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Sample records for opsin gene fusions

  1. Analysis of the opsin repertoire in the tardigrade Hypsibius dujardini provides insights into the evolution of opsin genes in panarthropoda.

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    Hering, Lars; Mayer, Georg

    2014-09-04

    Screening of a deeply sequenced transcriptome using Illumina sequencing as well as the genome of the tardigrade Hypsibius dujardini revealed a set of five opsin genes. To clarify the phylogenetic position of these genes and to elucidate the evolutionary history of opsins in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we reconstructed the phylogeny of broadly sampled metazoan opsin genes using maximum likelihood and Bayesian inference methods in conjunction with carefully selected substitution models. According to our findings, the opsin repertoire of H. dujardini comprises representatives of all three major bilaterian opsin clades, including one r-opsin, three c-opsins, and a Group 4 opsin (neuropsin/opsin-5). The identification of the tardigrade ortholog of neuropsin/opsin-5 is the first record of this opsin type in a protostome, but our screening of available metazoan genomes revealed that it is also present in other protostomes. Our opsin phylogeny further suggests that two r-opsins, including an "arthropsin," were present in the last common ancestor of Panarthropoda. Although both r-opsin lineages were retained in Onychophora and Arthropoda, the arthropsin was lost in Tardigrada. The single (most likely visual) r-opsin found in H. dujardini supports the hypothesis of monochromatic vision in the panarthropod ancestor, whereas two duplications of the ancestral panarthropod c-opsin have led to three c-opsins in tardigrades. Although the early-branching nodes are unstable within the metazoans, our findings suggest that the last common ancestor of Bilateria possessed six opsins: Two r-opsins, one c-opsin, and three Group 4 opsins, one of which (Go opsin) was lost in the ecdysozoan lineage.

  2. Analysis of the Opsin Repertoire in the Tardigrade Hypsibius dujardini Provides Insights into the Evolution of Opsin Genes in Panarthropoda

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    Hering, Lars; Mayer, Georg

    2014-01-01

    Screening of a deeply sequenced transcriptome using Illumina sequencing as well as the genome of the tardigrade Hypsibius dujardini revealed a set of five opsin genes. To clarify the phylogenetic position of these genes and to elucidate the evolutionary history of opsins in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we reconstructed the phylogeny of broadly sampled metazoan opsin genes using maximum likelihood and Bayesian inference methods in conjunction with carefully selected s...

  3. Extraordinary diversity of visual opsin genes in dragonflies.

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    Futahashi, Ryo; Kawahara-Miki, Ryouka; Kinoshita, Michiyo; Yoshitake, Kazutoshi; Yajima, Shunsuke; Arikawa, Kentaro; Fukatsu, Takema

    2015-03-17

    Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15-33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies.

  4. Visual sensitivities tuned by heterochronic shifts in opsin gene expression

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    McFarland William N

    2008-05-01

    Full Text Available Abstract Background Cichlid fishes have radiated into hundreds of species in the Great Lakes of Africa. Brightly colored males display on leks and vie to be chosen by females as mates. Strong discrimination by females causes differential male mating success, rapid evolution of male color patterns and, possibly, speciation. In addition to differences in color pattern, Lake Malawi cichlids also show some of the largest known shifts in visual sensitivity among closely related species. These shifts result from modulated expression of seven cone opsin genes. However, the mechanisms for this modulated expression are unknown. Results In this work, we ask whether these differences might result from changes in developmental patterning of cone opsin genes. To test this, we compared the developmental pattern of cone opsin gene expression of the Nile tilapia, Oreochromis niloticus, with that of several cichlid species from Lake Malawi. In tilapia, quantitative polymerase chain reaction showed that opsin gene expression changes dynamically from a larval gene set through a juvenile set to a final adult set. In contrast, Lake Malawi species showed one of two developmental patterns. In some species, the expressed gene set changes slowly, either retaining the larval pattern or progressing only from larval to juvenile gene sets (neoteny. In the other species, the same genes are expressed in both larvae and adults but correspond to the tilapia adult genes (direct development. Conclusion Differences in visual sensitivities among species of Lake Malawi cichlids arise through heterochronic shifts relative to the ontogenetic pattern of the tilapia outgroup. Heterochrony has previously been shown to be a powerful mechanism for change in morphological evolution. We found that altering developmental expression patterns is also an important mechanism for altering sensory systems. These resulting sensory shifts will have major impacts on visual communication and could help

  5. Visual sensitivities tuned by heterochronic shifts in opsin gene expression

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    Carleton, Karen L; Spady, Tyrone C; Streelman, J Todd; Kidd, Michael R; McFarland, William N; Loew, Ellis R

    2008-01-01

    Background Cichlid fishes have radiated into hundreds of species in the Great Lakes of Africa. Brightly colored males display on leks and vie to be chosen by females as mates. Strong discrimination by females causes differential male mating success, rapid evolution of male color patterns and, possibly, speciation. In addition to differences in color pattern, Lake Malawi cichlids also show some of the largest known shifts in visual sensitivity among closely related species. These shifts result from modulated expression of seven cone opsin genes. However, the mechanisms for this modulated expression are unknown. Results In this work, we ask whether these differences might result from changes in developmental patterning of cone opsin genes. To test this, we compared the developmental pattern of cone opsin gene expression of the Nile tilapia, Oreochromis niloticus, with that of several cichlid species from Lake Malawi. In tilapia, quantitative polymerase chain reaction showed that opsin gene expression changes dynamically from a larval gene set through a juvenile set to a final adult set. In contrast, Lake Malawi species showed one of two developmental patterns. In some species, the expressed gene set changes slowly, either retaining the larval pattern or progressing only from larval to juvenile gene sets (neoteny). In the other species, the same genes are expressed in both larvae and adults but correspond to the tilapia adult genes (direct development). Conclusion Differences in visual sensitivities among species of Lake Malawi cichlids arise through heterochronic shifts relative to the ontogenetic pattern of the tilapia outgroup. Heterochrony has previously been shown to be a powerful mechanism for change in morphological evolution. We found that altering developmental expression patterns is also an important mechanism for altering sensory systems. These resulting sensory shifts will have major impacts on visual communication and could help drive cichlid speciation

  6. Intra-retinal variation of opsin gene expression in the guppy (Poecilia reticulata).

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    Rennison, Diana J; Owens, Gregory L; Allison, W Ted; Taylor, John S

    2011-10-01

    Although behavioural experiments demonstrate that colouration influences mate choice in many species, a complete understanding of this form of signalling requires information about colour vision in the species under investigation. The guppy (Poecilia reticulata) has become a model species for the study of colour-based sexual selection. To investigate the role of opsin gene duplication and divergence in the evolution of colour-based mate choice, we used in situ hybridization to determine where the guppy's nine cone opsins are expressed in the retina. Long wavelength-sensitive (LWS) opsins were more abundant in the dorsal retina than in the ventral retina. One of the middle wavelength-sensitive opsins (RH2-1) exhibited the opposite pattern, while the other middle wavelength-sensitive opsin (RH2-2) and the short wavelength-sensitive opsins (SWS1, SWS2A and SWS2B) were expressed throughout the retina. We also found variation in LWS opsin expression among individuals. These observations suggest that regions of the guppy retina are specialized with respect to wavelength discrimination and/or sensitivity. Intra-retinal variability in opsin expression, which has been observed in several fish species, might be an adaptation to variation in the strength and spectral composition of light entering the eye from above and below. The discovery that opsin expression varies in the guppy retina may motivate new behavioural experiments designed to study its role in mate choice.

  7. Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African cichlid fishes

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    Streelman J Todd

    2011-05-01

    Full Text Available Abstract Background Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Among African cichlid fishes, the differential expression of seven opsin genes has produced adaptive differences in visual sensitivity. Quantitative genetic analysis suggests that cis-regulatory alleles near the SWS2-LWS opsins may contribute to this variation. Here, we sequence BACs containing the opsin genes of two cichlids, Oreochromis niloticus and Metriaclima zebra. We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory sequences that influence cichlid opsin expression. Results We identified 20 conserved non-coding elements surrounding the opsins of cichlids and other teleosts, including one known enhancer and a retinal microRNA. Most conserved elements contained computationally-predicted binding sites that correspond to transcription factors that function in vertebrate opsin expression; O. niloticus and M. zebra were significantly divergent in two of these. Similarly, we found a large number of relevant transcription factor binding sites within each opsin's proximal promoter, and identified five opsins that were considerably divergent in both expression and the number of transcription factor binding sites shared between O. niloticus and M. zebra. We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra. Finally, we examined interspecific divergence among 18 phenotypically diverse cichlids from Lake Malawi for one conserved non-coding element, two 3'-UTRs, and five opsin proximal promoters. We found that all regions were highly conserved with some evidence of CRX transcription

  8. Divergent selection for opsin gene variation in guppy (Poecilia reticulata) populations of Trinidad and Tobago.

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    Tezuka, A; Kasagi, S; van Oosterhout, C; McMullan, M; Iwasaki, W M; Kasai, D; Yamamichi, M; Innan, H; Kawamura, S; Kawata, M

    2014-11-01

    The guppy is known to exhibit remarkable interindividual variations in spectral sensitivity of middle to long wavelength-sensitive (M/LWS) cone photoreceptor cells. The guppy has four M/LWS-type opsin genes (LWS-1, LWS-2, LWS-3 and LWS-4) that are considered to be responsible for this sensory variation. However, the allelic variation of the opsin genes, particularly in terms of their absorption spectrum, has not been explored in wild populations. Thus, we examined nucleotide variations in the four M/LWS opsin genes as well as blue-sensitive SWS2-B and ultraviolet-sensitive SWS1 opsin genes for comparison and seven non-opsin nuclear loci as reference genes in 10 guppy populations from various light environments in Trinidad and Tobago. For the first time, we discovered a potential spectral variation (180 Ser/Ala) in LWS-1 that differed at an amino acid site known to affect the absorption spectra of opsins. Based on a coalescent simulation of the nucleotide variation of the reference genes, we showed that the interpopulation genetic differentiation of two opsin genes was significantly larger than the neutral expectation. Furthermore, this genetic differentiation was significantly related to differences in dissolved oxygen (DO) level, and it was not explained by the spatial distance between populations. The DO levels are correlated with eutrophication that possibly affects the color of aquatic environments. These results suggest that the population diversity of opsin genes is significantly driven by natural selection and that the guppy could adapt to various light environments through color vision changes.

  9. The opsin repertoire of Jenynsia onca: a new perspective on gene duplication and divergence in livebearers

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    Owens Gregory L

    2009-08-01

    Full Text Available Abstract Background Jenynsia onca, commonly known as the one sided livebearer, is a member of the family Anablepidae. The opsin gene repertoires of J. onca's close relatives, the four-eyed fish (Anableps anableps and the guppy (Poecilia reticulata, have been characterized and each found to include one unique LWS opsin. Currently, the relationship among LWS paralogs and orthologs in these species are unclear, making it difficult to test the hypotheses that link vision to morphology or life history traits. The phylogenetic signal appears to have been disrupted by gene conversion. Here we have sequenced the opsin genes of J. onca in order to resolve these relationships. Findings We identified nine visual opsins; LWS S180r, LWS S180, LWS P180, SWS1, SWS2A, SWS2B, RH1, RH2-1, and RH2-2. Key site analysis revealed only one unique haplotype, RH2-2, although this is unlikely to shift λmax significantly. LWS P180 was found to be a product of a gene conversion event with LWS S180, followed by convergence to a proline residue at the 180 site. Conclusion Jenynsia onca has at least 9 visual opsins: three LWS, one RH1, two RH2, one SWS1 and two SWS2. The presence of LWS P180 moves the location of the LWS P180-S180 tandem duplication event back to the base of the Poeciliidae-Anablepidae clade, expanding the number of species possessing this unusual blue shifted LWS opsin. The presence of the LWS P180 gene also confirms that gene conversion events have homogenized opsin paralogs in fish, just as they have in humans.

  10. Gene duplication and divergence of long wavelength-sensitive opsin genes in the guppy, Poecilia reticulata.

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    Watson, Corey T; Gray, Suzanne M; Hoffmann, Margarete; Lubieniecki, Krzysztof P; Joy, Jeffrey B; Sandkam, Ben A; Weigel, Detlef; Loew, Ellis; Dreyer, Christine; Davidson, William S; Breden, Felix

    2011-02-01

    Female preference for male orange coloration in the genus Poecilia suggests a role for duplicated long wavelength-sensitive (LWS) opsin genes in facilitating behaviors related to mate choice in these species. Previous work has shown that LWS gene duplication in this genus has resulted in expansion of long wavelength visual capacity as determined by microspectrophotometry (MSP). However, the relationship between LWS genomic repertoires and expression of LWS retinal cone classes within a given species is unclear. Our previous study in the related species, Xiphophorus helleri, was the first characterization of the complete LWS opsin genomic repertoire in conjunction with MSP expression data in the family Poeciliidae, and revealed the presence of four LWS loci and two distinct LWS cone classes. In this study we characterized the genomic organization of LWS opsin genes by BAC clone sequencing, and described the full range of cone cell types in the retina of the colorful Cumaná guppy, Poecilia reticulata. In contrast to X. helleri, MSP data from the Cumaná guppy revealed three LWS cone classes. Comparisons of LWS genomic organization described here for Cumaná to that of X. helleri indicate that gene divergence and not duplication was responsible for the evolution of a novel LWS haplotype in the Cumaná guppy. This lineage-specific divergence is likely responsible for a third additional retinal cone class not present in X. helleri, and may have facilitated the strong sexual selection driven by female preference for orange color patterns associated with the genus Poecilia.

  11. Spectral sensitivity in Onychophora (velvet worms) revealed by electroretinograms, phototactic behaviour and opsin gene expression.

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    Beckmann, Holger; Hering, Lars; Henze, Miriam J; Kelber, Almut; Stevenson, Paul A; Mayer, Georg

    2015-03-01

    Onychophorans typically possess a pair of simple eyes, inherited from the last common ancestor of Panarthropoda (Onychophora+Tardigrada+Arthropoda). These visual organs are thought to be homologous to the arthropod median ocelli, whereas the compound eyes probably evolved in the arthropod lineage. To gain insights into the ancestral function and evolution of the visual system in panarthropods, we investigated phototactic behaviour, opsin gene expression and the spectral sensitivity of the eyes in two representative species of Onychophora: Euperipatoides rowelli (Peripatopsidae) and Principapillatus hitoyensis (Peripatidae). Our behavioural analyses, in conjunction with previous data, demonstrate that both species exhibit photonegative responses to wavelengths ranging from ultraviolet to green light (370-530 nm), and electroretinograms reveal that the onychophoran eye is maximally sensitive to blue light (peak sensitivity ∼480 nm). Template fits to these sensitivities suggest that the onychophoran eye is monochromatic. To clarify which type of opsin the single visual pigment is based on, we localised the corresponding mRNA in the onychophoran eye and brain using in situ hybridization. Our data show that the r-opsin gene (onychopsin) is expressed exclusively in the photoreceptor cells of the eye, whereas c-opsin mRNA is confined to the optic ganglion cells and the brain. Together, our findings suggest that the onychopsin is involved in vision, whereas c-opsin might have a photoreceptive, non-visual function in onychophorans.

  12. A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

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    Taro Tsujimura

    2010-12-01

    Full Text Available A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the long member of double cones (LDCs in the retina. Expression of the longer-wave sensitive LWS-1 occurs later in development and is thus confined to the peripheral, especially ventral-nasal region of the adult retina, whereas expression of LWS-2 occurs earlier and is confined to the central region of the adult retina, shifted slightly to the dorsal-temporal region. In this study, we employed a transgenic reporter assay using fluorescent proteins and P1-artificial chromosome (PAC clones encompassing the two genes and identified a 0.6-kb "LWS-activating region" (LAR upstream of LWS-1, which regulates expression of both genes. Under the 2.6-kb flanking upstream region containing the LAR, the expression pattern of LWS-1 was recapitulated by the fluorescent reporter. On the other hand, when LAR was directly conjugated to the LWS-2 upstream region, the reporter was expressed in the LDCs but also across the entire outer nuclear layer. Deletion of LAR from the PAC clones drastically lowered the reporter expression of the two genes. These results suggest that LAR regulates both LWS-1 and LWS-2 by enhancing their expression and that interaction of LAR with the promoters is competitive between the two genes in a developmentally restricted manner. Sharing a regulatory region between duplicated genes could be a general way to facilitate the expression differentiation in duplicated visual opsins.

  13. Signatures of functional constraint at aye-aye opsin genes: the potential of adaptive color vision in a nocturnal primate.

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    Perry, George H; Martin, Robert D; Verrelli, Brian C

    2007-09-01

    While color vision perception is thought to be adaptively correlated with foraging efficiency for diurnal mammals, those that forage exclusively at night may not need color vision nor have the capacity for it. Indeed, although the basic condition for mammals is dichromacy, diverse nocturnal mammals have only monochromatic vision, resulting from functional loss of the short-wavelength sensitive opsin gene. However, many nocturnal primates maintain intact two opsin genes and thus have dichromatic capacity. The evolutionary significance of this surprising observation has not yet been elucidated. We used a molecular population genetics approach to test evolutionary hypotheses for the two intact opsin genes of the fully nocturnal aye-aye (Daubentonia madagascariensis), a highly unusual and endangered Madagascar primate. No evidence of gene degradation in either opsin gene was observed for any of 8 aye-aye individuals examined. Furthermore, levels of nucleotide diversity for opsin gene functional sites were lower than those for 15 neutrally evolving intergenic regions (>25 kb in total), which is consistent with a history of purifying selection on aye-aye opsin genes. The most likely explanation for these findings is that dichromacy is advantageous for aye-ayes despite their nocturnal activity pattern. We speculate that dichromatic nocturnal primates may be able to perceive color while foraging under moonlight conditions, and suggest that behavioral and ecological comparisons among dichromatic and monochromatic nocturnal primates will help to elucidate the specific activities for which color vision perception is advantageous.

  14. Light-controlled inhibition of malignant glioma by opsin gene transfer

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    Yang, F; Tu, J; Pan, J-Q; Luo, H-L; Liu, Y-H; Wan, J; Zhang, J; Wei, P-F; Jiang, T; Chen, Y-H; Wang, L-P

    2013-01-01

    Glioblastomas are aggressive cancers with low survival rates and poor prognosis because of their highly proliferative and invasive capacity. In the current study, we describe a new optogenetic strategy that selectively inhibits glioma cells through light-controlled membrane depolarization and cell death. Transfer of the engineered opsin ChETA (engineered Channelrhodopsin-2 variant) gene into primary human glioma cells or cell lines, but not normal astrocytes, unexpectedly decreased cell proliferation and increased mitochondria-dependent apoptosis, upon light stimulation. These optogenetic effects were mediated by membrane depolarization-induced reductions in cyclin expression and mitochondrial transmembrane potential. Importantly, the ChETA gene transfer and light illumination in mice significantly inhibited subcutaneous and intracranial glioma growth and increased the survival of the animals bearing the glioma. These results uncover an unexpected effect of opsin ion channels on glioma cells and offer the opportunity for the first time to treat glioma using a light-controllable optogenetic approach. PMID:24176851

  15. Mutational changes in S-cone opsin genes common to both nocturnal and cathemeral Aotus monkeys.

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    Levenson, David H; Fernandez-Duque, Eduardo; Evans, Sian; Jacobs, Gerald H

    2007-07-01

    Aotus is a platyrrhine primate that has been classically considered to be nocturnal. Earlier research revealed that this animal lacks a color vision capacity because, unlike all other platyrrhine monkeys, Aotus has a defect in the opsin gene that is required to produce short-wavelength sensitive (S) cone photopigment. Consequently, Aotus retains only a single type of cone photopigment. Other mammals have since been found to show similar losses and it has often been speculated that such change is in some fashion tied to nocturnality. Although most species of Aotus are indeed nocturnal, recent observations show that Aotus azarai, an owl monkey species native to portions of Argentina and Paraguay, displays a cathemeral activity pattern being active during daylight hours as frequently as during nighttime hours. We have sequenced portions of the S-cone opsin gene in A. azarai and Aotus nancymaae, the latter a typically nocturnal species. The S-cone opsin genes in both species contain the same fatal defects earlier detected for Aotus trivirgatus. On the basis of the phylogenetic relationships of these three species these results imply that Aotus must have lost a capacity for color vision early in its history and they also suggest that the absence of color vision is not compulsively linked to a nocturnal lifestyle.

  16. RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata - a species with color-based sexual selection and 11 visual-opsin genes

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    Taylor John S

    2011-03-01

    Full Text Available Abstract Background PCR-based surveys have shown that guppies (Poecilia reticulata have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system. Results Juvenile and adult (male and female guppies express 10 visual opsins at varying levels in the eye. Two opsin genes in juveniles, SWS2B and RH2-2, accounted for >85% of all visual-opsin transcripts in the eye, excluding RH1. This relative abundance (RA value dropped to about 65% in adults, as LWS-A180 expression increased from approximately 3% to 20% RA. The juvenile-to-female transition also showed LWS-S180 upregulation from about 1.5% to 7% RA. Finally, we found that expression in guppies' SWS2-LWS gene cluster is negatively correlated with distance from a candidate locus control region (LCR. Conclusions Selective pressures influencing visual-opsin gene expression appear to differ among age and sex. LWS upregulation in females is implicated in augmenting spectral discrimination of male coloration and courtship displays. In males, enhanced discrimination of carotenoid-rich food and possibly rival males are strong candidate selective pressures driving LWS upregulation. These developmental changes in expression suggest that adults possess better wavelength discrimination than juveniles. Opsin expression within the SWS2-LWS gene cluster appears to be regulated, in part, by a common LCR. Finally, by comparing our RT-qPCR data to MSP data, we

  17. Genomic and gene regulatory signatures of cryptozoic adaptation: Loss of blue sensitive photoreceptors through expansion of long wavelength-opsin expression in the red flour beetle Tribolium castaneum

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    Cook Tiffany A

    2007-12-01

    Full Text Available Abstract Background Recent genome sequence analysis in the red flour beetle Tribolium castaneum indicated that this highly crepuscular animal encodes only two single opsin paralogs: a UV-opsin and a long wavelength (LW-opsin; however, these animals do not encode a blue (B-opsin as most other insects. Here, we studied the spatial regulation of the Tribolium single LW- and UV-opsin gene paralogs in comparison to that of the five opsin paralogs in the retina of Drosophila melanogaster. Results In situ hybridization analysis reveals that the Tribolium retina, in contrast with other insect retinas, constitutes a homogenous field of ommatidia that have seven LW-opsin expressing photoreceptors and one UV-/LW-opsin co-expressing photoreceptor per eye unit. This pattern is consistent with the loss of photoreceptors sensitive to blue wavelengths. It also identifies Tribolium as the first example of a species in insects that co-expresses two different opsins across the entire retina in violation of the widely observed "one receptor rule" of sensory cells. Conclusion Broader studies of opsin evolution in darkling beetles and other coleopteran groups have the potential to pinpoint the permissive and adaptive forces that played a role in the evolution of vision in Tribolium castaneum.

  18. Differentially-expressed opsin genes identified in Sinocyclocheilus cavefish endemic to China

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    Fanwei MENG; Yahui ZHAO; John H.POSTLETHWAIT; Chunguang ZHANG

    2013-01-01

    Eye degeneration is a common troglomorphic character of cave-dwelling organisms.Comparing the morphology and molecular biology of cave species and their close surface relatives is a powerful tool for studying regressive eye evolution and other adaptive phenotypes.We compared two co-occurring and closely-related species of the fish genus Sinocyclocheilus,which is endemic to China and includes both surface-and cave-dwelling species.Sinocyclocheilus tileihornes,a cave species,had smaller eyes than Sinocyclocheilus angustiporus,a surface species.Histological and immunohistochemical analyses revealed that the cavefish had shorter cones and more disorderly rods than did the surface-dwelling species.Using quantitative PCR and in situ hybridization,we found that rhodopsin and a long-wavelength sensitive opsin had significantly lower expression levels in the cavefish.Furthermore,one of two short-wavelength-sensitive opsins was expressed at significantly higher levels in the cavefish.Changes in the expression ofopsin genes may have played a role in the degeneration of cavefish eyes.

  19. Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

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    Schnitzler Christine E

    2012-12-01

    Full Text Available Abstract Background Calcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria and comb jellies (Phylum Ctenophora. The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis. Results The Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light

  20. Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes.

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    Schnitzler, Christine E; Pang, Kevin; Powers, Meghan L; Reitzel, Adam M; Ryan, Joseph F; Simmons, David; Tada, Takashi; Park, Morgan; Gupta, Jyoti; Brooks, Shelise Y; Blakesley, Robert W; Yokoyama, Shozo; Haddock, Steven Hd; Martindale, Mark Q; Baxevanis, Andreas D

    2012-12-21

    Calcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis. The Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light

  1. The expression of three opsin genes from the compound eye of Helicoverpa armigera (Lepidoptera: Noctuidae) is regulated by a circadian clock, light conditions and nutritional status

    National Research Council Canada - National Science Library

    Yan, Shuo; Zhu, Jialin; Zhu, Weilong; Zhang, Xinfang; Li, Zhen; Liu, Xiaoxia; Zhang, Qingwen

    2014-01-01

    .... In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR...

  2. The effect of white light on normal and malignant murine melanocytes: A link between opsins, clock genes, and melanogenesis.

    Science.gov (United States)

    de Assis, L V M; Moraes, M N; da Silveira Cruz-Machado, S; Castrucci, A M L

    2016-06-01

    The skin possesses a photosensitive system comprised of opsins whose function is not fully understood, and clock genes which exert an important regulatory role in skin biology. Here, we evaluated the presence of opsins in normal (Melan-a cells) and malignant (B16-F10 cells) murine melanocytes. Both cell lines express Opn2, Opn4--for the first time reported in these cell types--as well as S-opsin. OPN4 protein was found in a small area capping the cell nuclei of B16-F10 cells kept in constant dark (DD); twenty-four hours after the white light pulse (WLP), OPN4 was found in the cell membrane. Despite the fact that B16-F10 cells expressed less Opn2 and Opn4 than Melan-a cells, our data indicate that the malignant melanocytes exhibited increased photoresponsiveness. The clock gene machinery is also severely downregulated in B16-F10 cells as compared to Melan-a cells. Per1, Per2, and Bmal1 expression increased in B16-F10 cells in response to WLP. Although no response in clock gene expression to WLP was observed in Melan-a cells, gene correlational data suggest a minor effect of WLP. In contrast to opsins and clock genes, melanogenesis is significantly upregulated in malignant melanocytes in comparison to Melan-a cells. Tyrosinase expression increased after WLP only in B16-F10 cells; however no increase in melanin content after WLP was seen in either cell line. Our findings may prove useful in the treatment and the development of new pharmacological approaches of depigmentation diseases and skin cancer.

  3. The expression of three opsin genes from the compound eye of Helicoverpa armigera (Lepidoptera: Noctuidae is regulated by a circadian clock, light conditions and nutritional status.

