved ortholog of the Ftz-F1/SF-1 family of nuclear receptors. In Drosophila, Ftz-F1 is required first as a cofactor of the homeotic protein Fushi tarazu (Ftz) for embryonic segmentation, later for the steroid-dependent molting and metamorphosis. Mammalian steroidogenic factor 1 (SF-1) regulates steroid hormone synthesis and is necessary for the development of adrenal glands and male gonads.
We and others have shown that NHR-25 is required for the nematode embryogenesis, molting, and the differentiation of the gonad and vulva (Asahina et al. 2000, Genes Cells).
How this single molecule exerts such diverse pleiotropic effects is not known, since NHR-25 has not yet been placed in any well-characterized signaling pathway and its target genes as well as specific cellular processes have also remained undisclosed. Our recent work (Silhankova et al. 2005, J Cell Sci) shows that NHR-25 ensures the proper development of C. elegans epidermis, where it is required for cell shape changes and mutual contacts between specific epidermal stem cells (see Figure). By preventing renewal of contacts between these stem cells, the loss of NHR-25 disrupts the subsequent fate determination of their epidermal daughters. We also have indications that NHR-25 interacts with a Hox gene, a ftz relative mab-5 downstream in the Wnt pathway. We are currently searching for genes regulated by NHR-25. | | | | Genetic transformation of the silkworm Bombyx mori | |  Insect science cannot advance without developing efficient methods for gene transfer, forced expression and gene silencing in vivo. To begin building our tools for gene manipulations in insects, we have utilized the commercial silkworm Bombyx mori as a model. In preparation for transgenic approaches including protein misexpression or RNA interference, we have realized a stable germ-line transformation of the silkworm with a vector based on the transposon piggyBac. Transformants are marked with a jellyfish gene encoding the green fluorescent protein (GFP), whose expression in the silkworm eyes is regulated by a CNS and photoreceptor-specific factor Pax-6 (see Figure). We have equipped piggyBac with the Drosophila hsp70 promoter and thus have been able to induce expression of chosen proteins in live silkworms by heat (Uhlirova et al. 2002, Dev Genes Evol). | | | | JH resistance gene Met controls entry to metamorphosis through BR-C | |  Metamorphosis of holometabolous insects such as beetles or butterflies is a marked change of form between juvenile and adult stages that enables the larva to efficiently utilize food sources and the flying adult to spread the species. The entry to metamorphosis depends on the morphogenesis-promoting ecdysteroids and the antagonistically acting juvenile hormone (JH), which precludes metamorphosis until a larva attains the appropriate size and developmental stage. JH has been known to prevent metamorphosis since the work of V.B. Wigglesworth for over 70 years. However, the mechanism of JH action has remained an enigma as neither a JH receptor nor its signaling pathway are known. By using the red flour beetle Tribolium castaneum, we showed that a gene Methoprene-tolerant (Met), originally uncovered as a mutation conferring resistance to JH in the fly Drosophila, mediates the anti-metamorphic JH effect. Loss of Met function renders Tribolium insensitive to JH and, unlike in Drosophila, it also causes the beetle larvae to metamorphose precociously (Konopova and Jindra 2007, PNAS USA). We further showed that in response to JH, Met controls metamorphosis by regulating expression of the Broad-Complex gene, which is required for the metamorphic changes (Konopova and Jindra 2008, Development). Our latest studies thus for the first time demonstrate the key role of Met in the regulation of insect metamorphosis by JH and support the disputed function of Met as a receptor or transducer of the JH signal. | | |
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