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1.
A Polycomb and Gaga Dependent Silencer Adjoins the Fab-7 Boundary in the Drosophila Bithorax Complex 总被引:5,自引:0,他引:5
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The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7(1)) dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-7(1) is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-7(1) deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a BXD, Ubx/lacZ reporter transgene. Both silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste(1) allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. 相似文献
2.
Kyrchanova O. V. Postika N. Y. Sokolov V. V. Georgiev P. G. 《Doklady. Biochemistry and biophysics》2022,502(1):21-24
Doklady Biochemistry and Biophysics - The segment-specific regulatory domains of the Bithorax complex (BX-C), which consists of three homeotic genes Ubx, abd-A and Abd-B, are separated by... 相似文献
3.
Iampietro C Cléard F Gyurkovics H Maeda RK Karch F 《Development (Cambridge, England)》2008,135(24):3983-3987
Although the boundary elements of the Drosophila Bithorax complex (BX-C) have properties similar to chromatin insulators, genetic substitution experiments have demonstrated that these elements do more than simply insulate adjacent cis-regulatory domains. Many BX-C boundaries lie between enhancers and their target promoter, and must modulate their activity to allow distal enhancers to communicate with their target promoter. Given this complex function, it is surprising that the numerous BX-C boundaries share little sequence identity. To determine the extent of the similarity between these elements, we tested whether different BX-C boundary elements can functionally substitute for one another. Using gene conversion, we exchanged the Fab-7 and Fab-8 boundaries within the BX-C. Although the Fab-8 boundary can only partially substitute for the Fab-7 boundary, we find that the Fab-7 boundary can almost completely replace the Fab-8 boundary. Our results suggest that although boundary elements are not completely interchangeable, there is a commonality to the mechanism by which boundaries function. This commonality allows different DNA-binding proteins to create functional boundaries. 相似文献
4.
Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene
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Kyrchanova O Toshchakov S Podstreshnaya Y Parshikov A Georgiev P 《Molecular and cellular biology》2008,28(12):4188-4195
Boundary elements have been found in the regulatory region of the Drosophila melanogaster Abdominal-B (Abd-B) gene, which is subdivided into a series of iab domains. The best-studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF (dCTCF) were identified in the Fab-8 boundary and upstream of Abd-B promoter A, with no binding of CTCF to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when its binding sites are separated by a 5-kb yellow gene, we have tested the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing a dCTCF binding site. It has been found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggests that additional, as yet unidentified proteins are involved in long-distance interactions between them. We have also shown that Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter. 相似文献
5.
Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import
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Michael J. Baker Chaille T. Webb David A. Stroud Catherine S. Palmer Ann E. Frazier Bernard Guiard Agnieszka Chacinska Jacqueline M. Gulbis Michael T. Ryan 《Molecular biology of the cell》2009,20(3):769-779
The Tim9–Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9–Tim10 hexameric assembly determined to 2.5 Å and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space. 相似文献
6.
Enhancer Traps in the Drosophila Bithorax Complex Mark Parasegmental Domains 总被引:3,自引:1,他引:3
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Eight P elements carrying a β-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. The enhancer traps inserted at the promoter of one of the genes, Ultrabithorax, express lacZ in patterns which mimic the Ultrabithorax protein pattern. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure. 相似文献
7.
8.
Transvection in the Ultrabithorax Domain of the Bithorax Complex of Drosophila Melanogaster 总被引:2,自引:0,他引:2
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D. Mathog 《Genetics》1990,125(2):371-382
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10.
Elisa M. LaBeau Damian L. Trujillo Richard M. Cripps 《Mechanisms of development》2009,126(5-6):478-486
Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. We found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. Double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles. 相似文献
11.
The bithorax complex in Drosophila melanogaster includes three homeobox-containing genes—Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)—which are required for the proper differentiation of the posterior 10 segments of the body. Each of these genes has multiple distinct regulatory regions; there is one for each segmental unit of the body plan where the genes are expressed. One additional protein- coding gene in the bithorax complex, Glut3, a sugar-transporter homolog, can be deleted without phenotype. We focus here on the upstream regulatory region for Ubx, the bithoraxoid (bxd) domain, and its border with the adjacent infraabdominal-2 (iab-2) domain, which controls abdA. These two domains can be defined by the phenotypes of rearrangement breakpoints, and by the expression patterns of enhancer traps. In D. virilis, the homeotic cluster is split between Ubx and abd-A, and so the border can also be located by a sequence comparison between species. When the border region is deleted in melanogaster, the flies show a dominant phenotype called Front-ultraabdominal (Fub); the first abdominal segment is transformed into a copy of the second abdominal segment. Thus, the border blocks the spread of activation from the bxd domain into the iab-2 domain. 相似文献
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13.