    Directory of Open Access Journals (Sweden)

    Shuo Yan

    Full Text Available Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV, blue (BL and long-wavelength-sensitive (LW opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths.

  4. Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African cichlid fishes

    National Research Council Canada - National Science Library

    O'Quin, Kelly E; Smith, Daniel; Naseer, Zan; Schulte, Jane; Engel, Samuel D; Loh, Yong-Hwee E; Streelman, J Todd; Boore, Jeffrey L; Carleton, Karen L

    2011-01-01

    .... We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory...

  5. Characterization of Opsin Gene Alleles Affecting Color Vision in a Wild Population of Titi Monkeys (Callicebus brunneus)

    Science.gov (United States)

    Bunce, John A.; Isbell, Lynne A.; Neitz, Maureen; Bonci, Daniela; Surridge, Alison K.; Jacobs, Gerald H.; Smith, David Glenn

    2011-01-01

    The color vision of most platyrrhine primates is determined by alleles at the polymorphic X-linked locus coding for the opsin responsible for the middle- to long-wavelength (M/L) cone photopigment. Females who are heterozygous at the locus have trichromatic vision while homozygous females and all males are dichromatic. This study characterized the opsin alleles in a wild population of the socially monogamous platyrrhine monkey Callicebus brunneus (the brown titi monkey), a primate that an earlier study suggests may possess an unusual number of alleles at this locus and thus may be a subject of special interest in the study of primate color vision. Direct sequencing of regions of the M/L opsin gene using feces-, blood-, and saliva-derived DNA obtained from 14 individuals yielded evidence for the presence of three functionally distinct alleles, corresponding to the most common M/L photopigment variants inferred from a physiological study of cone spectral sensitivity in captive Callicebus. PMID:20938927

  6. Multiple rod-cone and cone-rod photoreceptor transmutations in snakes: evidence from visual opsin gene expression.

    Science.gov (United States)

    Simões, Bruno F; Sampaio, Filipa L; Loew, Ellis R; Sanders, Kate L; Fisher, Robert N; Hart, Nathan S; Hunt, David M; Partridge, Julian C; Gower, David J

    2016-01-27

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor 'transmutation'. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels.

  7. Multiple rod–cone and cone–rod photoreceptor transmutations in snakes: Evidence from visual opsin gene expression

    Science.gov (United States)

    Simoe, Bruno F; Sampaio, Filipa L.; Loew, Ellis R.; Sanders, Kate L.; Fisher, Robert N.; Hart, Nathan S.; Hunt, David M.; Partridge, Julian C.; Gower, David J.

    2016-01-01

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor ‘transmutation’. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels.

  8. Myopia and Late-Onset Progressive Cone Dystrophy Associate to LVAVA/MVAVA Exon 3 Interchange Haplotypes of Opsin Genes on Chromosome X.

    Science.gov (United States)

    Orosz, Orsolya; Rajta, István; Vajas, Attila; Takács, Lili; Csutak, Adrienne; Fodor, Mariann; Kolozsvári, Bence; Resch, Miklós; Sényi, Katalin; Lesch, Balázs; Szabó, Viktória; Berta, András; Balogh, István; Losonczy, Gergely

    2017-03-01

    Rare interchange haplotypes in exon 3 of the OPN1LW and OPN1MW opsin genes cause X-linked myopia, color vision defect, and cone dysfunction. The severity of the disease varies on a broad scale from nonsyndromic high myopia to blue cone monochromatism. Here, we describe a new genotype-phenotype correlation attributed to rare exon 3 interchange haplotypes simultaneously present in the long- and middle-wavelength sensitive opsin genes (L- and M-opsin genes). A multigenerational family with X-linked high myopia and cone dystrophy was investigated. Affected male patients had infantile onset myopia with normal visual acuity and color vision until their forties. Visual acuity decreased thereafter, along with the development of severe protan and deutan color vision defects. A mild decrease in electroretinography response of cone photoreceptors was detected in childhood, which further deteriorated in middle-aged patients. Rods were also affected, however, to a lesser extent than cones. Clinical exome sequencing identified the LVAVA and MVAVA toxic haplotypes in the OPN1LW and OPN1MW opsin genes, respectively. Here, we show that LVAVA haplotype of the OPN1LW gene and MVAVA haplotype of the OPN1MW gene cause apparently nonsyndromic high myopia in young patients but lead to progressive cone-rod dystrophy with deuteranopia and protanopia in middle-aged patients corresponding to a previously unknown disease course. To the best of our knowledge, this is the first report on the joint effect of these toxic haplotypes in the two opsin genes on chromosome X.

  9. Gene conversion between red and defective green opsin gene in blue cone monochromacy

    Energy Technology Data Exchange (ETDEWEB)

    Reyniers, E.; Van Thienen, M.N.; De Boulle, K.; Willems, P.J. [Univ. of Antwerp (Belgium)] [and others

    1995-09-20

    Blue cone monochromacy is an X-linked condition in which the function of both the red pigment gene (RCP) and the green pigment gene (GCP) is impaired. Blue cone monochromacy can be due to a red/green gene array rearrangement existing of a single red/green hybrid gene and an inactivating C203R point mutation in both RCP and GCP. The flanking sequences of the C230R mutation in exon 4 of RCP were characteristic for GCP, indicating that this mutation was transferred from GCP into RCP by gene conversion. 23 refs., 3 figs., 1 tab.

  10. Deep Brain Photoreceptor (val-opsin) Gene Knockout Using CRISPR/Cas Affects Chorion Formation and Embryonic Hatching in the Zebrafish

    Science.gov (United States)

    Hang, Chong Yee; Moriya, Shogo; Ogawa, Satoshi; Parhar, Ishwar S.

    2016-01-01

    Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish. PMID:27792783

  11. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... patterns that increase activation of the innate immune system. Importantly, viral-vectored vaccines that act through the induction of one or more of these factors also may benefit from cytokine coadministration and increased antigen presentation. In order to increase immunogenicity to the level achieved...

  12. Losses of functional opsin genes, short-wavelength cone photopigments, and color vision--a significant trend in the evolution of mammalian vision.

    Science.gov (United States)

    Jacobs, Gerald H

    2013-03-01

    All mammalian cone photopigments are derived from the operation of representatives from two opsin gene families (SWS1 and LWS in marsupial and eutherian mammals; SWS2 and LWS in monotremes), a process that produces cone pigments with respective peak sensitivities in the short and middle-to-long wavelengths. With the exception of a number of primate taxa, the modal pattern for mammals is to have two types of cone photopigment, one drawn from each of the gene families. In recent years, it has been discovered that the SWS1 opsin genes of a widely divergent collection of eutherian mammals have accumulated mutational changes that render them nonfunctional. This alteration reduces the retinal complements of these species to a single cone type, thus rendering ordinary color vision impossible. At present, several dozen species from five mammalian orders have been identified as falling into this category, but the total number of mammalian species that have lost short-wavelength cones in this way is certain to be much larger, perhaps reaching as high as 10% of all species. A number of circumstances that might be used to explain this widespread cone loss can be identified. Among these, the single consistent fact is that the species so affected are nocturnal or, if they are not technically nocturnal, they at least feature retinal organizations that are typically associated with that lifestyle. At the same time, however, there are many nocturnal mammals that retain functional short-wavelength cones. Nocturnality thus appears to set the stage for loss of functional SWS1 opsin genes in mammals, but it cannot be the sole circumstance.

  13. Assessment of fusion gene status in sarcomas using a custom made fusion gene microarray.

    Directory of Open Access Journals (Sweden)

    Marthe Løvf

    Full Text Available Sarcomas are relatively rare malignancies and include a large number of histological subgroups. Based on morphology alone, the differential diagnoses of sarcoma subtypes can be challenging, but the identification of specific fusion genes aids correct diagnostication. The presence of individual fusion products are routinely investigated in Pathology labs. However, the methods used are time-consuming and based on prior knowledge about the expected fusion gene and often the most likely break-point. In this study, 16 sarcoma samples, representing seven different sarcoma subtypes with known fusion gene status from a diagnostic setting, were investigated using a fusion gene microarray. The microarray was designed to detect all possible exon-exon breakpoints between all known fusion genes in a single analysis. An automated scoring of the microarray data from the 38 known sarcoma-related fusion genes identified the correct fusion gene among the top-three hits in 11 of the samples. The analytical sensitivity may be further optimised, but we conclude that a sarcoma-fusion gene microarray is suitable as a time-saving screening tool to identify the majority of the correct fusion genes.

  14. Opsin clines in butterflies suggest novel roles for insect photopigments.

    Science.gov (United States)

    Frentiu, Francesca D; Yuan, Furong; Savage, Wesley K; Bernard, Gary D; Mullen, Sean P; Briscoe, Adriana D

    2015-02-01

    Opsins are ancient molecules that enable animal vision by coupling to a vitamin-derived chromophore to form light-sensitive photopigments. The primary drivers of evolutionary diversification in opsins are thought to be visual tasks related to spectral sensitivity and color vision. Typically, only a few opsin amino acid sites affect photopigment spectral sensitivity. We show that opsin genes of the North American butterfly Limenitis arthemis have diversified along a latitudinal cline, consistent with natural selection due to environmental factors. We sequenced single nucleotide (SNP) polymorphisms in the coding regions of the ultraviolet (UVRh), blue (BRh), and long-wavelength (LWRh) opsin genes from ten butterfly populations along the eastern United States and found that a majority of opsin SNPs showed significant clinal variation. Outlier detection and analysis of molecular variance indicated that many SNPs are under balancing selection and show significant population structure. This contrasts with what we found by analysing SNPs in the wingless and EF-1 alpha loci, and from neutral amplified fragment length polymorphisms, which show no evidence of significant locus-specific or genome-wide structure among populations. Using a combination of functional genetic and physiological approaches, including expression in cell culture, transgenic Drosophila, UV-visible spectroscopy, and optophysiology, we show that key BRh opsin SNPs that vary clinally have almost no effect on spectral sensitivity. Our results suggest that opsin diversification in this butterfly is more consistent with natural selection unrelated to spectral tuning. Some of the clinally varying SNPs may instead play a role in regulating opsin gene expression levels or the thermostability of the opsin protein. Lastly, we discuss the possibility that insect opsins might have important, yet-to-be elucidated, adaptive functions in mediating animal responses to abiotic factors, such as temperature or photoperiod.

  15. ETS fusion genes in prostate cancer.

    Science.gov (United States)

    Gasi Tandefelt, Delila; Boormans, Joost; Hermans, Karin; Trapman, Jan

    2014-06-01

    Prostate cancer is very common in elderly men in developed countries. Unravelling the molecular and biological processes that contribute to tumor development and progressive growth, including its heterogeneity, is a challenging task. The fusion of the genes ERG and TMPRSS2 is the most frequent genomic alteration in prostate cancer. ERG is an oncogene that encodes a member of the family of ETS transcription factors. At lower frequency, other members of this gene family are also rearranged and overexpressed in prostate cancer. TMPRSS2 is an androgen-regulated gene that is preferentially expressed in the prostate. Most of the less frequent ETS fusion partners are also androgen-regulated and prostate-specific. During the last few years, novel concepts of the process of gene fusion have emerged, and initial experimental results explaining the function of the ETS genes ERG and ETV1 in prostate cancer have been published. In this review, we focus on the most relevant ETS gene fusions and summarize the current knowledge of the role of ETS transcription factors in prostate cancer. Finally, we discuss the clinical relevance of TMRPSS2-ERG and other ETS gene fusions in prostate cancer.

  16. Palmitoylation stabilizes unliganded rod opsin

    OpenAIRE

    2010-01-01

    S-palmitoylation is a conserved feature in many G protein–coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm−/−) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound lig...

  17. In silico characterisation and chromosomal localisation of human RRH (peropsin – implications for opsin evolution

    Directory of Open Access Journals (Sweden)

    Foster Russell G

    2003-01-01

    Full Text Available Abstract Background The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue, RRH is unknown. Results By in silico analysis of the GenBank database we have determined that the human RRH comprises 7 exons spanning approximately 16.5 kb and is localised to chromosome 4q25 in the following gene sequence: cen-EGF-RRH-IF-qter – a position that excludes this gene as a candidate for the RP29 autosomal recessive retinitis pigmentosa locus. A comparison of opsin gene structures reveals that RRH and RGR share two common intron (introns 1 and 4 insertion positions which may reflect a shared ancestral gene. Conclusion The opsins comprise a diverse group of genes which appear to have arisen from three different lineages. These lineages comprise the "classical opsin superfamily" which includes the rod and cone opsins, pinopsin, VA-opsin, parapinopsin and encephalopsin; the RRH and RGR group; and the melanopsin line. A common lineage for RRH and RGR, together with their sites of expression in the RPE, indicates that peropsin may act as a retinal isomerase.

  18. Gene Fusion Markup Language: a prototype for exchanging gene fusion data.

    Science.gov (United States)

    Kalyana-Sundaram, Shanker; Shanmugam, Achiraman; Chinnaiyan, Arul M

    2012-10-16

    An avalanche of next generation sequencing (NGS) studies has generated an unprecedented amount of genomic structural variation data. These studies have also identified many novel gene fusion candidates with more detailed resolution than previously achieved. However, in the excitement and necessity of publishing the observations from this recently developed cutting-edge technology, no community standardization approach has arisen to organize and represent the data with the essential attributes in an interchangeable manner. As transcriptome studies have been widely used for gene fusion discoveries, the current non-standard mode of data representation could potentially impede data accessibility, critical analyses, and further discoveries in the near future. Here we propose a prototype, Gene Fusion Markup Language (GFML) as an initiative to provide a standard format for organizing and representing the significant features of gene fusion data. GFML will offer the advantage of representing the data in a machine-readable format to enable data exchange, automated analysis interpretation, and independent verification. As this database-independent exchange initiative evolves it will further facilitate the formation of related databases, repositories, and analysis tools. The GFML prototype is made available at http://code.google.com/p/gfml-prototype/. The Gene Fusion Markup Language (GFML) presented here could facilitate the development of a standard format for organizing, integrating and representing the significant features of gene fusion data in an inter-operable and query-able fashion that will enable biologically intuitive access to gene fusion findings and expedite functional characterization. A similar model is envisaged for other NGS data analyses.

  19. Euarchontan Opsin Variation Brings New Focus to Primate Origins

    OpenAIRE

    Melin, Amanda D.; Wells, Konstans; Moritz, Gillian L.; Kistler, Logan; Orkin, Joseph D.; Timm, Robert M.; Bernard, Henry; Lakim, Maklarin B.; Perry, George H.; Kawamura, Shoji; Dominy, Nathaniel J.

    2016-01-01

    Debate on the adaptive origins of primates has long focused on the functional ecology of the primate visual system. For example, it is hypothesized that variable expression of short- (SWS1) and middle-to-long-wavelength sensitive (M/LWS) opsins, which confer color vision, can be used to infer ancestral activity patterns and therefore selective ecological pressures. A problem with this approach is that opsin gene variation is incompletely known in the grandorder Euarchonta, that is, the orders...

  20. Reconstructing the ancestral butterfly eye: focus on the opsins.

    Science.gov (United States)

    Briscoe, Adriana D

    2008-06-01

    The eyes of butterflies are remarkable, because they are nearly as diverse as the colors of wings. Much of eye diversity can be traced to alterations in the number, spectral properties and spatial distribution of the visual pigments. Visual pigments are light-sensitive molecules composed of an opsin protein and a chromophore. Most butterflies have eyes that contain visual pigments with a wavelength of peak absorbance, lambda(max), in the ultraviolet (UV, 300-400 nm), blue (B, 400-500 nm) and long wavelength (LW, 500-600 nm) part of the visible light spectrum, respectively, encoded by distinct UV, B and LW opsin genes. In the compound eye of butterflies, each individual ommatidium is composed of nine photoreceptor cells (R1-9) that generally express only one opsin mRNA per cell, although in some butterfly eyes there are ommatidial subtypes in which two opsins are co-expressed in the same photoreceptor cell. Based on a phylogenetic analysis of opsin cDNAs from the five butterfly families, Papilionidae, Pieridae, Nymphalidae, Lycaenidae and Riodinidae, and comparative analysis of opsin gene expression patterns from four of the five families, I propose a model for the patterning of the ancestral butterfly eye that is most closely aligned with the nymphalid eye. The R1 and R2 cells of the main retina expressed UV-UV-, UV-B- or B-B-absorbing visual pigments while the R3-9 cells expressed a LW-absorbing visual pigment. Visual systems of existing butterflies then underwent an adaptive expansion based on lineage-specific B and LW opsin gene multiplications and on alterations in the spatial expression of opsins within the eye. Understanding the molecular sophistication of butterfly eye complexity is a challenge that, if met, has broad biological implications.

  1. Opsins have evolved under the permanent heterozygote model: insights from phylotranscriptomics of Odonata.

    Science.gov (United States)

    Suvorov, Anton; Jensen, Nicholas O; Sharkey, Camilla R; Fujimoto, M Stanley; Bodily, Paul; Wightman, Haley M Cahill; Ogden, T Heath; Clement, Mark J; Bybee, Seth M

    2017-03-01

    Gene duplication plays a central role in adaptation to novel environments by providing new genetic material for functional divergence and evolution of biological complexity. Several evolutionary models have been proposed for gene duplication to explain how new gene copies are preserved by natural selection, but these models have rarely been tested using empirical data. Opsin proteins, when combined with a chromophore, form a photopigment that is responsible for the absorption of light, the first step in the phototransduction cascade. Adaptive gene duplications have occurred many times within the animal opsins' gene family, leading to novel wavelength sensitivities. Consequently, opsins are an attractive choice for the study of gene duplication evolutionary models. Odonata (dragonflies and damselflies) have the largest opsin repertoire of any insect currently known. Additionally, there is tremendous variation in opsin copy number between species, particularly in the long-wavelength-sensitive (LWS) class. Using comprehensive phylotranscriptomic and statistical approaches, we tested various evolutionary models of gene duplication. Our results suggest that both the blue-sensitive (BS) and LWS opsin classes were subjected to strong positive selection that greatly weakens after multiple duplication events, a pattern that is consistent with the permanent heterozygote model. Due to the immense interspecific variation and duplicability potential of opsin genes among odonates, they represent a unique model system to test hypotheses regarding opsin gene duplication and diversification at the molecular level.

  2. Genome Fusion Detection: a novel method to detect fusion genes from SNP-array data.

    Science.gov (United States)

    Thieme, Sebastian; Groth, Philip

    2013-03-15

    Fusion genes result from genomic rearrangements, such as deletions, amplifications and translocations. Such rearrangements can also frequently be observed in cancer and have been postulated as driving event in cancer development. to detect them, one needs to analyze the transition region of two segments with different copy number, the location where fusions are known to occur. Finding fusion genes is essential to understanding cancer development and may lead to new therapeutic approaches. Here we present a novel method, the Genomic Fusion Detection algorithm, to predict fusion genes on a genomic level based on SNP-array data. This algorithm detects genes at the transition region of segments with copy number variation. With the application of defined constraints, certain properties of the detected genes are evaluated to predict whether they may be fused. We evaluated our prediction by calculating the observed frequency of known fusions in both primary cancers and cell lines. We tested a set of cell lines positive for the BCR-ABL1 fusion and prostate cancers positive for the TMPRSS2-ERG fusion. We could detect the fusions in all positive cell lines, but not in the negative controls.

  3. The Evolution and Expression of the Moth Visual Opsin Family

    Science.gov (United States)

    Fu, Xiaowei; Murphy, Robert W.; Wu, Kongming

    2013-01-01

    Because visual genes likely evolved in response to their ambient photic environment, the dichotomy between closely related nocturnal moths and diurnal butterflies forms an ideal basis for investigating their evolution. To investigate whether the visual genes of moths are associated with nocturnal dim-light environments or not, we cloned long-wavelength (R), blue (B) and ultraviolet (UV) opsin genes from 12 species of wild-captured moths and examined their evolutionary functions. Strong purifying selection appeared to constrain the functions of the genes. Dark-treatment altered the levels of mRNA expression in Helicoverpa armigera such that R and UV opsins were up-regulated after dark-treatment, the latter faster than the former. In contrast, B opsins were not significantly up-regulated. Diel changes of opsin mRNA levels in both wild-captured and lab-reared individuals showed no significant fluctuation within the same group. However, the former group had significantly elevated levels of expression compared with the latter. Consequently, environmental conditions appeared to affect the patterns of expression. These findings and the proportional expression of opsins suggested that moths potentially possessed color vision and the visual system played a more important role in the ecology of moths than previously appreciated. This aspect did not differ much from that of diurnal butterflies. PMID:24205129

  4. The evolution and expression of the moth visual opsin family.

    Directory of Open Access Journals (Sweden)

    Pengjun Xu

    Full Text Available Because visual genes likely evolved in response to their ambient photic environment, the dichotomy between closely related nocturnal moths and diurnal butterflies forms an ideal basis for investigating their evolution. To investigate whether the visual genes of moths are associated with nocturnal dim-light environments or not, we cloned long-wavelength (R, blue (B and ultraviolet (UV opsin genes from 12 species of wild-captured moths and examined their evolutionary functions. Strong purifying selection appeared to constrain the functions of the genes. Dark-treatment altered the levels of mRNA expression in Helicoverpa armigera such that R and UV opsins were up-regulated after dark-treatment, the latter faster than the former. In contrast, B opsins were not significantly up-regulated. Diel changes of opsin mRNA levels in both wild-captured and lab-reared individuals showed no significant fluctuation within the same group. However, the former group had significantly elevated levels of expression compared with the latter. Consequently, environmental conditions appeared to affect the patterns of expression. These findings and the proportional expression of opsins suggested that moths potentially possessed color vision and the visual system played a more important role in the ecology of moths than previously appreciated. This aspect did not differ much from that of diurnal butterflies.

  5. Identification of targetable FGFR gene fusions in diverse cancers.

    Science.gov (United States)

    Wu, Yi-Mi; Su, Fengyun; Kalyana-Sundaram, Shanker; Khazanov, Nickolay; Ateeq, Bushra; Cao, Xuhong; Lonigro, Robert J; Vats, Pankaj; Wang, Rui; Lin, Su-Fang; Cheng, Ann-Joy; Kunju, Lakshmi P; Siddiqui, Javed; Tomlins, Scott A; Wyngaard, Peter; Sadis, Seth; Roychowdhury, Sameek; Hussain, Maha H; Feng, Felix Y; Zalupski, Mark M; Talpaz, Moshe; Pienta, Kenneth J; Rhodes, Daniel R; Robinson, Dan R; Chinnaiyan, Arul M

    2013-06-01

    Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

  6. Evolutionary Dynamics of Rhodopsin Type 2 Opsins in Vertebrates

    Science.gov (United States)

    Yokoyama, Shozo; Tada, Takashi

    2010-01-01

    Among the five groups of visual pigments in vertebrates, the rhodopsin type 2 (RH2) group shows the largest number of gene duplication events. We have isolated three intact and one nonfunctional RH2 opsin genes each from Northern lampfish (Stenobrachius leucopsarus) and scabbardfish (Lepidopus fitchi). Using the deduced amino acid sequences of these and other representative RH2 opsin genes in vertebrates, we have estimated the divergence times and evolutionary rates of amino acid substitution at various stages of RH2 opsin evolution. The results show that the duplications of the lampfish and scabbardfish RH2 opsins have occurred ∼60 and ∼30 million years ago (Ma), respectively. The evolutionary rates of RH2 opsins in the early vertebrate ancestors were ∼0.25 × 10−9/site/year, which increased to ∼1 × 10−9 to 3 × 10−9/site/year in euteleost lineages and to ∼0.3 × 10−9 to 0.5 × 10−9/site/year in coelacanth and tetrapods. PMID:19759234

  7. Retention of duplicated long-wavelength opsins in mosquito lineages by positive selection and differential expression.

    Science.gov (United States)

    Giraldo-Calderón, Gloria I; Zanis, Michael J; Hill, Catherine A

    2017-03-21

    Opsins are light sensitive receptors associated with visual processes. Insects typically possess opsins that are stimulated by ultraviolet, short and long wavelength (LW) radiation. Six putative LW-sensitive opsins predicted in the yellow fever mosquito, Aedes aegypti and malaria mosquito, Anopheles gambiae, and eight in the southern house mosquito, Culex quinquefasciatus, suggest gene expansion in the Family Culicidae (mosquitoes) relative to other insects. Here we report the first detailed molecular and evolutionary analyses of LW opsins in three mosquito vectors, with a goal to understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control. Time of divergence estimates suggest that the mosquito LW opsins originated from 18 or 19 duplication events between 166.9/197.5 to 1.07/0.94 million years ago (MY) and that these likely occurred following the predicted divergence of the lineages Anophelinae and Culicinae 145-226 MY. Fitmodel analyses identified nine amino acid residues in the LW opsins that may be under positive selection. Of these, eight amino acids occur in the N and C termini and are shared among all three species, and one residue in TMIII was unique to culicine species. Alignment of 5' non-coding regions revealed potential Conserved Non-coding Sequences (CNS) and transcription factor binding sites (TFBS) in seven pairs of LW opsin paralogs. Our analyses suggest opsin gene duplication and residues possibly associated with spectral tuning of LW-sensitive photoreceptors. We explore two mechanisms - positive selection and differential expression mediated by regulatory units in CNS - that may have contributed to the retention of LW opsin genes in Culicinae and Anophelinae. We discuss the evolution of mosquito LW opsins in the context of major Earth events and possible adaptation of mosquitoes to LW-dominated photo environments, and implications for mosquito control strategies based on disrupting vision

  8. Molecular pathways: targeting ETS gene fusions in cancer.

    Science.gov (United States)

    Feng, Felix Y; Brenner, J Chad; Hussain, Maha; Chinnaiyan, Arul M

    2014-09-01

    Rearrangements, or gene fusions, involving the ETS family of transcription factors are common driving events in both prostate cancer and Ewing sarcoma. These rearrangements result in pathogenic expression of the ETS genes and trigger activation of transcriptional programs enriched for invasion and other oncogenic features. Although ETS gene fusions represent intriguing therapeutic targets, transcription factors, such as those comprising the ETS family, have been notoriously difficult to target. Recently, preclinical studies have demonstrated an association between ETS gene fusions and components of the DNA damage response pathway, such as PARP1, the catalytic subunit of DNA protein kinase (DNAPK), and histone deactylase 1 (HDAC1), and have suggested that ETS fusions may confer sensitivity to inhibitors of these DNA repair proteins. In this review, we discuss the role of ETS fusions in cancer, the preclinical rationale for targeting ETS fusions with inhibitors of PARP1, DNAPK, and HDAC1, as well as ongoing clinical trials targeting ETS gene fusions. ©2014 American Association for Cancer Research.

  9. Characterisation and localisation of the opsin protein repertoire in the brain and retinas of a spider and an onychophoran

    National Research Council Canada - National Science Library

    Eriksson, Bo Joakim; Fredman, David; Steiner, Gerhard; Schmid, Axel

    2013-01-01

    .... Opsins can be divided into three main groups: rhabdomeric opsins (r-opsins), ciliary opsins (c-opsins) and group 4 opsins. In arthropods, the main focus has been on the r-opsins involved in vision...

  10. Opsin evolution and expression in Arthropod compound Eyes and Ocelli: Insights from the cricket Gryllus bimaculatus

    Directory of Open Access Journals (Sweden)

    Henze Miriam J

    2012-08-01

    taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves.

  11. FGFR-TACC gene fusions in human glioma.

    Science.gov (United States)

    Lasorella, Anna; Sanson, Marc; Iavarone, Antonio

    2016-11-16

    Chromosomal translocations joining in-frame members of the fibroblast growth factor receptor-transforming acidic coiled-coil gene families (the FGFR-TACC gene fusions) were first discovered in human glioblastoma multiforme (GBM) and later in many other cancer types. Here, we review this rapidly expanding field of research and discuss the unique biological and clinical features conferred to isocitrate dehydrogenase wild-type glioma cells by FGFR-TACC fusions. FGFR-TACC fusions generate powerful oncogenes that combine growth-promoting effects with aneuploidy through the activation of as yet unclear intracellular signaling mechanisms. FGFR-TACC fusions appear to be clonal tumor-initiating events that confer strong sensitivity to FGFR tyrosine kinase inhibitors. Screening assays have recently been reported for the accurate identification of FGFR-TACC fusion variants in human cancer, and early clinical data have shown promising effects in cancer patients harboring FGFR-TACC fusions and treated with FGFR inhibitors. Thus, FGFR-TACC gene fusions provide a "low-hanging fruit" model for the validation of precision medicine paradigms in human GBM.

  12. Euarchontan Opsin Variation Brings New Focus to Primate Origins.

    Science.gov (United States)

    Melin, Amanda D; Wells, Konstans; Moritz, Gillian L; Kistler, Logan; Orkin, Joseph D; Timm, Robert M; Bernard, Henry; Lakim, Maklarin B; Perry, George H; Kawamura, Shoji; Dominy, Nathaniel J

    2016-04-01

    Debate on the adaptive origins of primates has long focused on the functional ecology of the primate visual system. For example, it is hypothesized that variable expression of short- (SWS1) and middle-to-long-wavelength sensitive (M/LWS) opsins, which confer color vision, can be used to infer ancestral activity patterns and therefore selective ecological pressures. A problem with this approach is that opsin gene variation is incompletely known in the grandorder Euarchonta, that is, the orders Scandentia (treeshrews), Dermoptera (colugos), and Primates. The ancestral state of primate color vision is therefore uncertain. Here, we report on the genes (OPN1SW and OPN1LW) that encode SWS1 and M/LWS opsins in seven species of treeshrew, including the sole nocturnal scandentian Ptilocercus lowii. In addition, we examined the opsin genes of the Central American woolly opossum (Caluromys derbianus), an enduring ecological analogue in the debate on primate origins. Our results indicate: 1) retention of ultraviolet (UV) visual sensitivity in C. derbianus and a shift from UV to blue spectral sensitivities at the base of Euarchonta; 2) ancient pseudogenization of OPN1SW in the ancestors of P. lowii, but a signature of purifying selection in those of C. derbianus; and, 3) the absence of OPN1LW polymorphism among diurnal treeshrews. These findings suggest functional variation in the color vision of nocturnal mammals and a distinctive visual ecology of early primates, perhaps one that demanded greater spatial resolution under light levels that could support cone-mediated color discrimination.

  13. Opsin Repertoire and Expression Patterns in Horseshoe Crabs: Evidence from the Genome of Limulus polyphemus (Arthropoda: Chelicerata).

    Science.gov (United States)

    Battelle, Barbara-Anne; Ryan, Joseph F; Kempler, Karen E; Saraf, Spencer R; Marten, Catherine E; Warren, Wesley C; Minx, Patrick J; Montague, Michael J; Green, Pamela J; Schmidt, Skye A; Fulton, Lucinda; Patel, Nipam H; Protas, Meredith E; Wilson, Richard K; Porter, Megan L

    2016-06-03

    Horseshoe crabs are xiphosuran chelicerates, the sister group to arachnids. As such, they are important for understanding the most recent common ancestor of Euchelicerata and the evolution and diversification of Arthropoda. Limulus polyphemus is the most investigated of the four extant species of horseshoe crabs, and the structure and function of its visual system have long been a major focus of studies critical for understanding the evolution of visual systems in arthropods. Likewise, studies of genes encoding Limulus opsins, the protein component of the visual pigments, are critical for understanding opsin evolution and diversification among chelicerates, where knowledge of opsins is limited, and more broadly among arthropods. In the present study, we sequenced and assembled a high quality nuclear genomic sequence of L. polyphemus and used these data to annotate the full repertoire of Limulus opsins. We conducted a detailed phylogenetic analysis of Limulus opsins, including using gene structure and synteny information to identify relationships among different opsin classes. We used our phylogeny to identify significant genomic events that shaped opsin evolution and therefore the visual system of Limulus We also describe the tissue expression patterns of the 18 opsins identified and show that transcripts encoding a number, including a peropsin, are present throughout the central nervous system. In addition to significantly extending our understanding of photosensitivity in Limulus and providing critical insight into the genomic evolution of horseshoe crab opsins, this work provides a valuable genomic resource for addressing myriad questions related to xiphosuran physiology and arthropod evolution.

  14. Multi-characteristic opsin enabled vision restoration

    Science.gov (United States)

    Wright, Weldon; Pradhan, Sanjay; Bhattacharya, Sulgana; Mahapatra, Vasu; Tripathy, Ashutosh; Gajjeraman, Sivakumar; Mohanty, Samarendra

    2017-02-01

    Photodegenerative retinal diseases such as retinitis pigmentosa (RP) and dry age related macular degeneration (dry- AMD) lead to loss of vision in millions of individuals. Currently, no surgical or medical treatment is available though optogenetic therapies are in clinical development. Here, we demonstrate vision restoration using Multi- Characteristics Opsin (MCO1) in animal models with photo-degenerated retina. MCO1 is reliably delivered to specific retinal cells via intravitreal injection of Adeno-Associated Virus, leading to significant improvement in visually guided behavior conducted using a radial-arm water maze. The time to reach platform significantly reduced after delivery of MCO1. Notably, the improvement in visually guided behavior was observed even at light intensity levels orders of magnitude lower than that required for Channelrhodopsin-2 opsin. Chronic light exposure study showed that chronic light exposure did not compromise viability of vMCO1-treated retina. Safe virus-mediated MCO1-delivery has potential for effective gene therapy of diverse retinal degenerations in patients.

  15. Rod monochromacy and the coevolution of cetacean retinal opsins.

    Science.gov (United States)

    Meredith, Robert W; Gatesy, John; Emerling, Christopher A; York, Vincent M; Springer, Mark S

    2013-04-01

    Cetaceans have a long history of commitment to a fully aquatic lifestyle that extends back to the Eocene. Extant species have evolved a spectacular array of adaptations in conjunction with their deployment into a diverse array of aquatic habitats. Sensory systems are among those that have experienced radical transformations in the evolutionary history of this clade. In the case of vision, previous studies have demonstrated important changes in the genes encoding rod opsin (RH1), short-wavelength sensitive opsin 1 (SWS1), and long-wavelength sensitive opsin (LWS) in selected cetaceans, but have not examined the full complement of opsin genes across the complete range of cetacean families. Here, we report protein-coding sequences for RH1 and both color opsin genes (SWS1, LWS) from representatives of all extant cetacean families. We examine competing hypotheses pertaining to the timing of blue shifts in RH1 relative to SWS1 inactivation in the early history of Cetacea, and we test the hypothesis that some cetaceans are rod monochomats. Molecular evolutionary analyses contradict the "coastal" hypothesis, wherein SWS1 was pseudogenized in the common ancestor of Cetacea, and instead suggest that RH1 was blue-shifted in the common ancestor of Cetacea before SWS1 was independently knocked out in baleen whales (Mysticeti) and in toothed whales (Odontoceti). Further, molecular evidence implies that LWS was inactivated convergently on at least five occasions in Cetacea: (1) Balaenidae (bowhead and right whales), (2) Balaenopteroidea (rorquals plus gray whale), (3) Mesoplodon bidens (Sowerby's beaked whale), (4) Physeter macrocephalus (giant sperm whale), and (5) Kogia breviceps (pygmy sperm whale). All of these cetaceans are known to dive to depths of at least 100 m where the underwater light field is dim and dominated by blue light. The knockout of both SWS1 and LWS in multiple cetacean lineages renders these taxa rod monochromats, a condition previously unknown among

  16. Rod monochromacy and the coevolution of cetacean retinal opsins.

    Directory of Open Access Journals (Sweden)

    Robert W Meredith

    2013-04-01

    Full Text Available Cetaceans have a long history of commitment to a fully aquatic lifestyle that extends back to the Eocene. Extant species have evolved a spectacular array of adaptations in conjunction with their deployment into a diverse array of aquatic habitats. Sensory systems are among those that have experienced radical transformations in the evolutionary history of this clade. In the case of vision, previous studies have demonstrated important changes in the genes encoding rod opsin (RH1, short-wavelength sensitive opsin 1 (SWS1, and long-wavelength sensitive opsin (LWS in selected cetaceans, but have not examined the full complement of opsin genes across the complete range of cetacean families. Here, we report protein-coding sequences for RH1 and both color opsin genes (SWS1, LWS from representatives of all extant cetacean families. We examine competing hypotheses pertaining to the timing of blue shifts in RH1 relative to SWS1 inactivation in the early history of Cetacea, and we test the hypothesis that some cetaceans are rod monochomats. Molecular evolutionary analyses contradict the "coastal" hypothesis, wherein SWS1 was pseudogenized in the common ancestor of Cetacea, and instead suggest that RH1 was blue-shifted in the common ancestor of Cetacea before SWS1 was independently knocked out in baleen whales (Mysticeti and in toothed whales (Odontoceti. Further, molecular evidence implies that LWS was inactivated convergently on at least five occasions in Cetacea: (1 Balaenidae (bowhead and right whales, (2 Balaenopteroidea (rorquals plus gray whale, (3 Mesoplodon bidens (Sowerby's beaked whale, (4 Physeter macrocephalus (giant sperm whale, and (5 Kogia breviceps (pygmy sperm whale. All of these cetaceans are known to dive to depths of at least 100 m where the underwater light field is dim and dominated by blue light. The knockout of both SWS1 and LWS in multiple cetacean lineages renders these taxa rod monochromats, a condition previously unknown among

  17. Opsin vs opsin: New materials for biotechnological applications

    Science.gov (United States)

    Alfinito, Eleonora; Reggiani, Lino

    2014-08-01

    The need of new diagnostic methods satisfying, as an early detection, a low invasive procedure and a cost-efficient value, is orienting the technological research toward the use of bio-integrated devices, in particular, bio-sensors. The set of know-why necessary to achieve this goal is wide, from biochemistry to electronics and is summarized in an emerging branch of electronics, called proteotronics. Proteotronics is here applied to state a comparative analysis of the electrical responses coming from type-1 and type-2 opsins. In particular, the procedure is used as an early investigation of a recently discovered family of opsins, the proteorhodopsins activated by blue light, BPRs. The results reveal some interesting and unexpected similarities between proteins of the two families, suggesting the global electrical response are not strictly linked to the class identity.

  18. Pinpointing disease genes through phenomic and genomic data fusion.

    Science.gov (United States)

    Jiang, Rui; Wu, Mengmeng; Li, Lianshuo

    2015-01-01

    Pinpointing genes involved in inherited human diseases remains a great challenge in the post-genomics era. Although approaches have been proposed either based on the guilt-by-association principle or making use of disease phenotype similarities, the low coverage of both diseases and genes in existing methods has been preventing the scan of causative genes for a significant proportion of diseases at the whole-genome level. To overcome this limitation, we proposed a rigorous statistical method called pgFusion to prioritize candidate genes by integrating one type of disease phenotype similarity derived from the Unified Medical Language System (UMLS) and seven types of gene functional similarities calculated from gene expression, gene ontology, pathway membership, protein sequence, protein domain, protein-protein interaction and regulation pattern, respectively. Our method covered a total of 7,719 diseases and 20,327 genes, achieving the highest coverage thus far for both diseases and genes. We performed leave-one-out cross-validation experiments to demonstrate the superior performance of our method and applied it to a real exome sequencing dataset of epileptic encephalopathies, showing the capability of this approach in finding causative genes for complex diseases. We further provided the standalone software and online services of pgFusion at http://bioinfo.au.tsinghua.edu.cn/jianglab/pgfusion. pgFusion not only provided an effective way for prioritizing candidate genes, but also demonstrated feasible solutions to two fundamental questions in the analysis of big genomic data: the comparability of heterogeneous data and the integration of multiple types of data. Applications of this method in exome or whole genome sequencing studies would accelerate the finding of causative genes for human diseases. Other research fields in genomics could also benefit from the incorporation of our data fusion methodology.

  19. Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field

    Science.gov (United States)

    Dalton, Brian E.; Loew, Ellis R.; Cronin, Thomas W.; Carleton, Karen L.

    2014-01-01

    Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions. PMID:25377457

  20. [The progress of TMPRSS2-ETS gene fusions and their mechanism in prostate cancer].

    Science.gov (United States)

    Guo, Xiao-Qiang; Gui, Yao-Ting; Cai, Zhi-Ming

    2011-02-01

    The gene fusions between transmembrane protease serine 2 (TMPRSS2) and E26 (ETS) transcription factors are present in over 50% of patients with prostate cancer. TMPRSS2-ERG is the most common gene fusion type. The ERG overexpression induced by TMPRSS2-ERG gene fusion contributes to the development of prostate cancer. Both androgen receptor binding and genotoxic stress induce chromosomal proximity and TMPRSS2-ETS gene fusions. TMPRSS2-ERG gene fusion functions as a biomarker for prostate cancer, which can be easily detected in urine. This review focuses on the characteristics, oncogenic and rearranged mechanism, and clinical application of TMPRSS2-ETS gene fusions.

  1. Optogenetics: opsins and optical interfaces in neuroscience.

    Science.gov (United States)

    Adamantidis, Antoine R; Zhang, Feng; de Lecea, Luis; Deisseroth, Karl

    2014-08-01

    Optogenetics is defined as the integration of optics and genetics to control well-defined events within specified cells of living tissue. In this introduction, we focus on the basic techniques necessary for employing microbial opsins as optogenetic tools in mammalian brains. We provide a guide for the fundamentals of optogenetic application-selecting an opsin, implementing expression of opsins based on the neuroscientific experimental requirements, and adapting the corresponding optical hardware for delivery of light into mammalian brains.

  2. Diversity of Active States in TMT Opsins.

    Directory of Open Access Journals (Sweden)

    Kazumi Sakai

    Full Text Available Opn3/TMT opsins belong to one of the opsin groups with vertebrate visual and non-visual opsins, and are widely distributed in eyes, brains and other internal organs in various vertebrates and invertebrates. Vertebrate Opn3/TMT opsins are further classified into four groups on the basis of their amino acid identities. However, there is limited information about molecular properties of these groups, due to the difficulty in preparing the recombinant proteins. Here, we successfully expressed recombinant proteins of TMT1 and TMT2 opsins of medaka fish (Oryzias latipes in cultured cells and characterized their molecular properties. Spectroscopic and biochemical studies demonstrated that TMT1 and TMT2 opsins functioned as blue light-sensitive Gi/Go-coupled receptors, but exhibited spectral properties and photo-convertibility of the active state different from each other. TMT1 opsin forms a visible light-absorbing active state containing all-trans-retinal, which can be photo-converted to 7-cis- and 9-cis-retinal states in addition to the original 11-cis-retinal state. In contrast, the active state of TMT2 opsin is a UV light-absorbing state having all-trans-retinal and does not photo-convert to any other state, including the original 11-cis-retinal state. Thus, TMT opsins are diversified so as to form a different type of active state, which may be responsible for their different functions.

  3. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  4. Gene Prioritization by Compressive Data Fusion and Chaining.

    Directory of Open Access Journals (Sweden)

    Marinka Žitnik

    2015-10-01

    Full Text Available Data integration procedures combine heterogeneous data sets into predictive models, but they are limited to data explicitly related to the target object type, such as genes. Collage is a new data fusion approach to gene prioritization. It considers data sets of various association levels with the prediction task, utilizes collective matrix factorization to compress the data, and chaining to relate different object types contained in a data compendium. Collage prioritizes genes based on their similarity to several seed genes. We tested Collage by prioritizing bacterial response genes in Dictyostelium as a novel model system for prokaryote-eukaryote interactions. Using 4 seed genes and 14 data sets, only one of which was directly related to the bacterial response, Collage proposed 8 candidate genes that were readily validated as necessary for the response of Dictyostelium to Gram-negative bacteria. These findings establish Collage as a method for inferring biological knowledge from the integration of heterogeneous and coarsely related data sets.

  5. ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer

    Science.gov (United States)

    2015-10-01

    Award Number: W81XWH-10-1-0582 TITLE: ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer PRINCIPAL...ETS gene fusion status associated with clinical outcomes following radiation therapy , by analyzing both the collected biomarker and clinical data...denotes absence of an ERG fusion). ETS gene fusions status did not predict outcomes following radiation therapy , as demonstrated by Kaplan Meier

  6. Overcoming the loss of blue sensitivity through opsin duplication in the largest animal group, beetles.

    Science.gov (United States)

    Sharkey, Camilla R; Fujimoto, M Stanley; Lord, Nathan P; Shin, Seunggwan; McKenna, Duane D; Suvorov, Anton; Martin, Gavin J; Bybee, Seth M

    2017-12-01

    Opsin proteins are fundamental components of animal vision whose structure largely determines the sensitivity of visual pigments to different wavelengths of light. Surprisingly little is known about opsin evolution in beetles, even though they are the most species rich animal group on Earth and exhibit considerable variation in visual system sensitivities. We reveal the patterns of opsin evolution across 62 beetle species and relatives. Our results show that the major insect opsin class (SW) that typically confers sensitivity to "blue" wavelengths was lost ~300 million years ago, before the origin of modern beetles. We propose that UV and LW opsin gene duplications have restored the potential for trichromacy (three separate channels for colour vision) in beetles up to 12 times and more specifically, duplications within the UV opsin class have likely led to the restoration of "blue" sensitivity up to 10 times. This finding reveals unexpected plasticity within the insect visual system and highlights its remarkable ability to evolve and adapt to the available light and visual cues present in the environment.

  7. Rooting the eukaryote tree by using a derived gene fusion.

    Science.gov (United States)

    Stechmann, Alexandra; Cavalier-Smith, Thomas

    2002-07-05

    Single-gene trees have failed to locate the root of the eukaryote tree because of systematic biases in sequence evolution. Structural genetic data should yield more reliable insights into deep phylogenetic relationships. We searched major protist groups for the presence or absence of a gene fusion in order to locate the root of the eukaryote tree. In striking contrast to previous molecular studies, we show that all eukaryote groups ancestrally with two cilia (bikonts) are evolutionarily derived. The root lies between bikonts and opisthokonts (animals, Fungi, Choanozoa). Amoebozoa either diverged even earlier or are sister of bikonts or (less likely) opisthokonts.

  8. The Microbial Opsin Family of Optogenetic Tools

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Feng; Vierock, Johannes; Yizhar, Ofer; Fenno, Lief E.; Tsunoda, Satoshi; Kianianmomeni, Arash; Prigge, Matthias; Berndt, Andre; Cushman, John C.; Polle, Juergen E.; Magnuson, Jon K.; Hegemann, Peter; Deisseroth, Karl

    2011-12-23

    The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

  9. Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives.

    Science.gov (United States)

    Wang, Qingguo; Xia, Junfeng; Jia, Peilin; Pao, William; Zhao, Zhongming

    2013-07-01

    Gene fusions are important genomic events in human cancer because their fusion gene products can drive the development of cancer and thus are potential prognostic tools or therapeutic targets in anti-cancer treatment. Major advancements have been made in computational approaches for fusion gene discovery over the past 3 years due to improvements and widespread applications of high-throughput next generation sequencing (NGS) technologies. To identify fusions from NGS data, existing methods typically leverage the strengths of both sequencing technologies and computational strategies. In this article, we review the NGS and computational features of existing methods for fusion gene detection and suggest directions for future development.

  10. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    Science.gov (United States)

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-09

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion.

  11. C-opsin expressing photoreceptors in echinoderms.

    Science.gov (United States)

    Ullrich-Lüter, Esther M; D'Aniello, Salvatore; Arnone, Maria I

    2013-07-01

    Today's progress in molecular analysis and, in particular, the increased availability of genome sequences have enabled us to investigate photoreceptor cells (PRCs) in organisms that were formerly inaccessible to experimental manipulation. Our studies of marine non-chordate deuterostomes thus aim to bridge a gap of knowledge regarding the evolution of deuterostome PRCs prior to the emergence of vertebrates' eyes. In this contribution, we will show evidence for expression of a c-opsin photopigment, which, according to our phylogenetic analysis, is closely related to an assemblage of chordate visual c-opsins. An antibody raised against sea urchins' c-opsin protein (Sp-Opsin1) recognizes epitopes in a variety of tissues of different echinoderms. While in sea urchins this c-opsin is expressed in locomotory and buccal tube feet, spines, pedicellaria, and epidermis, in brittlestars and starfish we found the immuno-reaction to be located exclusively in cells within the animals' spines. Structural characteristics of these c-opsin+ PRC types include the close vicinity/connection to nerve strands and a, so far unexplored, conspicuous association with the animals' calcite skeleton, which previously has been hypothesized to play a role in echinoderm photobiology. These features are discussed within the context of the evolution of photoreceptors in echinoderms and in deuterostomes generally.

  12. Transforming fusions of FGFR and TACC genes in human glioblastoma.

    Science.gov (United States)

    Singh, Devendra; Chan, Joseph Minhow; Zoppoli, Pietro; Niola, Francesco; Sullivan, Ryan; Castano, Angelica; Liu, Eric Minwei; Reichel, Jonathan; Porrati, Paola; Pellegatta, Serena; Qiu, Kunlong; Gao, Zhibo; Ceccarelli, Michele; Riccardi, Riccardo; Brat, Daniel J; Guha, Abhijit; Aldape, Ken; Golfinos, John G; Zagzag, David; Mikkelsen, Tom; Finocchiaro, Gaetano; Lasorella, Anna; Rabadan, Raul; Iavarone, Antonio

    2012-09-07

    The brain tumor glioblastoma multiforme (GBM) is among the most lethal forms of human cancer. Here, we report that a small subset of GBMs (3.1%; 3 of 97 tumors examined) harbors oncogenic chromosomal translocations that fuse in-frame the tyrosine kinase coding domains of fibroblast growth factor receptor (FGFR) genes (FGFR1 or FGFR3) to the transforming acidic coiled-coil (TACC) coding domains of TACC1 or TACC3, respectively. The FGFR-TACC fusion protein displays oncogenic activity when introduced into astrocytes or stereotactically transduced in the mouse brain. The fusion protein, which localizes to mitotic spindle poles, has constitutive kinase activity and induces mitotic and chromosomal segregation defects and triggers aneuploidy. Inhibition of FGFR kinase corrects the aneuploidy, and oral administration of an FGFR inhibitor prolongs survival of mice harboring intracranial FGFR3-TACC3-initiated glioma. FGFR-TACC fusions could potentially identify a subset of GBM patients who would benefit from targeted FGFR kinase inhibition.

  13. Fusion

    CERN Document Server

    Mahaffey, James A

    2012-01-01

    As energy problems of the world grow, work toward fusion power continues at a greater pace than ever before. The topic of fusion is one that is often met with the most recognition and interest in the nuclear power arena. Written in clear and jargon-free prose, Fusion explores the big bang of creation to the blackout death of worn-out stars. A brief history of fusion research, beginning with the first tentative theories in the early 20th century, is also discussed, as well as the race for fusion power. This brand-new, full-color resource examines the various programs currently being funded or p

  14. Fusion genes in solid tumors:an emerging target for cancer diagnosis and treatment

    Institute of Scientific and Technical Information of China (English)

    Brittany C. Parker; Wei Zhang

    2013-01-01

    Studies over the past decades have uncovered fusion genes, a class of oncogenes that provide immense diagnostic and therapeutic advantages because of their tumor-specific expression. Originally associated with hemotologic cancers, fusion genes have recently been discovered in a wide array of solid tumors, including sarcomas, carcinomas, and tumors of the central nervous system. Fusion genes are attractive as both therapeutic targets and diagnostic tools due to their inherent expression in tumor tissue alone. Therefore, the discovery and elucidation of fusion genes in various cancer types may provide more effective therapies in the future for cancer patients.

  15. Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels

    Directory of Open Access Journals (Sweden)

    Andrew S French

    2015-07-01

    Full Text Available Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1 was 100-1000 times more abundant than the other opsins (pGO2 and pUVO, while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR. Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi was achieved by injecting long (596-708 bp double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude seven days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction.

  16. Oncofuse: a computational framework for the prediction of the oncogenic potential of gene fusions.

    Science.gov (United States)

    Shugay, Mikhail; Ortiz de Mendíbil, Iñigo; Vizmanos, José L; Novo, Francisco J

    2013-10-15

    Gene fusions resulting from chromosomal aberrations are an important cause of cancer. The complexity of genomic changes in certain cancer types has hampered the identification of gene fusions by molecular cytogenetic methods, especially in carcinomas. This is changing with the advent of next-generation sequencing, which is detecting a substantial number of new fusion transcripts in individual cancer genomes. However, this poses the challenge of identifying those fusions with greater oncogenic potential amid a background of 'passenger' fusion sequences. In the present work, we have used some recently identified genomic hallmarks of oncogenic fusion genes to develop a pipeline for the classification of fusion sequences, namely, Oncofuse. The pipeline predicts the oncogenic potential of novel fusion genes, calculating the probability that a fusion sequence behaves as 'driver' of the oncogenic process based on features present in known oncogenic fusions. Cross-validation and extensive validation tests on independent datasets suggest a robust behavior with good precision and recall rates. We believe that Oncofuse could become a useful tool to guide experimental validation studies of novel fusion sequences found during next-generation sequencing analysis of cancer transcriptomes. Oncofuse is a naive Bayes Network Classifier trained and tested using Weka machine learning package. The pipeline is executed by running a Java/Groovy script, available for download at www.unav.es/genetica/oncofuse.html.