The Interaction of Two Complex Loci, Zeste and Bithorax in DROSOPHILA MELANOGASTER 总被引:27,自引:10,他引:17
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It has been found that certain alleles of the zeste locus (z(a) 1-1.0) have no phenotype of their own, but interact with certain alleles at the bithorax locus (bx 3-58.8). This interaction takes the form of an enhancement of the homeotic bx phenotype to a more extreme form-i.e., the metathorax is transformed into mesothorax in varying degrees depending on the bx allele used. This enhancement is somewhat reminiscent of the transvection effect described by Lewis (1954). The characterization of the interaction thus far has shown that the enhancement only effects bx alleles which arise spontaneously, whereas the origin of the z(a) allele is unimportant. The gene claret nondisjunctional was used for the production of gynandromorphs which showed that the enhancing ability of z(a), like the eye pigment change caused by z, is autonomous. The enhancement of one specific allele (bx(34e)), which is temperature-sensitive, has allowed a delineation of the temperature-sensitive period of the bithorax locus to a period extending from the middle of the second larval instar to the middle of the third larval instar. These results, as well as those of other enhancer and suppressor systems in Drosophila, have revealed the possibility of the involvement of heterocyclic compounds in the control of cell determination and fate in Drosophila melanogaster. 相似文献
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15.
Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase
Alexander Schwarze Marta J. Kopczak Matthias R?gner Oliver Lenz 《Applied and environmental microbiology》2010,76(8):2641-2651
The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H2 sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O2-tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBHPsaE) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H2 production capacity of the purified MBHPsaE fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBHPsaE with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS I-hydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBHPsaE-PS I complex formation and its light-driven H2 production capacity by means of spectroelectrochemistry.Molecular hydrogen (H2) is often discussed as an alternative source of energy (13, 22, 26, 41). It is a highly energetic, renewable, and zero-carbon dioxide emission fuel; however, it is produced mainly from fossil resources. One intriguing possibility for sustainable H2 production is the development of cellular systems in which the light-driven oxygenic photosynthesis is efficiently coupled to hydrogen production by hydrogenase (1, 21, 36).During the process of oxygenic photosynthesis, photosystem II (PS II), a thylakoid membrane (TM)-embedded multiprotein complex, utilizes solar energy to oxidize water into dioxygen (O2), protons, and electrons. The electrons released by PS II are further conducted through an electron transport chain consisting of plastoquinones, the cytochrome b6f complex, and either plastocyanin or cytochrome c6 to the chlorophyll (Chl) dimer P700 in photosystem I (PS I) (20, 48). During light-induced charge separation in PS I, P700 is oxidized, leading to the reduction of the adjacent cofactor A0 (Chl a). From there, the electrons are transmitted to the phylloquinone A1 and subsequently to the Fe4S4 clusters FX, FA, and FB (9) that are located at the acceptor site of PS I. The acceptor site is composed of the PsaC subunit, which harbors the iron-sulfur clusters FA and FB, and the two additional cofactor-free extrinsic subunits PsaD and PsaE. In the final step, the electrons are transferred from FB to the ferredoxin (PetF), which has a midpoint potential of −412 mV (see Fig. Fig.1B)1B) (8, 9).Open in a separate windowFIG. 1.Models of the hydrogenase and photosystem I complexes used in this study. (A) Membrane-bound hydrogenase (MBHwt) of Ralstonia eutropha H16. (B) Wild-type photosystem I (PS I) from Synechocystis sp. PCC 6803. (C) MBHstop protein lacking the C-terminal anchor domain of HoxK. (D) MBHPsaE and PS IΔPsaE.Hydrogenases of the NiFe and FeFe types catalyze the reversible cleavage of H2 into protons and electrons (18, 63). For most hydrogenases, this reaction is highly sensitive to O2 and leads to the reversible or even irreversible inactivation of the enzyme (49, 66, 67). A prominent exception is the oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16, which catalyzes H2 conversion in the presence of O2 (42, 65). The MBH consists of large subunit HoxG (67 kDa), harboring the NiFe active site, and small subunit HoxK (35 kDa), bearing three FeS clusters (Fig. (Fig.1)1) (32). Both cofactor-containing subunits are completely assembled within the cytoplasm and become subsequently translocated through the cytoplasmic membrane by the twin-arginine translocation (Tat) system. This transport is guided by a specific Tat signal peptide that is located at the N terminus of small subunit HoxK (53). The MBH is then connected to the membrane via the hydrophobic C-terminal “anchor” domain of HoxK, which provides the electronic connection to the diheme cytochrome b, HoxZ (5, 57). All structural, accessory, and regulatory genes for the synthesis of active MBH are arranged in a large, megaplasmid-borne operon (7, 11, 14, 29, 33, 38, 58).The concept of light-driven hydrogen production has been investigated in numerous studies (for reviews, see references 3, 21, and 23), including one involving direct electron transfer from PS I to the free form of hydrogenase in vitro (45). In a preliminary attempt, the MBH from R. eutropha was recently directly fused to PsaE (creating MBHPsaE) (28). The fusion protein was partially purified and subjected to in vitro reconstitution with PS I lacking PsaE (PS IΔPsaE) (54) for light-driven hydrogen production. This concept was based on the previous observation that PS I lacking the peripheral subunit PsaE is fully reconstituted in vitro simply by the addition of independently purified PsaE protein (12).In the present communication, we describe a novel purification procedure for R. eutropha MBHPsaE that yields homogeneous, functionally active MBHPsaE. Additionally, a new method for efficient and fast purification of Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) His-tagged PS I was established. Finally, the pure proteins MBHPsaE and PS IΔPsaE were successfully subjected to in vitro reconstitution. 相似文献
16.