  17. The human Müller cell line MIO-M1 expresses opsins.

    Science.gov (United States)

    Hollborn, Margrit; Ulbricht, Elke; Rillich, Katja; Dukic-Stefanovic, Sladjana; Wurm, Antje; Wagner, Lysann; Reichenbach, Andreas; Wiedemann, Peter; Limb, Gloria Astrid; Bringmann, Andreas; Kohen, Leon

    2011-01-01

    To determine whether the human Müller cell line Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) expresses opsins. The gene expression of opsins was determined by reverse-transcription PCR (RT-PCR). The presence of opsin proteins was determined by western blotting and immunocytochemistry. The light sensitivity of the cells was examined with imaging experiments using the calcium-sensitive dye Fluo-4. MIO-M1 cells express glial (glutamine synthase [GLUL], vimentin [VIM], glial fibrillary acidic protein [GFAP], cellular retinaldehyde-binding protein [RLBP1], glial high-affinity glutamate transporter [SLCA1], aquaporin-4 [AQP4], inwardly rectifying potassium channel Kir4.1 [Kir4.1]), neuronal (Thy-1 cell surface antigen [THY1], heavy neurofilament polypeptide [NEFH], microtubule-associated protein 2 [MAP2], neurogenic differentiation 1 [NEUROD1], neuronal nuclei [NEUN]), and neural progenitor markers (Nestin [NES], paired-type homeobox transcription factor [PAX6], neurogenic locus notch homolog 1 [NOTCH1]). The cells contain mRNA for the following opsins: blue opsin (OPN1SW), rhodopsin (OPN2), panopsin (OPN3), melanopsin (OPN4), neuropsin (OPN5), and peropsin (RRH), as well as for the transducins (guanine nucleotide binding protein [GNAZ], alpha transducing activity polypeptide 1 [GNAT1], alpha transducing activity polypeptide 2 [GNAT2]). The presence of blue opsin and melanopsin was confirmed with immunocytochemistry and western blotting. The immunoreactivity and mRNA of red-green opsin were found in some but not all cultures, while the immunoreactivity for rhodopsin was absent in all cultures investigated. Repetitive stimulation with 480 nm light evoked slow and fast transient calcium responses in the majority of cells investigated, while irradiation with 600 nm light was ineffective. The human Müller cell line MIO-M1 expresses opsins. This suggests immortalized Müller cells could be used as a cellular source to produce human opsins for their potential

  18. The microbial opsin homologue sop1 is involved in Sclerotinia sclerotiorum development and environmental stress response

    Directory of Open Access Journals (Sweden)

    Xueliang eLyu

    2016-01-01

    Full Text Available Microbial opsins play a crucial role in responses to various environmental signals. Here, we report that the microbial opsin gene sop1 in the necrotrophic phytopathogenic fungus Sclerotinia sclerotiorum was dramatically up-regulated during infection and sclerotial development compared with the vegetative growth stage. Further study showed sop1 was essential for growth, sclerotial development and full virulence of S. sclerotiorum. Sop1-silenced transformants were more sensitive to high salt stress, fungicides and high osmotic stress. However, they were more tolerant to oxidative stress compared with the wild-type strain, suggesting that sop1 is involved in different stress responses and fungicide resistance, which plays a role in the environmental adaptability of S. sclerotiorum. Furthermore, a Delta blast search showed that microbial opsins are not present in animals and almost all higher plants, indicating that as a predicted transmembrane protein, sop1 is a potential drug target for disease control of S. sclerotiorum.

  19. Cadmium-regulated gene fusions in Pseudomonas fluorescens.

    Science.gov (United States)

    Rossbach, S; Kukuk, M L; Wilson, T L; Feng, S F; Pearson, M M; Fisher, M A

    2000-08-01

    To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas

  20. Effects of light-emitting diode spectra on the vertebrate ancient long opsin and gonadotropin hormone in the goldfish Carassius auratus.

    Science.gov (United States)

    Song, Jin Ah; Kim, Na Na; Choi, Young Jae; Choi, Ji Yong; Kim, Bong-Seok; Choi, Cheol Young

    2016-08-05

    We determined the molecular mechanism underlying the environmental (photoperiodic) regulation of sexual maturation in fish, we examined the expression of sexual maturation-related hormones and vertebrate ancient long opsin (VAL-opsin) in goldfish (Carassius auratus) exposed to different light spectra (red and green light-emitting diodes). We further evaluated the effect of exogenous gonadotropin hormone (GTH) on the expression of VAL-opsin under different light conditions. Our results demonstrated that the expression of GTHs was higher in the fish exposed to green light, and VAL-opsin levels were increased in the fish receiving GTH injection. Therefore, we have uncovered a molecular mechanism underlying the environmental (light)-induced trigger for sexual maturation: VAL-opsin is activated by green light and GTH, which promotes the expression of sexual maturation genes. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

    OpenAIRE

    Francesca Micci; Ioannis Panagopoulos; Jim Thorsen; Ben Davidson; Claes Gøran Tropé; Sverre Heim

    2014-01-01

    The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15%) serous ovarian car...

  2. ETS gene fusions in prostate cancer: from discovery to daily clinical practice.

    NARCIS (Netherlands)

    Tomlins, S.A.; Bjartell, A.; Chinnaiyan, A.M.; Jenster, G.; Nam, R.K.; Rubin, M.A.; Schalken, J.A.

    2009-01-01

    CONTEXT: In 2005, fusions between the androgen-regulated transmembrane protease serine 2 gene, TMPRSS2, and E twenty-six (ETS) transcription factors were discovered in prostate cancer. OBJECTIVE: To review advances in our understanding of ETS gene fusions, focusing on challenges affecting

  3. ETS gene fusions in prostate cancer: from discovery to daily clinical practice.

    NARCIS (Netherlands)

    Tomlins, S.A.; Bjartell, A.; Chinnaiyan, A.M.; Jenster, G.; Nam, R.K.; Rubin, M.A.; Schalken, J.A.

    2009-01-01

    CONTEXT: In 2005, fusions between the androgen-regulated transmembrane protease serine 2 gene, TMPRSS2, and E twenty-six (ETS) transcription factors were discovered in prostate cancer. OBJECTIVE: To review advances in our understanding of ETS gene fusions, focusing on challenges affecting translatio

  4. Opsin-mediated inhibition of bacterioruberin synthesis in halophilic Archaea.

    Science.gov (United States)

    Peck, Ronald F; Pleşa, Alexandru M; Graham, Serena M; Angelini, David R; Shaw, Emily L

    2017-08-07

    Halophilic Archaea often inhabit environments with limited oxygen, and many produce ion-pumping rhodopsin complexes that allow them to maintain electrochemical gradients when aerobic respiration is inhibited. Rhodopsins require a protein, opsin, and an organic cofactor, retinal. We have previously demonstrated that, in Halobacterium salinarum, bacterioopsin (BO), when not bound by retinal, inhibits the production of bacterioruberin, a biochemical pathway that shares intermediates with retinal biosynthesis. In this work, we use heterologous expression in a related halophilic Archaeon, Haloferax volcanii, to demonstrate that BO is sufficient to inhibit bacterioruberin synthesis catalyzed by the H. salinarum lycopene elongase (Lye) enzyme. This inhibition was observed both in liquid cultures and in a novel colorimetric assay to quantify bacterioruberin abundance based on the colony color. Addition of retinal to convert BO to the bacteriorhodopsin complex resulted in a partial rescue of bacterioruberin production. To explore if this regulatory mechanism occurs in other organisms, we expressed a Lye homolog and an opsin from Haloarcula vallismortis in H. volcaniiH. vallismortis cruxopsin expression inhibited bacterioruberin synthesis catalyzed by H. vallismortis Lye, but had no effect when bacterioruberin synthesis was catalyzed by H. salinarum or H. volcanii Lye. Conversely, H. salinarum BO did not inhibit H. vallismortis Lye activity. Together, our data suggest that opsin-mediated inhibition of Lye is potentially widespread and represents an elegant regulatory mechanism that allows organisms to efficiently utilize ion-pumping rhodopsins obtained through lateral gene transfer.Importance Many enzymes are complexes of proteins and non-protein organic molecules called cofactors. To ensure efficient formation of functional complexes, organisms must regulate the production of proteins and cofactors. To study this regulation, we use bacteriorhodopsin from the Archaeon

  5. An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

    Directory of Open Access Journals (Sweden)

    Jonathan A Scolnick

    Full Text Available Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET, for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE tissue RNA in both normal tissue and cancer cells.

  6. An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

    Science.gov (United States)

    Scolnick, Jonathan A; Dimon, Michelle; Wang, I-Ching; Huelga, Stephanie C; Amorese, Douglas A

    2015-01-01

    Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.

  7. DNA immunization with fusion genes containing HCV core region and HBV core region

    Institute of Scientific and Technical Information of China (English)

    杨莉; 刘晶; 孔玉英; 汪垣; 李光地

    1999-01-01

    The eucaryotic expression plasmids were constructed to express the complete (HCc191) or the truncated (HCc69 and HCc40) HCV core genes, solely or fused with the HBV core gene (HBc144). These constructions were transiently expressed in COS cells under the control of the CMV promoter. The antigenicity of HBc and HCc could be detected in the expression products by ELISA and Western blot. The mice immunized with these expression plasmids efficiently produced the anti-HCc antibodies, and also anti-HBc antibodies when the plasmids contained the fusion genes. In addition, the antibodies induced by the fusion genes were more persistent than those induced by the non-fusion HCV core genes. These indicate that the fusion of HCc genes to HBc gene is in favor of the immunogenicity of HCc, while the immunogenicity of HBc is not affected.

  8. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Directory of Open Access Journals (Sweden)

    Sara Kangaspeska

    Full Text Available RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60% of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  9. Fusion

    Science.gov (United States)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  10. The Investigation of EWS-FLI-1 Fusion Gene in the Ewing Family of Tumors

    Institute of Scientific and Technical Information of China (English)

    GangFeng; ZhongquanZhao; DonglinWang

    2004-01-01

    There is evidence that 95% of the Ewing family of tumors (EFT) have a EWS-FLI-1 fusion gene. EWS-FL1-1 is a transcription factor with a pivotal function and it is known to bind to a special DNA sequence. Research has demonstrated that the EWS-FLI-1 fusion gene occurrence is related to the EFT, and it has been used to diagnose, treat and serve as a basis for EFT prognosis. We have briefly summarized the progress of the EWS-FLI-1 fusion gene in basic and clinical investigation within the past several years.

  11. Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...

  12. Origin and ascendancy of a chimeric fusion gene: the beta/delta-globin gene of paenungulate mammals.

    Science.gov (United States)

    Opazo, Juan C; Sloan, Angela M; Campbell, Kevin L; Storz, Jay F

    2009-07-01

    The delta-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked beta-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric beta/delta fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the beta/delta fusion gene in elephants is structurally similar to the "anti-Lepore" duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric beta/delta fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the beta-chain subunits of adult hemoglobin. A phylogenetic survey of beta-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric beta/delta fusion gene and the concomitant inactivation of the HBB gene predated the radiation of "Paenungulata," a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived beta/delta fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal delta/beta fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin.

  13. Engineering and Functional Characterization of Fusion Genes Identifies Novel Oncogenic Drivers of Cancer. | Office of Cancer Genomics

    Science.gov (United States)

    Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in TCGA datasets.

  14. Fusion of the NUP98 gene with the LEDGF/p52 gene defines a recurrent acute myeloid leukemia translocation

    Directory of Open Access Journals (Sweden)

    Nicola Mario

    2001-11-01

    Full Text Available Abstract Background The NUP98 gene is involved in multiple rearrangements in haematological malignancy. The leukemic cells in an acute myeloid leukemia (AML patient with a t(9;11(p22;p15 were recently shown to have a fusion between the NUP98 gene and the LEDGF gene but it was not demonstrated that this fusion was recurrent in other leukaemia patients with the same translocation. Results We used RT-PCR to analyse the leukemic cells from an AML patient who presented with a cytogenetically identical translocation as the sole chromosomal abnormality. A NUP98-LEDGF fusion transcript was observed and confirmed by sequencing. The reciprocal transcript was also observed. The fusion transcript was not detectable during remission and recurred at relapse. The breakpoints in the NUP98 and LEDGF genes were different to those previously reported. The NUP98 breakpoint occurs in the intron between exons 8 and 9. It is the most 5' breakpoint reported in a translocation involving the NUP98 gene. All of the LEDGF gene is included in the fusion except for exon 1 which codes for the first 24 amino terminal amino acids. Conclusions Our results show that fusion of the NUP98 and LEDGF genes is a new recurrent translocation in AML.

  15. Novel fusion genes and chimeric transcripts in ependymal tumors

    DEFF Research Database (Denmark)

    Olsen, Thale Kristin; Panagopoulos, Ioannis; Gorunova, Ludmila

    2016-01-01

    We have previously identified two ALK rearrangements in a subset of ependymal tumors using a combination of cytogenetic data and RNA sequencing. The aim of this study was to perform an unbiased search for fusion transcripts in our entire series of ependymal tumors. Fusion analysis was performed...... using the FusionCatcher algorithm on 12 RNA-sequenced ependymal tumors. Candidate transcripts were prioritized based on the software's filtering and manual visualization using the BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) tools. Genomic and reverse transcriptase PCR...... with subsequent Sanger sequencing was used to validate the potential fusions. Fluorescent in situ hybridization (FISH) using locus-specific probes was also performed. A total of 841 candidate chimeric transcripts were identified in the 12 tumors, with an average of 49 unique candidate fusions per tumor. After...

  16. Emergence of FGFR family gene fusions as therapeutic targets in a wide spectrum of solid tumours.

    Science.gov (United States)

    Parker, Brittany C; Engels, Manon; Annala, Matti; Zhang, Wei

    2014-01-01

    The emergence of fibroblast growth factor receptor (FGFR) family fusions across diverse cancers has brought attention to FGFR-derived cancer therapies. The discovery of the first recurrent FGFR fusion in glioblastoma was followed by discoveries of FGFR fusions in bladder, lung, breast, thyroid, oral, and prostate cancers. Drug targeting of FGFR fusions has shown promising results and should soon be translating into clinical trials. FGFR fusions form as a result of various mechanisms – predominantly deletion for FGFR1, translocation for FGFR2, and tandem duplication for FGFR3. The ability to exploit the unique targetability of FGFR fusions proves that FGFR-derived therapies could have a promising future in cancer therapeutics. Drug targeting of fusion genes has proven to be an extremely effective therapeutic approach for cancers such as the recurrent BCR–ABL1 fusion in chronic myeloid leukaemia. The recent discovery of recurrent FGFR family fusions in several cancer types has brought to attention the unique therapeutic potential for FGFR-positive patients. Understanding the diverse mechanisms of FGFR fusion formation and their oncogenic potential will shed light on the impact of FGFR-derived therapy in the future.

  17. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  18. Opsins in onychophora (velvet worms) suggest a single origin and subsequent diversification of visual pigments in arthropods.

    Science.gov (United States)

    Hering, Lars; Henze, Miriam J; Kohler, Martin; Kelber, Almut; Bleidorn, Christoph; Leschke, Maren; Nickel, Birgit; Meyer, Matthias; Kircher, Martin; Sunnucks, Paul; Mayer, Georg

    2012-11-01

    Multiple visual pigments, prerequisites for color vision, are found in arthropods, but the evolutionary origin of their diversity remains obscure. In this study, we explore the opsin genes in five distantly related species of Onychophora, using deep transcriptome sequencing and screening approaches. Surprisingly, our data reveal the presence of only one opsin gene (onychopsin) in each onychophoran species, and our behavioral experiments indicate a maximum sensitivity of onychopsin to blue-green light. In our phylogenetic analyses, the onychopsins represent the sister group to the monophyletic clade of visual r-opsins of arthropods. These results concur with phylogenomic support for the sister-group status of the Onychophora and Arthropoda and provide evidence for monochromatic vision in velvet worms and in the last common ancestor of Onychophora and Arthropoda. We conclude that the diversification of visual pigments and color vision evolved in arthropods, along with the evolution of compound eyes-one of the most sophisticated visual systems known.

  19. Structural and functional alterations associated with deutan N94K and R330Q mutations of green cone opsin.

    Science.gov (United States)

    Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Ramon, Eva; Garriga, Pere

    2017-07-01

    Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. LINE FUSION GENES: a database of LINE expression in human genes

    Directory of Open Access Journals (Sweden)

    Park Hong-Seog

    2006-06-01

    Full Text Available Abstract Background Long Interspersed Nuclear Elements (LINEs are the most abundant retrotransposons in humans. About 79% of human genes are estimated to contain at least one segment of LINE per transcription unit. Recent studies have shown that LINE elements can affect protein sequences, splicing patterns and expression of human genes. Description We have developed a database, LINE FUSION GENES, for elucidating LINE expression throughout the human gene database. We searched the 28,171 genes listed in the NCBI database for LINE elements and analyzed their structures and expression patterns. The results show that the mRNA sequences of 1,329 genes were affected by LINE expression. The LINE expression types were classified on the basis of LINEs in the 5' UTR, exon or 3' UTR sequences of the mRNAs. Our database provides further information, such as the tissue distribution and chromosomal location of the genes, and the domain structure that is changed by LINE integration. We have linked all the accession numbers to the NCBI data bank to provide mRNA sequences for subsequent users. Conclusion We believe that our work will interest genome scientists and might help them to gain insight into the implications of LINE expression for human evolution and disease. Availability http://www.primate.or.kr/line

  1. NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights.

    Science.gov (United States)

    Gough, Sheryl M; Slape, Christopher I; Aplan, Peter D

    2011-12-08

    Structural chromosomal rearrangements of the Nucleoporin 98 gene (NUP98), primarily balanced translocations and inversions, are associated with a wide array of hematopoietic malignancies. NUP98 is known to be fused to at least 28 different partner genes in patients with hematopoietic malignancies, including acute myeloid leukemia, chronic myeloid leukemia in blast crisis, myelodysplastic syndrome, acute lymphoblastic leukemia, and bilineage/biphenotypic leukemia. NUP98 gene fusions typically encode a fusion protein that retains the amino terminus of NUP98; in this context, it is important to note that several recent studies have demonstrated that the amino-terminal portion of NUP98 exhibits transcription activation potential. Approximately half of the NUP98 fusion partners encode homeodomain proteins, and at least 5 NUP98 fusions involve known histone-modifying genes. Several of the NUP98 fusions, including NUP98-homeobox (HOX)A9, NUP98-HOXD13, and NUP98-JARID1A, have been used to generate animal models of both lymphoid and myeloid malignancy; these models typically up-regulate HOXA cluster genes, including HOXA5, HOXA7, HOXA9, and HOXA10. In addition, several of the NUP98 fusion proteins have been shown to inhibit differentiation of hematopoietic precursors and to increase self-renewal of hematopoietic stem or progenitor cells, providing a potential mechanism for malignant transformation.

  2. The tumorigenic FGFR3-TACC3 gene fusion escapes miR-99a regulation in glioblastoma

    Science.gov (United States)

    Parker, Brittany C.; Annala, Matti J.; Cogdell, David E.; Granberg, Kirsi J.; Sun, Yan; Ji, Ping; Li, Xia; Gumin, Joy; Zheng, Hong; Hu, Limei; Yli-Harja, Olli; Haapasalo, Hannu; Visakorpi, Tapio; Liu, Xiuping; Liu, Chang-gong; Sawaya, Raymond; Fuller, Gregory N.; Chen, Kexin; Lang, Frederick F.; Nykter, Matti; Zhang, Wei

    2013-01-01

    Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Fusions can lead to production of oncogenic fusion proteins or to enhanced expression of oncogenes. Several recent studies have reported that some fusion genes can escape microRNA regulation via 3′–untranslated region (3′-UTR) deletion. We performed whole transcriptome sequencing to identify fusion genes in glioma and discovered FGFR3-TACC3 fusions in 4 of 48 glioblastoma samples from patients both of mixed European and of Asian descent, but not in any of 43 low-grade glioma samples tested. The fusion, caused by tandem duplication on 4p16.3, led to the loss of the 3′-UTR of FGFR3, blocking gene regulation of miR-99a and enhancing expression of the fusion gene. The fusion gene was mutually exclusive with EGFR, PDGFR, or MET amplification. Using cultured glioblastoma cells and a mouse xenograft model, we found that fusion protein expression promoted cell proliferation and tumor progression, while WT FGFR3 protein was not tumorigenic, even under forced overexpression. These results demonstrated that the FGFR3-TACC3 gene fusion is expressed in human cancer and generates an oncogenic protein that promotes tumorigenesis in glioblastoma. PMID:23298836

  3. A screening method for the ALK fusion gene in NSCLC

    Directory of Open Access Journals (Sweden)

    Yoshiko eMurakami

    2012-03-01

    Full Text Available Lung cancer research has recently made significant progress in understanding the molecular pathogenesis and even the treatment of lung cancer. Such achievements are directly utilized in clinical practice. Indeed, the EML4-ALK fusion in non-small cell lung cancer (NSCLC was first described in 2007, and a molecularly targeted drug against the fusion was approved in 2011. However, lung cancer with the anaplastic lymphoma kinase (ALK fusion constitutes only a small fraction of lung cancers; thus, efficient patient selection is crucial for successful treatment using the ALK inhibitor. Currently, RT-PCR, fluorescent in-situ hybridization (FISH and immunohistochemistry are commonly used to detect the ALK fusion. Although FISH is currently the gold standard, there are no perfect methods for detecting the genetic alterations. This article discusses the advantages and disadvantages of the individual methods and possible criteria for selecting patients more likely to have the ALK fusion. If we can successfully screen patients, then ALK inhibitor treatment will be the best example of personalized therapy in terms of selecting patients with an uncommon genotype from those with the same tumor phenotype. In other words, it may offer a new challenge for current clinical oncology.

  4. Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

    Science.gov (United States)

    Micci, Francesca; Panagopoulos, Ioannis; Thorsen, Jim; Davidson, Ben; Tropé, Claes Gøran; Heim, Sverre

    2014-02-01

    The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15%) serous ovarian carcinomas examined, a finding that holds great promise for our understanding of ovarian tumorigenesis as well as, potentially, for new treatment strategies. We wanted to test how frequent the ESRRA/C11orf20 fusion is in ovarian carcinomas of all subtypes, and therefore examined a series of 230 ovarian carcinomas of which 197 were of the serous subtype and 163 of the 197 were of stages III and IV--that is, the very same carcinoma subset where the fusion transcript had been found. We performed PCR and high-throughput sequencing analyses in search of the fusion transcript. We used the same primers described previously for the detection of the fusion and the same primer combination, but found no ESRRA/C11orf20 fusion in our series. A synthetic DNA plasmid containing the reported ESRRA/C11orf20 fusion was included as a positive control for our PCR experiments. Data from high-throughput sequencing of 23 ovarian carcinomas were screened in search of alternative partner(s) for the ESRRA and/or C11orf20 gene, but none was found. We conclude that the frequency of the ESRRA/C11orf20 gene fusion in serous ovarian carcinomas of stages III and IV must be considerable less than that reported previously (0/163 in our experience compared with 10/67 in the previous study). At the very least, it seems clear that the said fusion cannot be a common pathogenetic event in this tumor type.

  5. Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

    Directory of Open Access Journals (Sweden)

    Francesca Micci

    2014-02-01

    Full Text Available The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15% serous ovarian carcinomas examined, a finding that holds great promise for our understanding of ovarian tumorigenesis as well as, potentially, for new treatment strategies. We wanted to test how frequent the ESRRA/C11orf20 fusion is in ovarian carcinomas of all subtypes, and therefore examined a series of 230 ovarian carcinomas of which 197 were of the serous subtype and 163 of the 197 were of stages III and IV--that is, the very same carcinoma subset where the fusion transcript had been found. We performed PCR and high-throughput sequencing analyses in search of the fusion transcript. We used the same primers described previously for the detection of the fusion and the same primer combination, but found no ESRRA/C11orf20 fusion in our series. A synthetic DNA plasmid containing the reported ESRRA/C11orf20 fusion was included as a positive control for our PCR experiments. Data from high-throughput sequencing of 23 ovarian carcinomas were screened in search of alternative partner(s for the ESRRA and/or C11orf20 gene, but none was found. We conclude that the frequency of the ESRRA/C11orf20 gene fusion in serous ovarian carcinomas of stages III and IV must be considerable less than that reported previously (0/163 in our experience compared with 10/67 in the previous study. At the very least, it seems clear that the said fusion cannot be a common pathogenetic event in this tumor type.

  6. Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity

    Directory of Open Access Journals (Sweden)

    Fei Yao

    2015-07-01

    Full Text Available Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET whole-genome sequencing, we analyzed 15 gastric cancers (GCs from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT. Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H+ leakage, and the fusion might contribute to invasiveness once a cell is transformed.

  7. Model systems for understanding absorption tuning by opsin proteins

    DEFF Research Database (Denmark)

    Nielsen, Mogens Brøndsted

    2009-01-01

    This tutorial review reports on model systems that have been synthesised and investigated for elucidating how opsin proteins tune the absorption of the protonated retinal Schiff base chromophore. In particular, the importance of the counteranion is highlighted. In addition, the review advocates...... is avoided, and it becomes clear that opsin proteins induce blueshifts in the chromophore absorption rather than redshifts....

  8. Spectral sensitivity of cone photoreceptors and opsin expression in two colour-divergent lineages of the lizard Ctenophorus decresii.

    Science.gov (United States)

    Yewers, Madeleine S; McLean, Claire A; Moussalli, Adnan; Stuart-Fox, Devi; Bennett, Andrew T D; Knott, Ben

    2015-05-15

    Intraspecific differences in sensory perception are rarely reported but may occur when a species range extends across varying sensory environments, or there is coevolution between the sensory system and a varying signal. Examples in colour vision and colour signals are rare in terrestrial systems. The tawny dragon lizard Ctenophorus decresii is a promising candidate for such intraspecific variation, because the species comprises two geographically and genetically distinct lineages in which throat colour (a social signal used in intra- and inter-specific interactions) is locally adapted to the habitat and differs between lineages. Male lizards from the southern lineage have UV-blue throats, whereas males from the northern lineage are polymorphic with four discrete throat colours that all show minimal UV reflectance. Here, we determine the cone photoreceptor spectral sensitivities and opsin expression of the two lineages, to test whether they differ, particularly in the UV wavelengths. Using microspectrophotometry on retinal cone photoreceptors, we identified a long-wavelength-sensitive (LWS) visual pigment, a 'short' and 'long' medium-wavelength-sensitive (MWS) pigment and a short-wavelength-sensitive (SWS) pigment, all of which did not differ in λmax between lineages. Through transcriptome analysis of opsin genes we found that both lineages express four cone opsin genes, including the SWS1 opsin with peak sensitivity in the UV range, and that amino acid sequences did not differ between lineages with the exception of a single leucine to valine substitution in the RH2 opsin. Counts of yellow and transparent oil droplets associated with LWS+MWS and SWS+UVS cones, respectively, showed no difference in relative cone proportions between lineages. Therefore, contrary to predictions, we find no evidence of differences between lineages in single cone photoreceptor spectral sensitivity or opsin expression. However, we confirm the presence of four single cone classes

  9. Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

    Institute of Scientific and Technical Information of China (English)

    XIONG Ying; YUAN Yuan; JIA Min; YU Bing; HUANG Hanju

    2007-01-01

    To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein.Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

  10. The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications.

    Science.gov (United States)

    Lagman, David; Ocampo Daza, Daniel; Widmark, Jenny; Abalo, Xesús M; Sundström, Görel; Larhammar, Dan

    2013-11-02

    Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the

  11. ESRRA-C11orf20 is a recurrent gene fusion in serous ovarian carcinoma.

    Directory of Open Access Journals (Sweden)

    Julia Salzman

    2011-09-01

    Full Text Available Every year, ovarian cancer kills approximately 14,000 women in the United States and more than 140,000 women worldwide. Most of these deaths are caused by tumors of the serous histological type, which is rarely diagnosed before it has disseminated. By deep paired-end sequencing of mRNA from serous ovarian cancers, followed by deep sequencing of the corresponding genomic region, we identified a recurrent fusion transcript. The fusion transcript joins the 5' exons of ESRRA, encoding a ligand-independent member of the nuclear-hormone receptor superfamily, to the 3' exons of C11orf20, a conserved but uncharacterized gene located immediately upstream of ESRRA in the reference genome. To estimate the prevalence of the fusion, we tested 67 cases of serous ovarian cancer by RT-PCR and sequencing and confirmed its presence in 10 of these. Targeted resequencing of the corresponding genomic region from two fusion-positive tumor samples identified a nearly clonal chromosomal rearrangement positioning ESRRA upstream of C11orf20 in one tumor, and evidence of local copy number variation in the ESRRA locus in the second tumor. We hypothesize that the recurrent novel fusion transcript may play a role in pathogenesis of a substantial fraction of serous ovarian cancers and could provide a molecular marker for detection of the cancer. Gene fusions involving adjacent or nearby genes can readily escape detection but may play important roles in the development and progression of cancer.

  12. ESRRA-C11orf20 is a recurrent gene fusion in serous ovarian carcinoma.

    Science.gov (United States)

    Salzman, Julia; Marinelli, Robert J; Wang, Peter L; Green, Ann E; Nielsen, Julie S; Nelson, Brad H; Drescher, Charles W; Brown, Patrick O

    2011-09-01

    Every year, ovarian cancer kills approximately 14,000 women in the United States and more than 140,000 women worldwide. Most of these deaths are caused by tumors of the serous histological type, which is rarely diagnosed before it has disseminated. By deep paired-end sequencing of mRNA from serous ovarian cancers, followed by deep sequencing of the corresponding genomic region, we identified a recurrent fusion transcript. The fusion transcript joins the 5' exons of ESRRA, encoding a ligand-independent member of the nuclear-hormone receptor superfamily, to the 3' exons of C11orf20, a conserved but uncharacterized gene located immediately upstream of ESRRA in the reference genome. To estimate the prevalence of the fusion, we tested 67 cases of serous ovarian cancer by RT-PCR and sequencing and confirmed its presence in 10 of these. Targeted resequencing of the corresponding genomic region from two fusion-positive tumor samples identified a nearly clonal chromosomal rearrangement positioning ESRRA upstream of C11orf20 in one tumor, and evidence of local copy number variation in the ESRRA locus in the second tumor. We hypothesize that the recurrent novel fusion transcript may play a role in pathogenesis of a substantial fraction of serous ovarian cancers and could provide a molecular marker for detection of the cancer. Gene fusions involving adjacent or nearby genes can readily escape detection but may play important roles in the development and progression of cancer.

  13. A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

    Directory of Open Access Journals (Sweden)

    Etchells J Peter

    2009-10-01

    Full Text Available Abstract Background The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. Results Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. Conclusion The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.

  14. ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer

    Science.gov (United States)

    2016-05-01

    Award  Number:    W81XWH-10-1-0582 TITLE:       ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer...5a.  CONTRACT  NUMBER   ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer 5b.  GRANT  NUMBER   W81XWH...SUPPLEMENTARY  NOTES 14. ABSTRACT The  research  goals  of  this  grant  proposal  are  to:  1)  investigate  the  effect  of   ETS  gene  fusions  on  radiation

  15. Fusion and fission of genes define a metric between fungal genomes.

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    Pascal Durrens

    2008-10-01

    Full Text Available Gene fusion and fission events are key mechanisms in the evolution of gene architecture, whose effects are visible in protein architecture when they occur in coding sequences. Until now, the detection of fusion and fission events has been performed at the level of protein sequences with a post facto removal of supernumerary links due to paralogy, and often did not include looking for events defined only in single genomes. We propose a method for the detection of these events, defined on groups of paralogs to compensate for the gene redundancy of eukaryotic genomes, and apply it to the proteomes of 12 fungal species. We collected an inventory of 1,680 elementary fusion and fission events. In half the cases, both composite and element genes are found in the same species. Per-species counts of events correlate with the species genome size, suggesting a random mechanism of occurrence. Some biological functions of the genes involved in fusion and fission events are slightly over- or under-represented. As already noted in previous studies, the genes involved in an event tend to belong to the same functional category. We inferred the position of each event in the evolution tree of the 12 fungal species. The event localization counts for all the segments of the tree provide a metric that depicts the "recombinational" phylogeny among fungi. A possible interpretation of this metric as distance in adaptation space is proposed.

  16. Nuclear fusion occurs during mating in Candida albicans and is dependent on the KAR3 gene.

    Science.gov (United States)

    Bennett, Richard J; Miller, Mathew G; Chua, Penelope R; Maxon, Mary E; Johnson, Alexander D

    2005-02-01

    It is now well established that mating can occur between diploid a and alpha cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/alpha cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.

  17. A New Target in Non-small Cell Lung Cancer: EML4-ALK Fusion Gene

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    Huijuan WANG

    2011-06-01

    Full Text Available It was only 3 years ago that the fusion gene between echinoderm microtubule-associated protein-like4 (EML4 and anaplastic lymphoma kinase (ALK has been identified in a subset of non-small cell lung cancer (NSCLC. EML4-ALK is most often detected in never smokers with lung adenocarcinoma and has unique pathologic features. EML4-ALK fusion gene is oncogenic, which could be suppressed by ALK-inhibitor through blocking the downstream signaling passway of EML4-ALK. This review will focus on the molecular structure, function, biology, detection method and the diagnostic and therapeutic meaning of EML4-ALK of lung cancer.

  18. Construction of expression vector for NT4-ADNF-9 fusion gene

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subc...

  19. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    Science.gov (United States)

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  20. CRTC1-MAML2 gene fusion in mucoepidermoid carcinoma of the lacrimal gland

    DEFF Research Database (Denmark)

    von Holstein, Sarah Linea; Fehr, André; Heegaard, Steffen

    2012-01-01

    -grade MEC of the lacrimal gland. There were no signs of recurrence or metastases during a five-year follow-up. Using RT-PCR and FISH we demonstrated that the tumor was positive for the CRTC1-MAML2 gene fusion previously shown to be associated with in particular low-grade salivary MECs with favorable...... prognosis. By immunohistochemistry we showed that the majority of tumor cells, including epidermoid, intermediate and mucous producing cells, expressed the CRTC1-MAML2 fusion protein. In contrast, 15 non-MEC lacrimal neoplasm were fusion-negative. Our findings show that lacrimal MEC is not only clinically...... anatomical sites and organs. Moreover, our findings indicate that the CRTC1-MAML2 fusion may be a useful diagnostic and prognostic biomarker for lacrimal MEC....

  1. The molecular basis of color vision in colorful fish: Four Long Wave-Sensitive (LWS opsins in guppies (Poecilia reticulata are defined by amino acid substitutions at key functional sites

    Directory of Open Access Journals (Sweden)

    Ward Pam R

    2008-07-01

    Full Text Available Abstract Background Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS opsin genes in six species of the family Poeciliidae. Results Sequences of four LWS opsin genes were amplified from the guppy genome and from mRNA isolated from adult guppy eyes. Variation in expression was quantified using qPCR. Three of the four genes encode opsins predicted to be most sensitive to different wavelengths of light because they vary at key amino acid positions. This family of LWS opsin genes was produced by a diversity of duplication events. One, an intronless gene, was produced prior to the divergence of families Fundulidae and Poeciliidae. Between-gene PCR and DNA sequencing show that two of the guppy LWS opsins are linked in an inverted orientation. This inverted tandem duplication event occurred near the base of the poeciliid tree in the common ancestor of Poecilia and Xiphophorus. The fourth sequence has been uncovered only in the genus Poecilia. In the guppies surveyed here, this sequence is a hybrid, with the 5' end most similar to one of the tandem duplicates and the 3' end identical to the other. Conclusion Enhanced wavelength discrimination, a possible consequence of opsin gene duplication and divergence, might have been an evolutionary prerequisite for color-based sexual selection and have led to the extraordinary coloration now observed in male guppies and in many other poeciliids.

  2. ChimerDB 3.0: an enhanced database for fusion genes from cancer transcriptome and literature data mining

    Science.gov (United States)

    Lee, Myunggyo; Lee, Kyubum; Yu, Namhee; Jang, Insu; Choi, Ikjung; Kim, Pora; Jang, Ye Eun; Kim, Byounggun; Kim, Sunkyu; Lee, Byungwook; Kang, Jaewoo; Lee, Sanghyuk

    2017-01-01

    Fusion gene is an important class of therapeutic targets and prognostic markers in cancer. ChimerDB is a comprehensive database of fusion genes encompassing analysis of deep sequencing data and manual curations. In this update, the database coverage was enhanced considerably by adding two new modules of The Cancer Genome Atlas (TCGA) RNA-Seq analysis and PubMed abstract mining. ChimerDB 3.0 is composed of three modules of ChimerKB, ChimerPub and ChimerSeq. ChimerKB represents a knowledgebase including 1066 fusion genes with manual curation that were compiled from public resources of fusion genes with experimental evidences. ChimerPub includes 2767 fusion genes obtained from text mining of PubMed abstracts. ChimerSeq module is designed to archive the fusion candidates from deep sequencing data. Importantly, we have analyzed RNA-Seq data of the TCGA project covering 4569 patients in 23 cancer types using two reliable programs of FusionScan and TopHat-Fusion. The new user interface supports diverse search options and graphic representation of fusion gene structure. ChimerDB 3.0 is available at http://ercsb.ewha.ac.kr/fusiongene/. PMID:27899563

  3. 千年笛鲷早期发育及视蛋白基因表达规律分析%PRELIMINARY STUDIES ON THE EARLY DEVELOPMENT AND THE EXPRESSION PATTERN OF OPSIN GENES OF EMPEROR RED SNAPPER(LUTJANUS SEBAE)

    Institute of Scientific and Technical Information of China (English)

    谢子强; 王中铎; 郭昱嵩; 刘楚吾

    2015-01-01

    初步观察千年笛鲷早期发育各个时期的形态特征,同时使用实时荧光定量 PCR 方法对4种视蛋白基因在早期发育中的表达规律进行分析。研究观察到千年笛鲷卵为圆球形,属浮性卵,中心有一明显的油球。在水温(24.5±0.5)℃的条件下,千年笛鲷胚胎发育共经历6个发育阶段18个时期,从受精卵到孵化一共经历24h。仔鱼经历12—15d发育为稚鱼,30d—35d发育为幼鱼。同时对7个胚胎发育时期和2个仔鱼发育时期4种视蛋白(LWS、SWS1、SWS2、RH)基因的表达情况进行检测,在下包1/2、胚孔封闭、视囊这3个时期有显著性表达(P<0.05),尤其在胚孔封闭时期,表达量达到最高。其余时期4种基因的表达水平显著下降,但在2个仔鱼时期表达量比孵化期略有增加。结果表明千年笛鲷4种视蛋白基因在早期表达过程中与神经胚的形成有密切的联系。%In this study, we observed the morphological characteristics and used qRT-PCR to analyze the expression pattern of four opsin genes during the early development of Emperor red snapper. The eggs of Emperor red snapper were a type of buoyancy that had spherical shape and one oil globule. Fertilized eggs hatched in 24 hours at the temperature of (24.5±0.5)℃, and there were 6 phases and 18 stages in the development of the embryo. After 12—15 days, the larvae developed into juvenile. After 30—35 days, the juvenile developed into young fish. We detected the expression of four opsin genes (LWS,SWS1,SWS2 andRH) at 7 embryo stages and 2 larvae stages. All four genes were markedly ex-pressed at the 1/2 epiboly stage, the closure of blastopore stage and the optic capsule stage (P<0.05). The expression level of the four genes was the highest at the closure of blastopore stage. At other stages the expression was largely de-creased. However, the expression was slightly increased at 2 larvae stages compared to the hatching stage. Our results suggested that

  4. Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jian; ZHAO Dun-yan; SHEN Ai-guo; LIU Fan; ZHANG Feng; SUN Yu; WU Hong-fu; LU Chun-feng; SHI Hong-guang

    2007-01-01

    Objective: To determine whether an adenoviral construct containing bone morphogenetic protein-4 (BMP-4) gene can be used for lumbar spinal fusion. Methods: Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter and β-galactosidase gene (Ad-β-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9×108 pfu/ml ) was directly implanted on the surface of L5-L6 lamina in the experimental group, while Ad-β-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection.Results: In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified.Conclusions: In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.

  5. IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Luisa Cironi

    Full Text Available BACKGROUND: The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT, the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1 for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. METHODOLOGY/PRINCIPAL FINDINGS: To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. CONCLUSION/SIGNIFICANCE: Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

  6. Recurrent fusion of the genes FN1 and ALK in gastrointestinal leiomyomas.

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Lund-Iversen, Marius; Lobmaier, Ingvild; Bjerkehagen, Bodil; Heim, Sverre

    2016-11-01

    Leiomyomas of the gastrointestinal tract are mostly found in the esophagus, stomach, and colon. Genetic information about them is very limited and no fusion genes have been described. We present herein cytogenetic and molecular genetic analyses of two gastrointestinal leiomyomas found in the esophagus and small intestine. The esophageal leiomyoma had the karyotype 45,Y,der(X)t(X;6)(p22;p21),inv(2)(p23q35),add(6)(p21),-11[cp6]/46,XY[7]. The intestinal leiomyoma karyotype was 46,X,add(X)(q2?),der(2)add(2)(p23)add(2)(q33),add(4)(p14),add(14)(q22)[10]/47,XX,+12[2]/46,XX[1]. RNA-sequencing detected FN1-ALK fusion transcripts in both tumors. RT-PCR together with Sanger sequencing verified the presence of the FN1-ALK fusion transcripts. Fluorescence in situ hybridization using an ALK breakapart probe further confirmed the rearrangement of the ALK gene. Immunohistochemical investigation of ALK in the leiomyoma of the small intestine revealed positivity with strong granular cytoplasmatic staining in the tumor cells. This is the first ever ALK fusion reported in gastrointestinal leiomyomas. Our results are of potential clinical importance because crizotinib, a selective ALK inhibitor, has demonstrated effect in patients whose tumors harbor ALK rearrangements. Thus, ALK emerges as a possible therapeutic target in patients whose tumors, including gastrointestinal leiomyomas, carry ALK fusions.

  7. Developing transgenic maize (Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

    Institute of Scientific and Technical Information of China (English)

    SUN He; LANG Zhi-hong; LU Wei; ZHANG Jie; HE Kang-lai; ZHU Li; LIN Min; HUANG Da-fang

    2015-01-01

    Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacil us thuringiensis (Bt) cry gene and epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1Ah gene with the 2mG2-epsps gene and combined the wide-used manA gene as a selective marker to construct one coordinated expression vector cal ed p2EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40%of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing;meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

  8. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins.

    Science.gov (United States)

    Maeder, Morgan L; Angstman, James F; Richardson, Marcy E; Linder, Samantha J; Cascio, Vincent M; Tsai, Shengdar Q; Ho, Quan H; Sander, Jeffry D; Reyon, Deepak; Bernstein, Bradley E; Costello, Joseph F; Wilkinson, Miles F; Joung, J Keith

    2013-12-01

    Genome-wide studies have defined cell type-specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.

  9. Experimental evidence validating the computational inference of functional associations from gene fusion events: a critical survey.

    Science.gov (United States)

    Promponas, Vasilis J; Ouzounis, Christos A; Iliopoulos, Ioannis

    2014-05-01

    More than a decade ago, a number of methods were proposed for the inference of protein interactions, using whole-genome information from gene clusters, gene fusions and phylogenetic profiles. This structural and evolutionary view of entire genomes has provided a valuable approach for the functional characterization of proteins, especially those without sequence similarity to proteins of known function. Furthermore, this view has raised the real possibility to detect functional associations of genes and their corresponding proteins for any entire genome sequence. Yet, despite these exciting developments, there have been relatively few cases of real use of these methods outside the computational biology field, as reflected from citation analysis. These methods have the potential to be used in high-throughput experimental settings in functional genomics and proteomics to validate results with very high accuracy and good coverage. In this critical survey, we provide a comprehensive overview of 30 most prominent examples of single pairwise protein interaction cases in small-scale studies, where protein interactions have either been detected by gene fusion or yielded additional, corroborating evidence from biochemical observations. Our conclusion is that with the derivation of a validated gold-standard corpus and better data integration with big experiments, gene fusion detection can truly become a valuable tool for large-scale experimental biology.

  10. Two novel imatinib-responsive PDGFRA fusion genes in chronic eosinophilic leukaemia.

    Science.gov (United States)

    Curtis, Claire E; Grand, Francis H; Musto, Pellegrino; Clark, Andrew; Murphy, John; Perla, Gianni; Minervini, Maria M; Stewart, Janet; Reiter, Andreas; Cross, Nicholas C P

    2007-07-01

    We identified two patients with a t(2;4)(p24;q12) and a t(4;12)(q2?3;p1?2), respectively, in association with BCR-ABL and FIP1L1-PDGFRA negative chronic eosinophilic leukaemia. Molecular analysis revealed a novel STRN-PDGFRA fusion for the t(2;4) and ETV6-PDGFRA for the t(4;12). The fusions were confirmed by specific amplification of the genomic breakpoints, reverse transcription polymerase chain reaction and fluorescence in situ hybridisation. Both patients were treated with imatinib and, following a rapid haematological response, achieved cytogenetic remission and a major molecular response. In conclusion, PDGFRA fuses to diverse partner genes in myeloid disorders. Identification of these fusions is important as they are particularly sensitive to imatinib.

  11. Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

    Science.gov (United States)

    Kannan, Kalpana; Coarfa, Cristian; Chao, Pei-Wen; Luo, Liming; Wang, Yan; Brinegar, Amy E.; Hawkins, Shannon M.; Milosavljevic, Aleksandar; Matzuk, Martin M.; Yen, Laising

    2015-01-01

    High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient’s tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC. PMID:25733895

  12. Gene fusion analysis in the battle against the African endemic sleeping sickness.

    Directory of Open Access Journals (Sweden)

    Philip Trimpalis

    Full Text Available The protozoan Trypanosoma brucei causes African Trypanosomiasis or sleeping sickness in humans, which can be lethal if untreated. Most available pharmacological treatments for the disease have severe side-effects. The purpose of this analysis was to detect novel protein-protein interactions (PPIs, vital for the parasite, which could lead to the development of drugs against this disease to block the specific interactions. In this work, the Domain Fusion Analysis (Rosetta Stone method was used to identify novel PPIs, by comparing T. brucei to 19 organisms covering all major lineages of the tree of life. Overall, 49 possible protein-protein interactions were detected, and classified based on (a statistical significance (BLAST e-value, domain length etc., (b their involvement in crucial metabolic pathways, and (c their evolutionary history, particularly focusing on whether a protein pair is split in T. brucei and fused in the human host. We also evaluated fusion events including hypothetical proteins, and suggest a possible molecular function or involvement in a certain biological process. This work has produced valuable results which could be further studied through structural biology or other experimental approaches so as to validate the protein-protein interactions proposed here. The evolutionary analysis of the proteins involved showed that, gene fusion or gene fission events can happen in all organisms, while some protein domains are more prone to fusion and fission events and present complex evolutionary patterns.

  13. Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors.

    Directory of Open Access Journals (Sweden)

    Nadine Norton

    Full Text Available Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16. Specifically for lincRNAs, we observed superb Pearson correlation (0.988 between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads. Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol

  14. The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression.

    Directory of Open Access Journals (Sweden)

    Gerhard Fuka

    Full Text Available BACKGROUND: ETV6/RUNX1 (E/R (also known as TEL/AML1 is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines. FINDINGS: Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP or down-regulated (KD-DOWN, the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the "cell activation", "immune response", "apoptosis", "signal transduction" and "development and differentiation" categories, whereas in the E/R KD-DOWN set only the "PI3K/AKT/mTOR signaling" and "hematopoietic stem cells" categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories "stem cell properties", "B-cell differentiation", "immune response", "cell adhesion" and "DNA damage" with RT-qPCR. CONCLUSION: Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.

  15. Ant opsins: sequences from the Saharan silver ant and the carpenter ant.

    Science.gov (United States)

    Popp, M P; Grisshammer, R; Hargrave, P A; Smith, W C

    1996-03-01

    cDNA clones encoding opsins from compound eyes of carpenter ant, Camponotus abdominalis, and Saharan silver ant, Cataglyphis bombycina, were isolated from cDNA libraries. The opsin cDNAs from each species code for deduced proteins with 378 amino acids which are 92% identical. Of the 30 amino acid differences between the two proteins, 13 are non-conservative. Eight of these non-conservative substitutions are within the membrane spanning domain. The presence of a potential Schiff-base counterion in helix III in both species suggests that these opsins are the protein moiety of the visible range pigments. When compared to all known opsins, these opsins are most similar to the opsin from preying mantis (76% identity at the amino acid level). Phyletic comparisons group the two ant opsins with the other arthropod long wavelength opsins.

  16. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

  17. Construction of expression vector for NT4-ADNF-9 fusion gene

    Institute of Scientific and Technical Information of China (English)

    Guo-xi Zheng; Kang Zhu; Yang Jing; Jun-rong Wei; Hong-liang Zhu

    2009-01-01

    Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neuraseusory deafness. Methods By means of asymmetrical prince/ template, double stranded eDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 eDNA was ligated to the signal and leader peptides of nenrotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was suheluned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restrtction enzymes digestion showed that we recombined ADNF-9 eDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcluned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 cbicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivtty is satisfactory.

  18. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  19. Detection of EMI4-ALK fusion gene in non-small cell lung cancer and its clinicopathologic correlation

    Institute of Scientific and Technical Information of China (English)

    钟山

    2013-01-01

    Objective To investigate the frequency of EML4-ALK fusion gene in non small-cell lung cancer NSCLC patients,and its correlation with clinicopathologic features.Methods Real-time PCR was used to detect

  20. Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on

    Institute of Scientific and Technical Information of China (English)

    Ma Zhen; Qin-Hai Shen; Guo-Min Chen; Da-Zhi Zhang

    2008-01-01

    AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells.METHODS: The recombinant adenoviruses AdXC,Ad-X and Ad-C expressing HBV X-HCV C fusion gene,HBVX gene and HCV C gene were constructed,respectively.Hepatoma cells were infected with different recombinant adenoviruses.MTT,colonyforming experiment,FCM,TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells.RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells.Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells.FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of GIS phase in the HepG2 cell cycle.The apoptosis index of the Ad-XC,Ad-X,Ad-C group cells was significantly lower than that of the AdO and control group cells.Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in AdXC infected cells.Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells,but not in control mice.CONCLUSION: Ad-XC,Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro.The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C.Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.

  1. Discovery of CTCF-sensitive Cis-spliced fusion RNAs between adjacent genes in human prostate cells.

    Directory of Open Access Journals (Sweden)

    Fujun Qin

    2015-02-01

    Full Text Available Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1 the parental genes are same-strand-neighboring genes; 2 the distance between the genes is within 30kb; 3 the 5' genes are actively transcribing; and 4 the chimeras tend to have the second-to-last exon in the 5' genes joined to the second exon in the 3' genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.

  2. Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase

    CSIR Research Space (South Africa)

    James, ER

    2012-10-01

    Full Text Available Microbiology and Biotechnology October 2012/ Vol. 96, No.2 Recombinant hepatitis B surface antigen production in Aspergillus niger: evaluating the strategy of gene fusion to native glucoamylase ER James a,c & WH van Zyl b & PJ van Zyl c & JF Görgens..., Pretoria 0001, South Africa Abstract This study demonstrates the potential of Aspergillus niger as a candidate expression system for virus- like particle production using gene fusion. Hepatitis B surface antigen (HBsAg) production, targeted...

  3. Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study.

    Science.gov (United States)

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2016-12-24

    Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5'-kinase fusion genes, combinatorial effects between 3'-KDR kinases and their 5'-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3'-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of 'effective' (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3'-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs' clinical implications.

  4. Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.

    Science.gov (United States)

    Tada, S; Gomi, K; Kitamoto, K; Takahashi, K; Tamura, G; Hara, S

    1991-10-01

    Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

  5. Analysis of EML4-ALK Gene Fusion Mutation in Patients 
with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xuzhou WANG

    2015-02-01

    Full Text Available Background and objective Non-small cell lung cancer (NSCLC is the main type of lung cancer, and the related locus mutation detection research has become a hot direction of molecular targeted therapy, studying on gene mutation status of echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase (EML4-ALK and epidermal growth factor receptor (EGFR, detecting the sensitivity of EML4-ALK gene fusion and gene mutation of EGFR. Methods EML4-ALK gene fusion in 85 cases of paraffin embedded tumor tissue and adjacent lung tissue was detected with the application of immunohistochemistry (IHC, Scorpions amplification refractory mutation system (Scorpions ARMS fluorescence quantitative PCR and fluorescence in situ hybridization (FISH technology, and EGFR gene in 18, 19, 20 and 21 exon mutation status was detected with the application of ARMS method. Results In 115 cases of NSCLC, IHC showed 32 cases with ALK (D5F3 expression, the expression rate was 27.8%; ARMS showed 27 cases with EML4-ALK fusion gene mutation, the mutation detection rate was 23.5%; 53 cases were detected with EGFR mutation, the mutation rate was 46%. While FISH showed 23 cases with EML4-ALK fusion gene mutation, the detection rate was 20%, slightly lower than the ARMS detection results, suggesting that ARMS more sensitive. Conclusion The application of IHC, ARMS fluorescence quantitative PCR and FISH technology can make a rapid and accurate evaluation of EML4-ALK gene fusion.

  6. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  7. Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans.

    Science.gov (United States)

    Ahmed, Zubair M; Masmoudi, Saber; Kalay, Ersan; Belyantseva, Inna A; Mosrati, Mohamed Ali; Collin, Rob W J; Riazuddin, Saima; Hmani-Aifa, Mounira; Venselaar, Hanka; Kawar, Mayya N; Tlili, Abdelaziz; van der Zwaag, Bert; Khan, Shahid Y; Ayadi, Leila; Riazuddin, S Amer; Morell, Robert J; Griffith, Andrew J; Charfedine, Ilhem; Caylan, Refik; Oostrik, Jaap; Karaguzel, Ahmet; Ghorbel, Abdelmonem; Riazuddin, Sheikh; Friedman, Thomas B; Ayadi, Hammadi; Kremer, Hannie

    2008-11-01

    Many proteins necessary for sound transduction have been identified through positional cloning of genes that cause deafness. We report here that mutations of LRTOMT are associated with profound nonsyndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, detected by protein blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, new transcripts can arise through partial or complete coalescence of genes. We provide evidence that in the primate lineage LRTOMT evolved from the fusion of two neighboring ancestral genes, which exist as separate genes (Lrrc51 and Tomt) in rodents.