Transvection in the Iab-5,6,7 Region of the Bithorax Complex of Drosophila: Homology Independent Interactions in Trans 总被引:2,自引:2,他引:0
The Abdominal-B (Abd-B) gene of the bithorax complex (BX-C) of Drosophila controls the identities of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14). Here we focus on iab-5, iab-6 and iab-7, regulatory regions of Abd-B that control expression in the fifth, sixth and seventh abdominal segments (parasegments 10-12). By analysis of partial BX-C deficiencies, we show that these regions are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. We establish that this ability does not result from cis-regulation of the adjacent abd-A or Ubx genes of the BX-C but rather occurs because the iab-5,6,7 region is able to interact with Abd-B in trans. We demonstrate that this interaction is proximity dependent and is, therefore, a case of what E. B. LEWIS has called transvection. Interactions of this type are presumably facilitated by the synapsis of homologues that occurs in somatic cells of Dipterans. Although transvection has been detected in a number of Drosophila genes, transvection of the iab-5,6,7 region is exceptional in two ways. First, interaction in trans with Abd-B does not require that homologues share homologous sequences within, or for some distance to either side of, the BX-C. This is the first case of transvection shown to be independent of local synapsis. A second unusual feature of iab-5,6,7 transvection is that it is remarkably difficult to disrupt by heterozygosity for chromosome rearrangements. The lack of requirement for local synapsis and the tenacity of trans-interaction argue that the iab-5,6,7 region can locate and interact with Abd-B over considerable distance. This is consistent with the normal role of iab-5,6,7, which must act over some 20-60 kb to influence its regulatory target in cis at the Abd-B promoter. Evidence is presented that trans-action of iab-5,6,7 requires, and may be mediated by, the region between distal iab-7 and Abd-B. Also, we show that iab-5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7 and Abd-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Transvection may, therefore, be of more general significance than previously suspected. 相似文献
17.
Guan-Feng Wang Jiabing Ji Farid EI-Kasmi Jeffery L. Dangl Guri Johal Peter J. Balint-Kurti 《PLoS pathogens》2015,11(2)
Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance. 相似文献
18.
In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like. 相似文献
19.
The Fab-7 boundary is required to ensure that the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax complex can function autonomously. Though Fab-7 functions as a boundary from early embryogenesis through to the adult stage, this constitutive boundary activity depends on subelements whose activity is developmentally restricted. In the studies reported here, we have identified a factor, called early boundary activity (Elba), that confers Fab-7 boundary activity during early embryogenesis. The Elba factor binds to a recognition sequence within a Fab-7 subelement that has enhancer-blocking activity during early embryogenesis, but not during mid-embryogenesis or in the adult. We found that the Elba factor is present in early embryos but largely disappears during mid-embryogenesis. We show that mutations in the Elba recognition sequence that eliminate Elba binding in nuclear extracts disrupt the early boundary activity of the Fab-7 subelement. Conversely, we find that early boundary activity can be reconstituted by multimerizing the Elba recognition site. 相似文献
20.
Julia Gansler Miriam Jaax Silke Leiting Bettina Appel Andreas Greinacher Silvia Fischer Klaus T. Preissner 《PloS one》2012,7(11)
Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects. 相似文献