  8. Origin of the plant Tm-1-like gene via two independent horizontal transfer events and one gene fusion event.

    Science.gov (United States)

    Yang, Zefeng; Liu, Li; Fang, Huimin; Li, Pengcheng; Xu, Shuhui; Cao, Wei; Xu, Chenwu; Huang, Jinling; Zhou, Yong

    2016-01-01

    The Tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a direct inhibitor of ToMV RNA replication to protect tomato from infection. The plant Tm-1-like (Tm-1L) protein is predicted to contain an uncharacterized N-terminal UPF0261 domain and a C-terminal TIM-barrel signal transduction (TBST) domain. Homologous searches revealed that proteins containing both of these two domains are mainly present in charophyte green algae and land plants but absent from glaucophytes, red algae and chlorophyte green algae. Although Tm-1 homologs are widely present in bacteria, archaea and fungi, UPF0261- and TBST-domain-containing proteins are generally encoded by different genes in these linages. A co-evolution analysis also suggested a putative interaction between UPF0261- and TBST-domain-containing proteins. Phylogenetic analyses based on homologs of these two domains revealed that plants have acquired UPF0261- and TBST-domain-encoding genes through two independent horizontal gene transfer (HGT) events before the origin of land plants from charophytes. Subsequently, gene fusion occurred between these two horizontally acquired genes and resulted in the origin of the Tm-1L gene in streptophytes. Our results demonstrate a novel evolutionary mechanism through which the recipient organism may acquire genes with functional interaction through two different HGT events and further fuse them into one functional gene.

  9. Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples

    Directory of Open Access Journals (Sweden)

    Nacu Serban

    2011-01-01

    Full Text Available Abstract Background Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs, have been estimated using expressed sequence tag (EST libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. Methods We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays. Results Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%. We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal

  10. Analysis of API2-MALT1 fusion, trisomies, and immunoglobulin VH genes in pulmonary mucosa-associated lymphoid tissue lymphoma.

    Science.gov (United States)

    Xia, Hongjing; Nakayama, Takahisa; Sakuma, Hidenori; Yamada, Seiji; Sato, Fumihiko; Takino, Hisashi; Okabe, Mitsukuni; Fujiyoshi, Yukio; Hattori, Hideo; Inagaki, Hiroshi

    2011-09-01

    Pulmonary mucosa-associated lymphoid tissue lymphoma is unique in that chronic inflammation is rare and that API2-MALT1 fusion, resulting from t(11;18)(q21;q21), occurs frequently. In this study, we examined 20 cases for API2-MALT1 fusion using the multiplex reverse-transcription polymerase chain reaction and looked for trisomy 3, trisomy 18, and abnormalities of MALT1 and IGH genes using fluorescence in situ hybridization. In addition, we analyzed VH genes by subcloning of the monoclonal polymerase chain reaction products. Of 20 cases studied, we detected gene abnormalities in 16: API2-MALT1 fusion in 9, trisomy 3 in 5, trisomy 18 in 4, MALT1 abnormality in 13, and IGH abnormality in 1. MALT1 gene abnormalities were concordant with API2-MALT1 fusion or trisomy 18. One case showed API2-MALT1 fusion and trisomy 3. On detection of API2-MALT1 fusion and trisomies, we were able to divide our cases into 3 groups, API2-MALT1 positive, trisomy positive, and no detectable gene abnormality, suggesting that tumor development had processed along different genetic pathways. All 20 cases were analyzed for VH genes. Most of the VH genes selected by the lymphomas belonged to the VH3 family, but there was no restriction to any particular VH fragment. Of interest, VH genes were unmutated in 7 cases, suggesting that T-cell-independent extrafollicular B-cell maturation may be important in the development of this lymphoma. In addition, both mutated and unmutated tumor cases were found to carry the API2-MALT1 fusion and trisomy 3. This observation suggests that these gene abnormalities may occur in microenvironments found before or outside of follicular germinal centers.

  11. FN1-EGF gene fusions are recurrent in calcifying aponeurotic fibroma.

    Science.gov (United States)

    Puls, Florian; Hofvander, Jakob; Magnusson, Linda; Nilsson, Jenny; Haywood, Elaine; Sumathi, Vaiyapuri P; Mangham, D Chas; Kindblom, Lars-Gunnar; Mertens, Fredrik

    2016-03-01

    Calcifying aponeurotic fibroma (CAF) is a soft tissue neoplasm with a predilection for the hands and feet in children and adolescents. Its molecular basis is unknown. We used chromosome banding analysis, fluorescence in situ hybridization (FISH), mRNA sequencing (RNA-seq), RT-PCR, and immunohistochemistry to characterize a series of CAFs. An insertion ins(2;4)(q35;q25q?) was identified in the index case. Fusion of the FN1 and EGF genes, mapping to the breakpoint regions on chromosomes 2 and 4, respectively, was detected by RNA-seq and confirmed by RT-PCR in the index case and two additional cases. FISH on five additional tumours identified FN1-EGF fusions in all cases. CAFs analysed by RT-PCR showed that FN1 exon 23, 27 or 42 was fused to EGF exon 17 or 19. High-level expression of the entire FN1 gene in CAF suggests that strong FN1 promoter activity drives inappropriate expression of the biologically active portion of EGF, which was detected immunohistochemically in 8/9 cases. The FN1-EGF fusion, which has not been observed in any other neoplasm, appears to be the main driver mutation in CAF. Although further functional studies are required to understand the exact pathogenesis of CAF, the composition of the chimera suggests an autocrine/paracrine mechanism of transformation. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  12. First Insights into the Subterranean Crustacean Bathynellacea Transcriptome: Transcriptionally Reduced Opsin Repertoire and Evidence of Conserved Homeostasis Regulatory Mechanisms

    Science.gov (United States)

    Kim, Bo-Mi; Kang, Seunghyun; Ahn, Do-Hwan; Kim, Jin-Hyoung; Ahn, Inhye; Lee, Chi-Woo; Cho, Joo-Lae; Min, Gi-Sik; Park, Hyun

    2017-01-01

    Bathynellacea (Crustacea, Syncarida, Parabathynellidae) are subterranean aquatic crustaceans that typically inhabit freshwater interstitial spaces (e.g., groundwater) and are occasionally found in caves and even hot springs. In this study, we sequenced the whole transcriptome of Allobathynella bangokensis using RNA-seq. De novo sequence assembly produced 74,866 contigs including 28,934 BLAST hits. Overall, the gene sequences were most similar to those of the waterflea Daphnia pulex. In the A. bangokensis transcriptome, no opsin or related sequences were identified, and no contig aligned to the crustacean visual opsins and non-visual opsins (i.e. arthropsins, peropsins, and melaopsins), suggesting potential regressive adaptation to the dark environment. However, A. bangokensis expressed conserved gene family sets, such as heat shock proteins and those related to key innate immunity pathways and antioxidant defense systems, at the transcriptional level, suggesting that this species has evolved adaptations involving molecular mechanisms of homeostasis. The transcriptomic information of A. bangokensis will be useful for investigating molecular adaptations and response mechanisms to subterranean environmental conditions. PMID:28107438

  13. Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes

    Directory of Open Access Journals (Sweden)

    Schulte Ina

    2012-12-01

    Full Text Available Abstract Background It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. Results We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing—55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads. From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer—in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. Conclusions This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports

  14. Construction and Expression of GST and Carbon Skeleton of Amatoxins Fusion Gene

    Institute of Scientific and Technical Information of China (English)

    Zhao Jian(赵建); Cao Mei; Zhang Jie; Hou Ruotong; Chen Yaofeng; Yang Zhirong

    2004-01-01

    The primer, AT For1, AT For2 and AT Back, are designed and synthesized for adding-PCR that is used to construct the fusion GST-AT gene. Depending on two-time adding-PCR amplification and the insertion of coding sequence of octapeptide carbon skeleton into the 3 terminus of GST gene, the authors get the masculine recon, pGAT-1, confirmed by sequence determination and analysis, whose ORF is read-through. After IPTG induction and partial purification, SDS-PAGE electrophoresis is employed to detect the gene expression. The expressions of total bacterial proteins are about 21.3%, 22.5%, 19.32%, 21.73% in E. coli BL21, DH5α, JM109 and Top10 strains, respectively. Considering the quantity of induced total bacteria, the E. coli BL21 is the best one among the four recipient strains. This article supplies an academic foundation for producing biological active amatoxins.

  15. Obesity and Prostate Cancer Risk According to Tumor TMPRSS2:ERG Gene Fusion Status.

    Science.gov (United States)

    Egbers, Lieke; Luedeke, Manuel; Rinckleb, Antje; Kolb, Suzanne; Wright, Jonathan L; Maier, Christiane; Neuhouser, Marian L; Stanford, Janet L

    2015-05-01

    The T2E gene fusion, formed by fusion of the transmembrane protease, serine 2, gene (TMPRSS2) with the erythroblast transformation-specific (ETS)-related gene (ERG), is found in approximately 50% of prostate cancers and may characterize distinct molecular subtypes of prostate cancer with different etiologies. We investigated the relationship between body mass index (BMI; weight (kg)/height (m)(2)) and prostate cancer risk by T2E status. Study participants were residents of King County, Washington, recruited for 2 population-based case-control studies conducted in 1993-1996 and 2002-2005. Tumor T2E status was determined for 563 prostate cancer patients who underwent radical prostatectomy. Information on weight, height, and covariables was obtained through in-person interviews. We performed polytomous logistic regression to calculate odds ratios and 95% confidence intervals for T2E-positive and -negative prostate cancer. Comparing the highest BMI quartile with the lowest, inverse associations were observed between recent (≥29.7 vs. prostate cancer. No significant associations were seen for men with T2E-negative tumors. This study provides evidence that obesity is specifically associated with reduced risk of developing androgen-responsive T2E fusion-positive tumors. The altered steroid hormone profile in obese men may contribute to this inverse association. © The Author 2015. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Opsin stability and folding: modulation by phospholipid bicelles.

    Science.gov (United States)

    McKibbin, Craig; Farmer, Nicola A; Jeans, Chris; Reeves, Philip J; Khorana, H Gobind; Wallace, B A; Edwards, Patricia C; Villa, Claudio; Booth, Paula J

    2007-12-14

    Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-alpha-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to approximately 70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability

  17. Molecular Characterization of Inflammatory Myofibroblastic Tumors With Frequent ALK and ROS1 Gene Fusions and Rare Novel RET Rearrangement

    NARCIS (Netherlands)

    Antonescu, Cristina R.; Suurmeijer, Albert J. H.; Zhang, Lei; Sung, Yun-Shao; Jungbluth, Achim A.; Travis, William D.; Al-Ahmadie, Hikmat; Fletcher, Christopher D. M.; Alaggio, Rita

    2015-01-01

    Approximately 50% of conventional inflammatory myofibroblastic tumors (IMTs) harbor ALK gene rearrangement and overexpress ALK. Recently, gene fusions involving other kinases have been implicated in the pathogenesis of IMT, including ROS1 and in 1 patient PDGFRB. However, it remains uncertain whethe

  18. Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Cole, G.M.; Schild, D.; Lovett, S.T.; Mortimer, R.K.

    1987-03-01

    The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.

  19. Extraordinarily low evolutionary rates of short wavelength-sensitive opsin pseudogenes

    Science.gov (United States)

    Yokoyama, Shozo; Starmer, William T.; Liu, Yang; Tada, Takashi; Britt, Lyle

    2013-01-01

    Aquatic organisms such as cichlids, coelacanths, seals, and cetaceans are active in UV-blue color environments, but many of them mysteriously lost their abilities to detect these colors. The loss of these functions is a consequence of the pseudogenization of their short wavelength-sensitive (SWS1) opsin genes without gene duplication. We show that the SWS1 gene (BdenS1ψ) of the deep-sea fish, pearleye (Benthalbella dentata), became a pseudogene in a similar fashion about 130 million years ago (Mya) yet it is still transcribed. The rates of nucleotide substitution (~1.4 × 10−9 /site/year) of the pseudogenes of these aquatic species as well as some prosimian and bat species are much smaller than the previous estimates for the globin and immunoglobulin pseudogenes. PMID:24125953

  20. Limited inter- and intra-patient sequence diversity of the genetic lineage A human metapneumovirus fusion gene

    DEFF Research Database (Denmark)

    Winther, Thilde Nordmann; Madsen, Chris D; Pedersen, Anders

    2005-01-01

    Human metapneumovirus (hMPV) is associated with respiratory tract illness especially in young children. Two hMPV genetic lineages, A and B, and four sublineages A1, A2 and B1, B2 have been defined. Infection with hMPV occurs through membrane fusion mediated by the hMPV fusion (F) protein. In this...... diversity observed lay emphasis on the hMPV F gene as a putative target for future vaccine development....

  1. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    Science.gov (United States)

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.

  2. High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine.

    Science.gov (United States)

    Koo, Kevin M; Wee, Eugene J H; Trau, Matt

    2017-03-15

    Aberrant chromosal rearrangements, such as the multiple variants of TMPRSS2:ERG fusion gene mutations in prostate cancer (PCa), are promising diagnostic and prognostic biomarkers due to their specific expression in cancerous tissue only. Additionally, TMPRSS2:ERG variants are detectable in urine to provide non-invasive PCa diagnostic sampling as an attractive surrogate for needle biopsies. Therefore, rapid and simplistic assays for identifying multiple urinary TMPRSS2:ERG variants are potentially useful to aid in early cancer detection, immediate patient risk stratification, and prompt personalized treatment. However, current strategies for simultaneous detection of multiple gene fusions are limited by tedious and prolonged experimental protocols, thus limiting their use as rapid clinical screening tools. Herein, we report a simple and rapid gene fusion strategy which expliots the specificity of DNA ligase and the speed of isothermal amplification to simultaneously detect multiple fusion gene RNAs within a short sample-to-answer timeframe of 60min. The method has a low detection limit of 2 amol (1000 copies), and was successfully applied for non-invasive fusion gene profiling in patient urine samples with subsequent validation by a PCR-based gold standard approach.

  3. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    Science.gov (United States)

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  4. Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.

    Science.gov (United States)

    Feng, Weiguo; Leach, Sonia M; Tipney, Hannah; Phang, Tzulip; Geraci, Mark; Spritz, Richard A; Hunter, Lawrence E; Williams, Trevor

    2009-12-16

    Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions - the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5-E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional

  5. Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.

    Directory of Open Access Journals (Sweden)

    Weiguo Feng

    Full Text Available Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions - the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5-E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of

  6. Convergent evolution of SWS2 opsin facilitates adaptive radiation of threespine stickleback into different light environments.

    Science.gov (United States)

    Marques, David A; Taylor, John S; Jones, Felicity C; Di Palma, Federica; Kingsley, David M; Reimchen, Thomas E

    2017-04-01

    Repeated adaptation to a new environment often leads to convergent phenotypic changes whose underlying genetic mechanisms are rarely known. Here, we study adaptation of color vision in threespine stickleback during the repeated postglacial colonization of clearwater and blackwater lakes in the Haida Gwaii archipelago. We use whole genomes from 16 clearwater and 12 blackwater populations, and a selection experiment, in which stickleback were transplanted from a blackwater lake into an uninhabited clearwater pond and resampled after 19 y to test for selection on cone opsin genes. Patterns of haplotype homozygosity, genetic diversity, site frequency spectra, and allele-frequency change support a selective sweep centered on the adjacent blue- and red-light sensitive opsins SWS2 and LWS. The haplotype under selection carries seven amino acid changes in SWS2, including two changes known to cause a red-shift in light absorption, and is favored in blackwater lakes but disfavored in the clearwater habitat of the transplant population. Remarkably, the same red-shifting amino acid changes occurred after the duplication of SWS2 198 million years ago, in the ancestor of most spiny-rayed fish. Two distantly related fish species, bluefin killifish and black bream, express these old paralogs divergently in black- and clearwater habitats, while sticklebacks lost one paralog. Our study thus shows that convergent adaptation to the same environment can involve the same genetic changes on very different evolutionary time scales by reevolving lost mutations and reusing them repeatedly from standing genetic variation.

  7. On the origin of protein synthesis factors: a gene duplication/fusion model.

    Science.gov (United States)

    Cousineau, B; Leclerc, F; Cedergren, R

    1997-12-01

    Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor. We evaluate this proposition and whether the relationship can be extended to other factors of protein synthesis. Analysis of amino acid sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G and suggests further that this association is a result of gene duplication/fusion events. In support of this mechanism, the three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity. The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes. The root of this protein superfamily tree would be an ancestor of the modern IF-1 gene sequence. The repeated fundamental motif of this protein superfamily is a small RNA binding domain composed of two alpha-helices packed along side of an antiparallel beta-sheet.

  8. Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia.

    Science.gov (United States)

    Alcalay, M; Zangrilli, D; Fagioli, M; Pandolfi, P P; Mencarelli, A; Lo Coco, F; Biondi, A; Grignani, F; Pelicci, P G

    1992-01-01

    Two chimeric genes, PML-RAR alpha and RAR alpha-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal translocation of chromosomes 15 and 17 [t(15;17)]. PML-RAR alpha is expressed as a fusion protein. We investigated the organization and expression pattern of the RAR alpha-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RAR alpha-PML mRNA junctions (RAR alpha exon 2/PML exon 4 or RAR alpha exon 2/PML exon 7) that maintain the RAR alpha and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (ii) 10 different RAR alpha-PML fusion transcripts that differ for the assembly of their PML coding exons. A RAR alpha-PML transcript was present in most, but not all, APL patients. Images PMID:1317574

  9. PL1 fusion gene: a novel visual selectable marker gene that confers tolerance to multiple abiotic stresses in transgenic tomato.

    Science.gov (United States)

    Jin, Feng; Li, Shu; Dang, Lijie; Chai, Wenting; Li, Pengli; Wang, Ning Ning

    2012-10-01

    Visual selectable markers, including the purple color caused by the accumulation of anthocyanins, have been proposed for use as antibiotic-free alternatives. However, the excessive accumulation of anthocyanins seriously inhibits the growth and development of transgenic plants. In our study, the AtDWF4 promoter from Arabidopsis and the tomato LeANT1 gene, encoding a MYB transcription factor, were used to construct the PL1 fusion gene to test whether it could be used as a visual selectable marker gene for tomato transformation. All the PL1 transgenic shoots exhibited intense purple color on shoot induction medium. In the transgenic tomato plants, PL1 was highly expressed in the cotyledons, but expressed only slightly in the true leaves and other organs. The expression of PL1 had no significantly adverse effects on the growth or development of the transgenic tomato plants, and conferred tolerance to multiple abiotic stresses in them. With the “cut off green shoots” method, multiple independent 35S::GFP transgenic tomato lines were successfully obtained using PL1 as the selectable marker gene. These results suggest that PL1 has potential application of visual selectable marker gene for tomato transformation.

  10. Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review).

    Science.gov (United States)

    Sinkovics, Joseph G

    2009-09-01

    Evolving young genomes of archaea, prokaryota and unicellular eukaryota were wide open for the acceptance of alien genomic sequences, which they often preserved and vertically transferred to their descendants throughout three billion years of evolution. Established complex large genomes, although seeded with ancestral retroelements, have come to regulate strictly their integrity. However, intruding retroelements, especially the descendents of Ty3/Gypsy, the chromoviruses, continue to find their ways into even the most established genomes. The simian and hominoid-Homo genomes preserved and accommodated a large number of endogenous retroviral genomic segments. These retroelements may mature into exogenous retroviruses, or into functional new genes. Phages and viruses have been instrumental in incorporating and transferring host cell genes. These events profoundly influenced and altered the course of evolution. Horizontal (lateral) gene transfers (HGT) overwhelmed the genomes of the ancient protocells and the evolving unicellular microorganisms, actually leading to their Cambrian explosion. While the rigidly organized genomes of multicellular organisms increasingly resist H/LGT, de-differentiated cells assuming the metabolism of their onto- or phylogenetic ancestors, open up widely to the practice of H/LGT by direct transfer, or to transfers mediated by viruses, or by cell fusions. This activity is intensified in malignantly transformed cells, thus rendering these subjects receptive to therapy with oncolytic viruses and with viral vectors of tumor-suppressive or immunogenic genetic materials. Naturally formed hybrids of dendritic and tumor cells are often tolerogenic, whereas laboratory products of these unisons may be immunogenic in the hosts of origin. As human breast cancer stem cells are induced by a treacherous class of CD8+ T cells to undergo epithelial to mesenchymal (ETM) transition and to yield to malignant transformation by the omnipresent proto

  11. NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.

    Science.gov (United States)

    Kirby, J; Kavanagh, T A

    2002-11-01

    GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

  12. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

    Science.gov (United States)

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2017-01-01

    The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection.

  13. Evolution by Pervasive Gene Fusion in Antibiotic Resistance and Antibiotic Synthesizing Genes

    Directory of Open Access Journals (Sweden)

    Orla Coleman

    2015-03-01

    Full Text Available Phylogenetic (tree-based approaches to understanding evolutionary history are unable to incorporate convergent evolutionary events where two genes merge into one. In this study, as exemplars of what can be achieved when a tree is not assumed a priori, we have analysed the evolutionary histories of polyketide synthase genes and antibiotic resistance genes and have shown that their history is replete with convergent events as well as divergent events. We demonstrate that the overall histories of these genes more closely resembles the remodelling that might be seen with the children’s toy Lego, than the standard model of the phylogenetic tree. This work demonstrates further that genes can act as public goods, available for re-use and incorporation into other genetic goods.

  14. Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation.

    Science.gov (United States)

    Sun, He; Lang, Zhihong; Zhu, Li; Huang, Dafang

    2012-10-01

    The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG ( 2 ) -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG(2), were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.

  15. Anti-colorectal cancer effect of interleukin-2 and interferon-β fusion gene driven by carcinoembryonic antigen promoter

    Directory of Open Access Journals (Sweden)

    Wang Y

    2016-05-01

    Full Text Available Yan Wang, Mengchun Wang, Yan LiDepartment of Gastroenterology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of ChinaAbstract: This study was designed to investigate the antitumor effects of combined interleukin-2/interferon-β-based gene therapy in colorectal cancer. Transfection of the fusion gene expression plasmid induced significant apoptosis of Lovo cells. Additionally, the fusion gene exhibited strong inhibitory activity against tumor growth and apoptosis when being injected into the nude mice implanted with human colon cancer cells. Furthermore, the tail-vein injection showed a more notable effect than direct injection into tumor. These results suggest that the combined interleukin-2/interferon-β-based gene therapy with the carcinoembryonic antigen promoter might be an effective antitumor strategy.Keywords: apoptosis, interferon-β, interleukin-2, antitumor, combined gene therapy

  16. A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy

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    Yumei Luo

    2015-01-01

    Full Text Available Recent progress in neural stem cell- (NSC- based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is “switched off” by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is “switched on” by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.

  17. The co-chaperone and reductase ERdj5 facilitates rod opsin biogenesis and quality control.

    Science.gov (United States)

    Athanasiou, Dimitra; Bevilacqua, Dalila; Aguila, Monica; McCulley, Caroline; Kanuga, Naheed; Iwawaki, Takao; Chapple, J Paul; Cheetham, Michael E

    2014-12-15

    Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations, such as P23H, lead to misfolding of rod opsin with detrimental effects on photoreceptor function and viability. Misfolded P23H rod opsin and other mutations in the intradiscal domain are characterized by the formation of an incorrect disulphide bond between C185 and C187, as opposed to the correct and highly conserved C110-C187 disulphide bond. Therefore, we tested the hypothesis that incorrect disulphide bond formation might be a factor that affects the biogenesis of rod opsin by studying wild-type (WT) or P23H rod opsin in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds involving C185. These mutants had altered traffic dynamics, suggesting a requirement for regulation of disulphide bond formation/reduction during rod opsin biogenesis. Here, we show that the BiP co-chaperone and reductase protein ERdj5 (DNAJC10) regulates this process. ERdj5 overexpression promoted the degradation, improved the endoplasmic reticulum mobility and prevented the aggregation of P23H rod opsin. ERdj5 reduction by shRNA delayed rod opsin degradation and promoted aggregation. The reductase and co-chaperone activity of ERdj5 were both required for these effects on P23H rod opsin. Furthermore, mutations in these functional domains acted as dominant negatives that affected WT rod opsin biogenesis. Collectively, these data identify ERdj5 as a member of the proteostasis network that regulates rod opsin biogenesis and supports a role for disulphide bond formation/reduction in rod opsin biogenesis and disease.

  18. Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.

    Directory of Open Access Journals (Sweden)

    Manuel A F V Gonçalves

    Full Text Available Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and

  19. Fusion gene and splice variant analyses in liquid biopsies of lung cancer patients

    Science.gov (United States)

    Giménez-Capitán, Ana; Karachaliou, Niki; Pérez-Rosado, Ana; Viteri, Santiago; Morales-Espinosa, Daniela; Rosell, Rafael

    2016-01-01

    Obtaining a biopsy of solid tumors requires invasive procedures that strongly limit patient compliance. In contrast, a blood extraction is safe, can be performed at many time points during the course disease and encourages appropriate therapy modifications, potentially improving the patient’s clinical outcome and quality of life. Fusion of the tyrosine kinase genes anaplastic lymphoma kinase (ALK), C-ROS oncogen 1 (ROS 1), rearranged during transfection (RET) and neurotrophic tyrosine kinase 1 (NTRK1) occur in 1–5% of lung adenocarcinomas and constitute therapeutic targets for tyrosine kinase inhibitors. In addition, a MET splicing variant of exon 14, has been reported in 2–4% of lung adenocarcinoma and recent studies suggests that targeted therapies inhibiting MET signaling would be beneficial for patients with this alteration. In this review, we will summarize the new techniques recently developed to detect ALK, RET, ROS and NTRK1 fusions and MET exon 14 splicing variant in liquid biopsy using plasma, serum, circulating tumor cells (CTCs), platelets and exosomes as starting material. PMID:27826534

  20. Short wavelength-sensitive opsins from the Saharan silver and carpenter ants.

    Science.gov (United States)

    Smith, W C; Ayers, D M; Popp, M P; Hargrave, P A

    1997-06-01

    We have previously cloned the opsins coding for the long-wavelength visual pigments from the Saharan silver ant and carpenter ant. Here we report two new cDNA clones isolated from cDNA libraries which also code for opsin proteins. These cDNAs code for deduced proteins with 369 amino acids which are 91% identical to each other, but only 38% identical to the previously cloned opsins. Phyletic comparisons suggest that these opsins are likely the ultraviolet sensitive visual pigments, a conclusion that is supported by the presence of a phenylalanine at the counterion position in the third transmembrane segment.

  1. Construction of hpaA gene from a clinical isolate of Helicobacter pyloriand identification of fusion protein

    Institute of Scientific and Technical Information of China (English)

    Ya-Fei Mao; Jie Yan; Li-Wei Li; Shu-Ping Li

    2003-01-01

    AIM: To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein.METHODS: The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E. coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE.Western blot with commercial antibody against whole cell of H. pylorias well as immunodiffusion assay with selfprepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H. pylori and to examine HpaA expression of 109 clinical isolates of H. pylori.RESULTS: In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32ahpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H. pyloriand to induce rabbit producing specific antiserum with 1:4immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125patients infected with H.pylori(102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori(109/109) were detectable for HpaA.CONCLUSION: A prokaryotic expression system with high efficiency of H.pylorihpaA gene was successfully established.The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H. pyloriclinicalstrains

  2. Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein.

    Science.gov (United States)

    Mao, Ya-Fei; Yan, Jie; Li, Li-Wei; Li, Shu-Ping

    2003-07-01

    To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein. The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H.pylori as well as immunodiffusion assay with self-prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H.pylori and to examine HpaA expression of 109 clinical isolates of H.pylori. In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32a-hpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H.pylori and to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125 patients infected with H.pylori (102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori (109/109) were detectable for HpaA. A prokaryotic expression system with high efficiency of H.pylori hpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H.pylori clinical strains and specific antibody

  3. Discovery and characterization of a novel CCND1/MRCK gene fusion in mantle cell lymphoma

    Directory of Open Access Journals (Sweden)

    Chioniso Patience Masamha

    2016-03-01

    Full Text Available Abstract The t(11;14 translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3′UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (CCND1 and a 3′UTR consisting of sequences from both the CCND1 3′UTR and myotonic dystrophy kinase-related Cdc42-binding kinase's (MRCK intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3′UTR with siRNA results in decreased CCND1 levels.

  4. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

    Science.gov (United States)

    Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca

    2014-04-01

    Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

  5. Species based synonymous codon usage in fusion protein gene of Newcastle disease virus.

    Directory of Open Access Journals (Sweden)

    Chandra Shekhar Kumar

    Full Text Available Newcastle disease is highly pathogenic to poultry and many other avian species. However, the Newcastle disease virus (NDV has also been reported from many non-avian species. The NDV fusion protein (F is a major determinant of its pathogenicity and virulence. The functionalities of F gene have been explored for the development of vaccine and diagnostics against NDV. Although the F protein is well studied but the codon usage and its nucleotide composition from NDV isolated from different species have not yet been explored. In present study, we have analyzed the factors responsible for the determination of codon usage in NDV isolated from four major avian host species. The F gene of NDV is analyzed for its base composition and its correlation with the bias in codon usage. Our result showed that random mutational pressure is responsible for codon usage bias in F protein of NDV isolates. Aromaticity, GC3s, and aliphatic index were not found responsible for species based synonymous codon usage bias in F gene of NDV. Moreover, the low amount of codon usage bias and expression level was further confirmed by a low CAI value. The phylogenetic analysis of isolates was found in corroboration with the relatedness of species based on codon usage bias. The relationship between the host species and the NDV isolates from the host does not represent a significant correlation in our study. The present study provides a basic understanding of the mechanism involved in codon usage among species.

  6. Drosophila Erect wing (Ewg) controls mitochondrial fusion during muscle growth and maintenance by regulation of the Opa1-like gene.

    Science.gov (United States)

    Rai, Mamta; Katti, Prasanna; Nongthomba, Upendra

    2014-01-01

    Mitochondrial biogenesis and morphological changes are associated with tissue-specific functional demand, but the factors and pathways that regulate these processes have not been completely identified. A lack of mitochondrial fusion has been implicated in various developmental and pathological defects. The spatiotemporal regulation of mitochondrial fusion in a tissue such as muscle is not well understood. Here, we show in Drosophila indirect flight muscles (IFMs) that the nuclear-encoded mitochondrial inner membrane fusion gene, Opa1-like, is regulated in a spatiotemporal fashion by the transcription factor/co-activator Erect wing (Ewg). In IFMs null for Ewg, mitochondria undergo mitophagy and/or autophagy accompanied by reduced mitochondrial functioning and muscle degeneration. By following the dynamics of mitochondrial growth and shape in IFMs, we found that mitochondria grow extensively and fuse during late pupal development to form the large tubular mitochondria. Our evidence shows that Ewg expression during early IFM development is sufficient to upregulate Opa1-like, which itself is a requisite for both late pupal mitochondrial fusion and muscle maintenance. Concomitantly, by knocking down Opa1-like during early muscle development, we show that it is important for mitochondrial fusion, muscle differentiation and muscle organization. However, knocking down Opa1-like, after the expression window of Ewg did not cause mitochondrial or muscle defects. This study identifies a mechanism by which mitochondrial fusion is regulated spatiotemporally by Ewg through Opa1-like during IFM differentiation and growth.

  7. Molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B.

    Science.gov (United States)

    Kimura, Hirokazu; Nagasawa, Koo; Kimura, Ryusuke; Tsukagoshi, Hiroyuki; Matsushima, Yuki; Fujita, Kiyotaka; Hirano, Eiko; Ishiwada, Naruhiko; Misaki, Takako; Oishi, Kazunori; Kuroda, Makoto; Ryo, Akihide

    2017-08-01

    In this study, we examined the molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B (HRSV-B). First, we performed time-scale evolution analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. Next, we performed genetic distance, linear B-cell epitope prediction, N-glycosylation, positive/negative selection site, and Bayesian skyline plot analyses. We also constructed a structural model of the F protein and mapped the amino acid substitutions and the predicted B-cell epitopes. The MCMC-constructed phylogenetic tree indicated that the HRSV F gene diverged from the bovine respiratory syncytial virus gene approximately 580years ago and had a relatively low evolutionary rate (7.14×10(-4)substitutions/site/year). Furthermore, a common ancestor of HRSV-A and -B diverged approximately 290years ago, while HRSV-B diverged into three clusters for approximately 60years. The genetic similarity of the present strains was very high. Although a maximum of 11 amino acid substitutions were observed in the structural model of the F protein, only one strain possessed an amino acid substitution located within the palivizumab epitope. Four epitopes were predicted, although these did not correspond to the neutralization sites of the F protein including the palivizumab epitope. In addition, five N-glycosylation sites of the present HRSV-B strains were inferred. No positive selection sites were identified; however, many sites were found to be under negative selection. The effective population size of the gene has remained almost constant. On the basis of these results, it can be concluded that the HRSV-B F gene is highly conserved, as is the F protein of HRSV-A. Moreover, our prediction of B-cell epitopes does not show that the palivizumab reaction site may be recognized as an epitope during naturally occurring infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Evolutionary Origins of the Eukaryotic Shikimate Pathway: Gene Fusions, Horizontal Gene Transfer, and Endosymbiotic Replacements†

    OpenAIRE

    2006-01-01

    Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These ...

  9. Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing.

    Science.gov (United States)

    Walther, Charles; Hofvander, Jakob; Nilsson, Jenny; Magnusson, Linda; Domanski, Henryk A; Gisselsson, David; Tayebwa, Johnbosco; Doyle, Leona A; Fletcher, Christopher D M; Mertens, Fredrik

    2015-09-01

    Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues.

  10. Synovial Sarcoma Microvesicles Harbor the SYT-SSX Fusion Gene Transcript: Comparison of Different Methods of Detection and Implications in Biomarker Research.

    Science.gov (United States)

    Fricke, A; Ullrich, P V; Cimniak, A F V; Follo, M; Nestel, S; Heimrich, B; Nazarenko, I; Stark, G B; Bannasch, H; Braig, D; Eisenhardt, S U

    2016-01-01

    Background. Synovial sarcoma is an aggressive soft-tissue malignancy. This study examines the presence of the SYT-SSX fusion transcript in synovial sarcoma microvesicles as well as its potential role as a biomarker for synovial sarcoma. Patients and Methods. Microvesicle release of synovial sarcoma cells was examined by transmission electron microscopy. RNA-content was analyzed by qPCR, nested PCR, nested qPCR, and droplet digital PCR to compare their sensitivity for detection of the SYT-SSX fusion gene transcript. Whole blood RNA, RNA of mononuclear cells, and microvesicle RNA of synovial sarcoma patients were analyzed for the presence of the fusion gene transcripts. Results. Electron microscopic analysis revealed synovial sarcoma cells releasing membrane-enclosed microvesicles. In vitro, the SYT-SSX fusion gene transcript was detected in both synovial sarcoma cells and microvesicles. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA. In contrast, the fusion gene transcript was not detected in peripheral blood cells and microvesicles of synovial sarcoma patients. Conclusion. Synovial sarcoma cells release microvesicles harboring the SYT-SSX fusion transcript. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA; however, more sensitive assays are needed to detect cancer-specific microvesicles in the peripheral blood of cancer patients.

  11. Hh/Gli antagonist in acute myeloid leukemia with CBFA2T3-GLIS2 fusion gene

    Directory of Open Access Journals (Sweden)

    Riccardo Masetti

    2017-01-01

    Full Text Available Abstract Background CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7 and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB subtypes. Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2 is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. Methods We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. Results As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. Conclusions Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.

  12. Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP.

    Science.gov (United States)

    Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi; Miyata, Maiko; Huang, Peng; Ishiguro, Naoki; Hamaguchi, Michinari; Iwamoto, Takashi

    2008-08-08

    Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.

  13. Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations.

    Science.gov (United States)

    Bueno, Raphael; Stawiski, Eric W; Goldstein, Leonard D; Durinck, Steffen; De Rienzo, Assunta; Modrusan, Zora; Gnad, Florian; Nguyen, Thong T; Jaiswal, Bijay S; Chirieac, Lucian R; Sciaranghella, Daniele; Dao, Nhien; Gustafson, Corinne E; Munir, Kiara J; Hackney, Jason A; Chaudhuri, Amitabha; Gupta, Ravi; Guillory, Joseph; Toy, Karen; Ha, Connie; Chen, Ying-Jiun; Stinson, Jeremy; Chaudhuri, Subhra; Zhang, Na; Wu, Thomas D; Sugarbaker, David J; de Sauvage, Frederic J; Richards, William G; Seshagiri, Somasekar

    2016-04-01

    We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score ≥ 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (∼2%; 4/216) and TRAF7 (∼2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

  14. The Study on BCR/ABL Fusion Gene in Adult Acute Lymphoblastic Leukemia

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to assess the significance of BCR/ABC fusion gene in adult acute lymphoblastic Leukemia (ALL), 28 patients who were diagnosed as ALL were enrolled to detect BCR/ABC gene using nested-RT PCR. The results showed that 9 cases (31.25%) were BCR/ABL positive ,and expressed P210 subtype. A mong them 7 cases were B-ALL, and one was T-ALL. The diagnosis was proved by monoclonal antibodies recognition by indirect immunofluorescence. Adult patients with BCR/ABL positive ALL were significantly older (p<0. 01) and had higher WBC count (p<0. 01) as compared with BCR/ABL-negative patients. There was no significant difference in sex, hemoglobin and splenomegaly between two group (p>0. 05). The induc tion failure rate was high in BCR/ABL positive patients and those who achieved complete remission usually relapsed earlier. In conclusion, adult ALL patients with BCR/ABL-positive have poorer prognosis.

  15. Limited inter- and intra-patient sequence diversity of the genetic lineage a human metapneumovirus fusion gene

    DEFF Research Database (Denmark)

    Winther, T.N.; Madsen, C.D.; Pedersen, Anders Gorm;

    2005-01-01

    Human metapneumovirus (hMPV) is associated with respiratory tract illness especially in young children. Two hMPV genetic lineages, A and B, and four sublineages A1, A2 and B1, B2 have been defined. Infection with hMPV occurs through membrane fusion mediated by the hMPV fusion (F) protein. In this...... diversity observed lay emphasis on the hMPV F gene as a putative target for future vaccine development....... been infected with at least two viruses. Several independent viruses contained premature stop codons in exactly identical positions resulting in truncated fusion proteins. Possibly this is a mechanism for immune system evasion. The F protein is a major antigenic determinant, and the limited sequence...

  16. Epigenomic profiling of prostate cancer identifies differentially methylated genes in TMPRSS2:ERG fusion-positive versus fusion-negative tumors.

    Science.gov (United States)

    Geybels, Milan S; Alumkal, Joshi J; Luedeke, Manuel; Rinckleb, Antje; Zhao, Shanshan; Shui, Irene M; Bibikova, Marina; Klotzle, Brandy; van den Brandt, Piet A; Ostrander, Elaine A; Fan, Jian-Bing; Feng, Ziding; Maier, Christiane; Stanford, Janet L

    2015-01-01

    About half of all prostate cancers harbor the TMPRSS2:ERG (T2E) gene fusion. While T2E-positive and T2E-negative tumors represent specific molecular subtypes of prostate cancer (PCa), previous studies have not yet comprehensively investigated how these tumor subtypes differ at the epigenetic level. We therefore investigated epigenome-wide DNA methylation profiles of PCa stratified by T2E status. The study included 496 patients with clinically localized PCa who had a radical prostatectomy as primary treatment for PCa. Fluorescence in situ hybridization (FISH) "break-apart" assays were used to determine tumor T2E-fusion status, which showed that 266 patients (53.6 %) had T2E-positive PCa. The study showed global DNA methylation differences between tumor subtypes. A large number of differentially methylated CpG sites were identified (false-discovery rate [FDR] Q-value <0.00001; n = 27,876) and DNA methylation profiles accurately distinguished between tumor T2E subgroups. A number of top-ranked differentially methylated CpGs in genes (FDR Q-values ≤1.53E-29) were identified: C3orf14, CACNA1D, GREM1, KLK10, NT5C, PDE4D, RAB40C, SEPT9, and TRIB2, several of which had a corresponding alteration in mRNA expression. These genes may have various roles in the pathogenesis of PCa, and the calcium-channel gene CACNA1D is a known ERG-target. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings. This study identified substantial differences in DNA methylation profiles of T2E-positive and T2E-negative tumors, thereby providing further evidence that different underlying oncogenic pathways characterize these molecular subtypes.

  17. Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples

    Science.gov (United States)

    Kojima, Takahiro; Nishimura, Kouichi; Kandori, Shuya; Kawahara, Takashi; Yoshino, Takayuki; Ueno, Satoshi; Iizumi, Yuichi; Mitsuzuka, Koji; Arai, Yoichi; Tsuruta, Hiroshi; Habuchi, Tomonori; Kobayashi, Takashi; Matsui, Yoshiyuki; Ogawa, Osamu; Sugimoto, Mikio; Kakehi, Yoshiyuki; Nagumo, Yoshiyuki; Tsutsumi, Masakazu; Oikawa, Takehiro; Kikuchi, Koji; Nishiyama, Hiroyuki

    2016-01-01

    Introduction and Objectives Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples. Materials and Methods The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections. Results FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive. Conclusions We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors. PMID:27930669

  18. Construction and Expression of Methionine-rich and Lysine-rich Fusion Gene inBacillus natto

    Institute of Scientific and Technical Information of China (English)

    Zhang Shuang; Luo Chao-chao; Wu Cai-xia; Gao Xue-jun

    2015-01-01

    Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene (zein) from maize endosperm protein with lysine-rich gene (Cflr) from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred intoBacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombinedBacillus nattoas feed additive.

  19. Fusion of ZMYND8 and RELA genes in acute erythroid leukemia

    DEFF Research Database (Denmark)

    Panagopoulos, Ioannis; Micci, Francesca; Thorsen, Jim

    2013-01-01

    Acute erythroid leukemia was diagnosed in a 4-month-old boy. Cytogenetic analysis of bone marrow (BM) cells showed a t(11;20)(p11;q11) translocation. RNA extracted from the BM was sequenced and analyzed for fusion transcripts using the software FusionMap. A ZMYND8-RELA fusion was ranked first. RT...... the translocation. The putative ZMYND8-RELA fusion protein contains the Zinc-PHD finger domain, a bromodomain, a PWWP domain, a MYND type of zinc finger of ZMYND8, and the entire RELA protein, indicating that it might act leukemogenically by influencing several cellular processes including the NF-kappa-B pathway....

  20. Opsin gene analysis in the cultured kawakawa Euthynnus affinis

    National Research Council Canada - National Science Library

    松本, 太朗; 阿川, 泰夫; 岡田, 貴彦; 澤田, 好史; 石橋, 泰典

    2015-01-01

    スマはインド洋から西太平洋の亜熱帯海域に分布する回遊性資源の一種である。本種は水産資源としての商業的価値が高いにもかかわらず,生理・生態学的特性に不明な点が多い。そこで本研究は,生態学的特性や養殖時の光環境等に関係する網膜の視物質オプシン遺伝子を解析した。その結果,11種類[Rh1(桿体); Rh2A1,...

  1. Visual sensitivities tuned by heterochronic shifts in opsin gene expression

    OpenAIRE

    McFarland William N; Kidd Michael R; Streelman J Todd; Spady Tyrone C; Carleton Karen L; Loew Ellis R

    2008-01-01

    Abstract Background Cichlid fishes have radiated into hundreds of species in the Great Lakes of Africa. Brightly colored males display on leks and vie to be chosen by females as mates. Strong discrimination by females causes differential male mating success, rapid evolution of male color patterns and, possibly, speciation. In addition to differences in color pattern, Lake Malawi cichlids also show some of the largest known shifts in visual sensitivity among closely related species. These shif...

  2. Gene fusions and gene duplications: relevance to genomic annotation and functional analysis

    Directory of Open Access Journals (Sweden)

    Riley Monica

    2005-03-01

    Full Text Available Abstract Background Escherichia coli a model organism provides information for annotation of other genomes. Our analysis of its genome has shown that proteins encoded by fused genes need special attention. Such composite (multimodular proteins consist of two or more components (modules encoding distinct functions. Multimodular proteins have been found to complicate both annotation and generation of sequence similar groups. Previous work overstated the number of multimodular proteins in E. coli. This work corrects the identification of modules by including sequence information from proteins in 50 sequenced microbial genomes. Results Multimodular E. coli K-12 proteins were identified from sequence similarities between their component modules and non-fused proteins in 50 genomes and from the literature. We found 109 multimodular proteins in E. coli containing either two or three modules. Most modules had standalone sequence relatives in other genomes. The separated modules together with all the single (un-fused proteins constitute the sum of all unimodular proteins of E. coli. Pairwise sequence relationships among all E. coli unimodular proteins generated 490 sequence similar, paralogous groups. Groups ranged in size from 92 to 2 members and had varying degrees of relatedness among their members. Some E. coli enzyme groups were compared to homologs in other bacterial genomes. Conclusion The deleterious effects of multimodular proteins on annotation and on the formation of groups of paralogs are emphasized. To improve annotation results, all multimodular proteins in an organism should be detected and when known each function should be connected with its location in the sequence of the protein. When transferring functions by sequence similarity, alignment locations must be noted, particularly when alignments cover only part of the sequences, in order to enable transfer of the correct function. Separating multimodular proteins into module units makes

  3. ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype

    Science.gov (United States)

    Hirabayashi, Shinsuke; Ohki, Kentaro; Nakabayashi, Kazuhiko; Ichikawa, Hitoshi; Momozawa, Yukihide; Okamura, Kohji; Yaguchi, Akinori; Terada, Kazuki; Saito, Yuya; Yoshimi, Ai; Ogata-Kawata, Hiroko; Sakamoto, Hiromi; Kato, Motohiro; Fujimura, Junya; Hino, Moeko; Kinoshita, Akitoshi; Kakuda, Harumi; Kurosawa, Hidemitsu; Kato, Keisuke; Kajiwara, Ryosuke; Moriwaki, Koichi; Morimoto, Tsuyoshi; Nakamura, Kozue; Noguchi, Yasushi; Osumi, Tomoo; Sakashita, Kazuo; Takita, Junko; Yuza, Yuki; Matsuda, Koich; Yoshida, Teruhiko; Matsumoto, Kenji; Hata, Kenichiro; Kubo, Michiaki; Matsubara, Yoichi; Fukushima, Takashi; Koh, Katsuyoshi; Manabe, Atsushi; Ohara, Akira; Kiyokawa, Nobutaka

    2017-01-01

    Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384. Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners. PMID:27634205

  4. Cloned s-Lap Gene Coding Area, Expression and Localizationof s-Lap/GFP Fusion Protein in Mammal Cells

    Institute of Scientific and Technical Information of China (English)

    SONG Yi-shu; SONG Zhi-yu; LI Hong-jun; Wu Yin; BAO Yong-li; TAN Da-peng; LI Yu-xin

    2005-01-01

    s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.

  5. Analysis of KIAA1549-BRAF fusion gene expression and IDH1/IDH2 mutations in low grade pediatric astrocytomas.

    Science.gov (United States)

    Cruz, Gabriela Rampazzo; Dias Oliveira, Indhira; Moraes, Laís; Del Giudice Paniago, Mário; de Seixas Alves, Maria Teresa; Capellano, Andrea Maria; Saba-Silva, Nasjla; Cavalheiro, Sérgio; Cerutti, Janete Maria; Toledo, Silvia Regina Caminada

    2014-04-01

    Low-grade astrocytomas comprise about 30 % of the central nervous system tumors in children. Several investigations have searched a correlation between the BRAF gene fusions alterations and mutations at IDH1 and IDH2 genes in low grade pediatric astrocytomas. This study identified the expression of KIAA1549-BRAF fusion gene and BRAF V600E mutation, mutations at exon 4 of the IDH1 and IDH2 genes in samples of pilocytic astrocytomas (PA) and grade-II astrocytomas (A-II) pediatric patients. The correlation between these alterations and the clinical profile of the patients was also evaluated. Eighty-two samples of low-grade astrocytomas (65 PA and 17 A-II) were analyzed by PCR and sequencing for each of the targets identified. We identified the KIAA1549-BRAF fusion transcript in 45 % of the samples. BRAF V600E and BRAFins598T mutations were detected in 7 and 1 % of the samples, respectively. Mutations in the R132/R172 residues of the IDH1/IDH2 genes were detected in only two samples, and the G105G polymorphism (rs11554137:C>T) was identified in ten patients. Additionally, we observed two mutations out of the usual hotspots at IDH1 and IDH2 genes. We observed a smaller frequency of mutations in IDHs genes than previously described, but since the prior studies were composed of adult or mixed (adults and children) samples, we believe that our results represent a relevant contribution to the growing knowledge in low grade childhood astrocytomas.

  6. Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice

    Directory of Open Access Journals (Sweden)

    Mitsuru Ando

    2014-01-01

    Full Text Available Sustained gene delivery of interferon (IFN γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

  7. Evaluation of reverse transcription-PCR protocols based on the fusion gene for diagnosis of bovine respiratory syncytial virus infections

    OpenAIRE

    Selim A.; Gaede W.

    2013-01-01

    Bovine respiratory syncytial virus (BRSV) is a pneumovirus in the family paramyxoviridae, is an important cause of acute respiratory disease in postweaning calves and feedlot cattle. The real-time reverse transcriptase PCR protocols were developed to detect BRSV infection in infected animals. The sensitivity of RT-PCR protocols based on fusion gene were evaluated using different Mastermixes such as Qiagen One Step RT-PCR (Qiagen) for conventional RT-PCR, Su...

  8. A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

    Institute of Scientific and Technical Information of China (English)

    Clara Bueno; Agustin F Femández; Mario F Fraga; Inmaculada Moreno-Gimeno; Deborah Burks; Maria del Carmen Plaza-Calonge; Juan C Rodríguez-Manzaneque; Pablo Menendez; Rosa Montes; Gustavo J Melen; Verónica Ramos-Mejia; Pedro J Real; Verónica Ayllón; Laura Sanchez; Gertrudis Ligero; Iván Gutierrez-Aranda

    2012-01-01

    The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in inants.Although it is well established that MLL-AF4 arises prenatally during human development,its effects on hematopoieric development in utero remain unexplored.We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs).Functional studies,clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic,functional and gene expression impact.MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs.Functionally,MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate.MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation,as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis.Furthermore,we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells.This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes,known to arise prenatally,regulate human embryonic hematopoietic specification.

  9. [Expression of SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia and its clinical significance].

    Science.gov (United States)

    Dai, Hai-Ping; Wang, Qian; Wu, Li-Li; Ping, Na-Na; Wu, Chun-Xiao; Xie, Jun-Dan; Pan, Jin-Lan; Xue, Yong-Quan; Wu, De-Pei; Chen, Su-Ning

    2012-10-01

    This study was aimed to investigate the occurrence and clinical significance of the SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia (T-ALL), analyse clinical and biological characteristics in this disease. RT-PCR was used to detect the expression of SET-NUP214 fusion gene in 58 T-ALL cases. Interphase FISH and Array-CGH were used to detect the deletion of 9q34. Direct sequencing was applied to detect mutations of PHF6 and NOTCH1. The results showed that 6 out of 58 T-ALL cases (10.3%) were detected to have the SET-NUP214 fusion gene by RT-PCR. Besides T-lineage antigens, expression of CD13 and(or) CD33 were detected in all the 6 cases. Deletions of 9q34 were detected in 4 out of the 6 patients by FISH. Array-CGH results of 3 SET-NUP214 positive T-ALL patients confirmed that this fusion gene was resulted from a cryptic deletion of 9q34.11q34.13. PHF6 and NOTCH1 gene mutations were found in 4 and 5 out of 6 SET-NUP214 positive T-ALL patients, respectively. It is concluded that SET-NUP214 fusion gene is often resulted from del(9)(q34). PHF6 and NOTCH1 mutations may be potential leukemogenic event in SET-NUP214 fusion gene.

  10. Clinicopathological differences between variants of the NAB2-STAT6 fusion gene in solitary fibrous tumors of the meninges and extra-central nervous system.

    Science.gov (United States)

    Nakada, Satoko; Minato, Hiroshi; Nojima, Takayuki

    2016-07-01

    Investigations on the NAB2-STAT6 fusion gene in solitary fibrous tumors (SFTs) and hemangiopericytomas (HPCs) have increased since its discovery in 2013. Although several SFTs reported without NAB2-STAT6 fusion gene analysis, we reviewed 546 SFTs/HPCs with NAB2-STAT6 fusion gene analysis in this study and investigated differences between the gene variants. In total, 452 cases tested positive for the NAB2-STAT6 fusion gene, with more than 40 variants being detected. The most frequent of these were NAB2 exon 6-STAT6 exon 16/17/18 and NAB2 exon 4-STAT6 exon 2/3, with the former occurring most frequently in SFTs in meninges, soft tissues, and head and neck; the latter predominated in SFTs in the pleura and lung. There was no difference between the histology of SFTs and fusion gene variants. A follow-up analysis of SFTs showed that 51 of 202 cases had a recurrence, with 18 of 53 meningeal SFTs having a local recurrence and/or metastasis within 0-19 years. In meninges and soft tissue, SFTs with the NAB2 exon 6-STAT6 exon 16/17/18 tended to recur more frequently than SFTs with the NAB2 exon 4-STAT6 exon 2/3. Clinicopathological data, including yearly follow-ups, are required for meningeal SFTs/HPCs to define the correlation of variants of NAB2-STAT6 fusion gene.

  11. Identification of mutations, gene expression changes and fusion transcripts by whole transcriptome RNAseq in docetaxel resistant prostate cancer cells.

    Science.gov (United States)

    Ma, Yuanjun; Miao, Yali; Peng, Zhuochun; Sandgren, Johanna; De Ståhl, Teresita Díaz; Huss, Mikael; Lennartsson, Lena; Liu, Yanling; Nistér, Monica; Nilsson, Sten; Li, Chunde

    2016-01-01

    Docetaxel has been the standard first-line therapy in metastatic castration resistant prostate cancer. The survival benefit is, however, limited by either primary or acquired resistance. In this study, Du145 prostate cancer cells were converted to docetaxel-resistant cells Du145-R and Du145-RB by in vitro culturing. Next generation RNAseq was employed to analyze these cell lines. Forty-two genes were identified to have acquired mutations after the resistance development, of which thirty-four were found to have mutations in published sequencing studies using prostate cancer samples from patients. Fourteen novel and 2 previously known fusion genes were inferred from the RNA-seq data, and 13 of these were validated by RT-PCR and/or re-sequencing. Four in-frame fusion transcripts could be transcribed into fusion proteins in stably transfected HEK293 cells, including MYH9-EIF3D and LDLR-RPL31P11, which were specific identified or up-regulated in the docetaxel resistant DU145 cells. A panel of 615 gene transcripts was identified to have significantly changed expression profile in the docetaxel resistant cells. These transcriptional changes have potential for further study as predictive biomarkers and as targets of docetaxel treatment.

  12. Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells

    Science.gov (United States)

    Looney, Timothy J.; Zhang, Li; Chen, Chih-Hsin; Lee, Jae Hyun; Chari, Sheila; Mao, Frank Fuxiang; Pelizzola, Mattia; Zhang, Lu; Lister, Ryan; Baker, Samuel W.; Fernandes, Croydon J.; Gaetz, Jedidiah; Foshay, Kara M.; Clift, Kayla L.; Zhang, Zhenyu; Li, Wei-Qiang; Vallender, Eric J.; Wagner, Ulrich; Qin, Jane Yuxia; Michelini, Katelyn J.; Bugarija, Branimir; Park, Donghyun; Aryee, Emmanuel; Stricker, Thomas; Zhou, Jie; White, Kevin P.; Ren, Bing; Schroth, Gary P.; Ecker, Joseph R.; Xiang, Andy Peng; Lahn, Bruce T.

    2014-01-01

    Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed “cis-silenced” (or “occluded”) genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease. PMID:24310002

  13. Visual adaptation in Lake Victoria cichlid fishes: depth-related variation of color and scotopic opsins in species from sand/mud bottoms.

    Science.gov (United States)

    Terai, Yohey; Miyagi, Ryutaro; Aibara, Mitsuto; Mizoiri, Shinji; Imai, Hiroo; Okitsu, Takashi; Wada, Akimori; Takahashi-Kariyazono, Shiho; Sato, Akie; Tichy, Herbert; Mrosso, Hillary D J; Mzighani, Semvua I; Okada, Norihiro

    2017-08-22

    For Lake Victoria cichlid species inhabiting rocky substrates with differing light regimes, it has been proposed that adaptation of the long-wavelength-sensitive (LWS) opsin gene triggered speciation by sensory drive through color signal divergence. The extensive and continuous sand/mud substrates are also species-rich, and a correlation between male nuptial coloration and the absorption of LWS pigments has been reported. However, the factors driving genetic and functional diversity of LWS pigments in sand/mud habitats are still unresolved. To address this issue, nucleotide sequences of eight opsin genes were compared in ten Lake Victoria cichlid species collected from sand/mud bottoms. Among eight opsins, the LWS and rod-opsin (RH1) alleles were diversified and one particular allele was dominant or fixed in each species. Natural selection has acted on and fixed LWS alleles in each species. The functions of LWS and RH1 alleles were measured by absorption of reconstituted A1- and A2-derived visual pigments. The absorption of pigments from RH1 alleles most common in deep water were largely shifted toward red, whereas those of LWS alleles were largely shifted toward blue in both A1 and A2 pigments. In both RH1 and LWS pigments, A2-derived pigments were closer to the dominant light in deep water, suggesting the possibility of the adaptation of A2-derived pigments to depth-dependent light regimes. The RH1 and LWS sequences may be diversified for adaptation of A2-derived pigments to different light environments in sand/mud substrates. Diversification of the LWS alleles may have originally taken place in riverine environments, with a new mutation occurring subsequently in Lake Victoria.

  14. 融合基因构建方法的建立%Establishment of fusion gene construction

    Institute of Scientific and Technical Information of China (English)

    张丽丽; 李振军; 李景富; 王傲雪

    2012-01-01

    Overlap extension PCR(OE-PCR) is frequently used for gene fragments fusion based on polymerase chain reaction. PCR, restriction enzyme digestion and ligation are primarily applied to the construction of recombinant vectors. These two techniques are in widespread application in the fields of gene engineering and molecular biology. LhCBF gene (Lhirsutum CRT-binding factor 1), EGFP gene and promoter RD29A sequence were taken as example and successfully obtained LhCBF1-EGFP andRD29A..EGFP fusion genes by using one step and two step OE-PCR, PCR-digestion-ligation method and optimized PCR protocol and system. This study provides significant theoretical basis for OE-PCR, PCR-digestion-ligation and fusion gene construction techniques.%重叠PCR技术是以聚合酶链式反应为基础,将多条DNA片段进行融合;PCR-醇切连接主要应用于基因片段连接及载体构建,这两种方法在基因工程与分子生物学领域已得到广泛应用.以多毛番茄抗冷转录因子LhCBF1基因、EGFP基因及RD29A启动子为例,分别采用一步法重叠PCR、二步法重叠PCR及PCR-酶切连接技术,优化PCR体系及反应条件,成功构建LhCBF1-EGFP及RD29A::EGFP融合基因.研究为重叠PCR及PCR-酶切连接技术的改进及构建融合基因方法的选择提供了有益参考.

  15. TBL1XR1/TP63: a novel recurrent gene fusion in B-cell non-Hodgkin lymphoma | Office of Cancer Genomics

    Science.gov (United States)

    Recently, the landscape of single base mutations in diffuse large B-cell lymphoma (DLBCL) was described. Here we report the discovery of a gene fusion between TBL1XR1 and TP63, the only recurrent somatic novel gene fusion identified in our analysis of transcriptome data from 96 DLBCL cases. Based on this cohort and a further 157 DLBCL cases analyzed by FISH, the incidence in de novo germinal center B cell-like (GCB) DLBCL is 5% (6 of 115).

  16. The role of FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, in Ewing sarcoma

    OpenAIRE

    Elzi, David J.; Song, Meihua; Houghton, Peter J.; Chen, Yidong; Shiio, Yuzuru

    2015-01-01

    Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly FLI-1. The EWS-FLI-1 fusion oncogene is widely believed to play a central role in Ewing sarcoma. The EWS-FLI-1 gene product regulates the expression of a number of genes important for cancer progression, can transform mouse cells such as NIH3T3 and C3H10T1/2, and is necessary for proliferation and tumorigenicity ...

  17. Identification of a novel SEPT9-ABL1 fusion gene in a patient with T-cell prolymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Rikio Suzuki

    2014-01-01

    Full Text Available T-cell prolymphocytic leukemia (T-PLL, a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.

  18. Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    De-Hua Wu; Li Liu; Long-Hua Chen

    2005-01-01

    AIM: To investigate the killing effect and radiosensitization of double suicide gene mediated by adenovirus on colorectal carcinoma cells.METHODS: Colorectal carcinoma cell line SW480 was transfected with adenovirus expression vector containing cytosine deaminase (CD) and thymidine kinase (Tk) fusion gene. The expression of CD-TK fusion gene was detected by reverse transcriptase-polymerase chain reaction. The toxic effect of ganciclovir (GCV) and 5-fiuorocytosine (5FC) on infected cells was determined by MTT assay. The radiosensitization of double suicide gene was evaluated by clonogenic assay.RESULTS: After prodrugs were used, the survival rate of colorectal carcinoma cells was markedly decreased. When GCV and 5-FC were used in combination, the cytotoxicity and bystandereffect were markedly superior to a single prodrug (x2 = 30.371, P<0.01). Both GCV and 5-FC could sensitize colorectal carcinoma cells to the toxic effect of radiation, and greater radiosensitization was achieved when both prodrug were used in combination. CONCLUSION: CD-TK double suicide gene can kill and radiosensitize colorectal carcinoma cells.

  19. The anti-tumour effect of a DNA vaccine carrying a fusion gene of human VEGFR2 and IL-12

    Directory of Open Access Journals (Sweden)

    Sha Wen

    2016-09-01

    Full Text Available Because of tumour dependence on angiogenesis, anti-angiogenic therapy has become the most attractive area of basic and clinical study in the field of cancer research. In order to create a synergistic effect on angiogenesis and immune regulation, we designed and constructed a new type of DNA vaccine that can express VEGFR2 (vascular endothelial growth factor receptor 2 and the prostate cancer antigen IL-12 (interleukin 12 in the same reading frame. The aim of this study was to investigate the anti-tumour activity of a eukaryotic expression plasmid carrying a fusion gene of human VEGFR2 and IL-12. According to the gene sequences in GenBank, we synthesized the human VEGFR2 and IL-12 genes. VEGFR2 and IL-12 were joined by a sequence encoding a Furin recognition site and a 2A cleavage site, and the resulting fusion gene was cloned into the eukaryotic expression vector pVAX1 to construct the expression plasmid pVAX1-VEGFR2-F2A-IL-12. The expression of VEGFR2 and IL-12 could be detected in 293T cells transfected with pVAX1-VEGFR2-F2A-IL-12 by enzyme-linked immunosorbent assay. Each of these proteins, and in particular co-expression of both proteins, can result in humoral and cellular immune responses in C57BL/6 mice. After injection into the tumour-bearing mouse model, the plasmid showed stronger inhibition of tumour growth than a plasmid expressing VEGFR2 alone. Our results demonstrate that a DNA vaccine carrying a fusion gene of human VEGFR2 and IL-12 could represent a promising approach for tumour immunotherapy.

  20. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  1. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  2. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Strebel, K.; Beck, E.; Strohmaier, K.; Schaller, H.

    1986-03-01

    Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambdaPL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in /sup 35/S-labeled extracts from foot-and-mouth disease virus-infected BHK cells. This allowed us to locate unequivocally all mature foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and mouth disease virus gene products not previously identified in vivo or in vitro.

  3. TMPRSS2-ERG Gene Fusion Causing ERG Overexpression Precedes Chromosome Copy Number Changes in Prostate Carcinomas, Paired HGPIN Lesions

    Directory of Open Access Journals (Sweden)

    Nuno Cerveira

    2006-10-01

    Full Text Available TMPRSS2-ETS gene fusions have been found recurrently in prostate carcinomas, but not in the presumed precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN. However, HGPIN lesions may share chromosomal changes with prostate cancer. To determine the relative order of genetic events in prostate carcinogenesis, we have analyzed 34 prostate carcinomas, 19 paired HGPIN lesions, 14 benign prostate hyperplasias, 11 morphologically normal prostatic tissues for TMPRSS2-ERG, TMPRSS2-ETV1 rearrangements, genomic imbalances. TMPRSS2 exon 1 was fused in-frame with ERG exon 4 in 17 of 34 (50% prostate carcinomas, in 4 of 19 (21% HGPIN lesions, but in none of controls. The findings were further validated by sequencing analysis, by the real-time polymerase chain reaction quantification of TMPRSS2-ERG fusion transcript, the ERG exons 5/6:exons 1/2 expression ratio. Chromosome copy number changes were detected by comparative genomic hybridization in 42% of clinically confined carcinomas, in none of the 16 HGPIN lesions analyzed. We demonstrate for the first time that the TMPRSS2-ERG fusion gene can be detected in a proportion of HGPIN lesions, that this molecular rearrangement is an early event that may precede chromosome-level alterations in prostate carcinogenesis.

  4. Analysis of NAB2-STAT6 Gene Fusion in 17 Cases of Meningeal Solitary Fibrous Tumor/Hemangiopericytoma: Review of the Literature.

    Science.gov (United States)

    Yuzawa, Sayaka; Nishihara, Hiroshi; Wang, Lei; Tsuda, Masumi; Kimura, Taichi; Tanino, Mishie; Tanaka, Shinya

    2016-08-01

    Solitary fibrous tumor/hemangiopericytoma (SFT/HPC) is a mesenchymal tumor that can affect virtually any region of the body. SFT/HPC of the thoracic cavity and soft tissue has been histologically considered a single biological entity termed SFT; in fact, NAB2-STAT6 gene fusion was recently identified in both diseases. In contrast, meningeal SFT and HPC still need to be investigated in detail with regard to gene fusion variants. The aim of this study was to verify the frequency of NAB2-STAT6 fusion and the relationship between fusion variants and clinicopathologic findings of SFT/HPC, especially meningeal SFT/HPC. We examined the NAB2-STAT6 fusion by reverse transcription polymerase chain reaction with 4 cases of meningeal SFT and 13 cases of meningeal HPC. NAB2-STAT6 fusion transcripts were identified in 12 of 17 cases, including NAB2ex6-STAT6ex17 (4/17, 24%), NAB2ex6-STAT6ex16 and NAB2ex4-STAT6ex2 (3/17, 18%, respectively), and NAB2ex5-STAT6ex16 (2/17, 12%). Three cases showed a pseudopapillary pattern, and 2 of them carried NAB2ex6-STAT6ex17. In addition, our meta-analysis revealed that the major fusion variant in meningeal SFT/HPC was NAB2ex6-STAT6ex16/17 (29/54, 54%), which was also common in soft tissue and intraperitoneum/retroperitoneum but rare in thoracic SFT. Fusion variant significantly correlated with age and histologic diagnosis in meningeal SFT/HPC but not with prognosis. Our results represented that meningeal SFT and HPC were in a single biological spectrum with NAB2-STAT6 gene fusion as was nonmeningeal SFT and further confirmed the organ-specific tumorigenic process and morphologic differences on the basis of fusion variants in meningeal SFT/HPC.

  5. Optogenetics: 10 years of microbial opsins in neuroscience.

    Science.gov (United States)

    Deisseroth, Karl

    2015-09-01

    Over the past 10 years, the development and convergence of microbial opsin engineering, modular genetic methods for cell-type targeting and optical strategies for guiding light through tissue have enabled versatile optical control of defined cells in living systems, defining modern optogenetics. Despite widespread recognition of the importance of spatiotemporally precise causal control over cellular signaling, for nearly the first half (2005-2009) of this 10-year period, as optogenetics was being created, there were difficulties in implementation, few publications and limited biological findings. In contrast, the ensuing years have witnessed a substantial acceleration in the application domain, with the publication of thousands of discoveries and insights into the function of nervous systems and beyond. This Historical Commentary reflects on the scientific landscape of this decade-long transition.

  6. The EWSR1/NR4A3 fusion protein of extraskeletal myxoid chondrosarcoma activates the PPARG nuclear receptor gene.

    Science.gov (United States)

    Filion, C; Motoi, T; Olshen, A B; Laé, M; Emnett, R J; Gutmann, D H; Perry, A; Ladanyi, M; Labelle, Y

    2009-01-01

    The NR4A3 nuclear receptor is implicated in the development of extraskeletal myxoid chondrosarcoma (EMC), primitive sarcoma unrelated to conventional chondrosarcomas, through a specific fusion with EWSR1 resulting in an aberrant fusion protein that is thought to disrupt the transcriptional regulation of specific target genes. We performed an expression microarray analysis of EMC tumours expressing the EWSR1/NR4A3 fusion protein, comparing their expression profiles to those of other sarcoma types. We thereby identified a set of genes significantly overexpressed in EMC relative to other sarcomas, including PPARG and NDRG2. Western blot or immunohistochemical analyses confirm that PPARG and NDRG2 are expressed in tumours positive for EWSR1/NR4A3. Bioinformatic analysis identified a DNA response element for EWSR1/NR4A3 in the PPARG promoter, and band-shift experiments and transient transfections indicate that EWSR1/NR4A3 can activate transcription through this element. Western blots further show that an isoform of the native NR4A3 receptor lacking the C-terminal domain is very highly expressed in tumours positive for EWSR1/NR4A3, and co-transfections of this isoform along with EWSR1/NR4A3 indicate that it may negatively regulate the activity of the fusion protein on the PPARG promoter. These results suggest that the overall expression of PPARG in EMC may be regulated in part by the balance between EWSR1/NR4A3 and NR4A3, and that PPARG may play a crucial role in the development of these tumours. The specific up-regulation of PPARG by EWSR1/NR4A3 may also have potential therapeutic implications.

  7. The photochemical determinants of color vision: revealing how opsins tune their chromophore's absorption wavelength.

    Science.gov (United States)

    Wang, Wenjing; Geiger, James H; Borhan, Babak

    2014-01-01

    The evolution of a variety of important chromophore-dependent biological processes, including microbial light sensing and mammalian color vision, relies on protein modifications that alter the spectral characteristics of a bound chromophore. Three different color opsins share the same chromophore, but have three distinct absorptions that together cover the entire visible spectrum, giving rise to trichromatic vision. The influence of opsins on the absorbance of the chromophore has been studied through methods such as model compounds, opsin mutagenesis, and computational modeling. The recent development of rhodopsin mimic that uses small soluble proteins to recapitulate the binding and wavelength tuning of the native opsins provides a new platform for studying protein-regulated spectral tuning. The ability to achieve far-red shifted absorption in the rhodopsin mimic system was attributed to a combination of the lack of a counteranion proximal to the iminium, and a uniformly neutral electrostatic environment surrounding the chromophore.

  8. Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model

    Institute of Scientific and Technical Information of China (English)

    Florian Graepler; Ulrike A Lauer; Reinhard Vonthein; Michael Gregor; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer; Marie-Luise Lemken; Wolfgang A Wybranietz; Ulrike Schmidt; Irina Smirnow; Christine D Groβ; Martin Spiegel; Andrea Schenk; Hansj(o)rg Graf

    2005-01-01

    AIM: To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model.METHODS: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene.RESULTS: In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH ceils stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin(MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application,whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>>YCD > > BCD > > > negative control) was defi ned as a result of a directin vivo comparison of all three suicide genes.CONCLUSION: Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model,thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

  9. Expression of Chlamydomonas actin-gfp fusion gene in to-bacco suspension cell and polymerization of the actin-gfp protein in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The fusion gene of actin (cDNA of Chlamydo- monas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed in E. coli and tobacco suspension cells BY2. The correct expression was observed in E. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was dis-tributed around nucleus and cell plate, while the fusion pro-tein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin in Chlamydomonas was similar with those of animals and higher plants.

  10. FGFR3–TACC3: A novel gene fusion in cervical cancer

    Directory of Open Access Journals (Sweden)

    Benedito A. Carneiro

    2015-08-01

    Full Text Available Cervical cancer epitomizes the success of cancer prevention through the human papillomavirus (HPV vaccine, but significant challenges remain in the treatment of advanced disease. We report the first three cases of cervical carcinoma harboring an FGFR3–TACC3 fusion, which serves as a novel therapeutic target. The fusion, identified by comprehensive genomic profiling, activates the FGFR pathway that has been implicated in HPV-driven carcinogenesis. One of the patients whose tumor contained the FGFR3–TACC3 fusion was treated with an investigational FGFR tyrosine kinase inhibitor. Concomitant molecular alterations involving the PI3K/AKT/mTOR and RAF/MEK pathways were also identified and suggest other treatment strategies that deserve investigation. This case series highlights the role of comprehensive genomic profiling in the identification of new therapeutic targets and in targeted therapy selection for patients with cervical cancer.

  11. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  12. A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene.

    Science.gov (United States)

    Konishi, Yuko; Karnan, Sivasundaram; Takahashi, Miyuki; Ota, Akinobu; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

    2012-09-01

    Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.

  13. Analysis of a MULE-cyanide hydratase gene fusion in Verticillium dahliae

    Science.gov (United States)

    The genome of the phytopathogenic fungus Verticillium dahliae encodes numerous Class II “cut-and-paste” transposable elements, including those of a small group of MULE transposons. We have previously identified a fusion event between a MULE transposon sequence and sequence encoding a cyanide hydrata...

  14. Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Guo; Zhi-Ming Hao; Jin-Yan Luo; Jun-Hong Wang

    2005-01-01

    AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles

  15. Fusion of the homeobox gene HLXB9 and the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13).

    Science.gov (United States)

    Beverloo, H B; Panagopoulos, I; Isaksson, M; van Wering, E; van Drunen, E; de Klein, A; Johansson, B; Slater, R

    2001-07-15

    Recently, we and others reported a recurrent t(7;12)(q36;p13) found in myeloid malignancies in children < or =18 months of age and associated with a poor prognosis. Fluorescence in situ hybridization studies mapped the 12p13 breakpoint to the first intron of ETV6 and narrowed down the region of 7q36 involved. By using the sequences made public recently by the Human Genome Project, two candidate genes in 7q36 were identified: the homeobox gene HLXB9 and c7orf3, a gene with unknown function. Reverse transcription-PCR of two cases with t(7;12), using primers for c7orf3 and ETV6, was negative. However, reverse transcription-PCR for HLXB9-ETV6 demonstrated alternative splicing; the two major bands corresponded to fusion of exon 1 of HLXB9 to exons 2 and 3, respectively, of ETV6. The reciprocal ETV6-HLXB9 transcript was not detected. It remains to be elucidated if the leukemic phenotype is attributable to the formation of the HLXB9-ETV6 fusion protein, which includes the helix-loop-helix and E26 transformation-specific DNA binding domains of ETV6 or to the disruption of the normal ETV6 protein.

  16. Biosensing of BCR/ABL fusion gene using an intensity-interrogation surface plasmon resonance imaging system

    Science.gov (United States)

    Wu, Jiangling; Huang, Yu; Bian, Xintong; Li, DanDan; Cheng, Quan; Ding, Shijia

    2016-10-01

    In this work, a custom-made intensity-interrogation surface plasmon resonance imaging (SPRi) system has been developed to directly detect a specific sequence of BCR/ABL fusion gene in chronic myelogenous leukemia (CML). The variation in the reflected light intensity detected from the sensor chip composed of gold islands array is proportional to the change of refractive index due to the selective hybridization of surface-bound DNA probes with target ssDNA. SPRi measurements were performed with different concentrations of synthetic target DNA sequence. The calibration curve of synthetic target sequence shows a good relationship between the concentration of synthetic target and the change of reflected light intensity. The detection limit of this SPRi measurement could approach 10.29 nM. By comparing SPRi images, the target ssDNA and non-complementary DNA sequence are able to be distinguished. This SPRi system has been applied for assay of BCR/ABL fusion gene extracted from real samples. This nucleic acid-based SPRi biosensor therefore offers an alternative high-effective, high-throughput label-free tool for DNA detection in biomedical research and molecular diagnosis.

  17. Imaging of dihydrofolate reductase fusion gene expression in xenografts of human liver metastases of colorectal cancer in living rats

    Energy Technology Data Exchange (ETDEWEB)

    Mayer-Kuckuk, Philipp; Bertino, Joseph R.; Banerjee, Debabrata [Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); The Cancer Institute of New Jersey, Robert Wood Johnson Medical School/UMDNJ, 195 Little Albany Street, NJ 08903, New Brunswick (United States); Doubrovin, Mikhail; Blasberg, Ronald; Tjuvajev, Juri Gelovani [Department of Neurooncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gusani, Niraj J.; Fong, Yuman [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gade, Terence; Koutcher, Jason A. [Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Balatoni, Julius; Finn, Ronald [Radiochemistry/Cyclotron Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Akhurst, Tim; Larson, Steven [Nuclear Medicine Service, Memorial Sloan-Kettering Cancer Center, New York, NY (United States)

    2003-09-01

    Radionuclide imaging has been demonstrated to be feasible to monitor transgene expression in vivo. We hypothesized that a potential application of this technique is to non-invasively detect in deep tissue, such as cancer cells metastatic to the liver, a specific molecular response following systemic drug treatment. Utilizing human colon adenocarcinoma cells derived from a patient's liver lesion we first developed a nude rat xenograft model for colorectal cancer metastatic to the liver. Expression of a dihydrofolate reductase-herpes simplex virus 1 thymidine kinase fusion (DHFR-HSV1 TK) transgene in the hepatic tumors was monitored in individual animals using the tracer [{sup 124}I]2'-fluoro-2'-deoxy-5-iodouracil-{beta}-d-arabinofuranoside (FIAU) and a small animal micro positron emission tomograph (microPET), while groups of rats were imaged using the tracer [{sup 131}I]FIAU and a clinical gamma camera. Growth of the human metastatic colorectal cancer cells in the rat liver was detected using magnetic resonance imaging and confirmed by surgical inspection. Single as well as multiple lesions of different sizes and sites were observed in the liver of the animals. Next, using a subset of rats bearing hepatic tumors, which were retrovirally bulk transduced to express the DHFR-HSV1 TK transgene, we imaged the fusion protein expression in the hepatic tumor of living rats using the tracer [{sup 124}I]FIAU and a microPET. The observed deep tissue signals were highly specific for the tumors expressing the DHFR-HSV1 TK fusion protein compared with parental untransduced tumors and other tissues as determined by gamma counting of tissue samples. A subsequent study used the tracer [{sup 131}I]FIAU and a gamma camera to monitor two groups of transduced hepatic tumor-bearing rats. Prior to imaging, one group was treated with trimetrexate to exploit DHFR-mediated upregulation of the fusion gene product. Imaging in the living animal as well as subsequent gamma

  18. LAMTOR1-PRKCD and NUMA1-SFMBT1 fusion genes identified by RNA sequencing in aneurysmal benign fibrous histiocytoma with t(3;11)(p21;q13).

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Bjerkehagen, Bodil; Lobmaier, Ingvild; Heim, Sverre

    2015-11-01

    RNA sequencing of an aneurysmal benign fibrous histiocytoma with the karyotype 46,XY,t(3;11)(p21;q13),del(6)(p23)[17]/46,XY[2] showed that the t(3;11) generated two fusion genes: LAMTOR1-PRKCD and NUMA1-SFMBT1. RT-PCR together with Sanger sequencing verified the presence of fusion transcripts from both fusion genes. In the LAMTOR1-PRKCD fusion, the part of the PRKCD gene coding for the catalytic domain of the serine/threonine kinase is under control of the LAMTOR1 promoter. In the NUMA1-SFMBT1 fusion, the part of the SFMBT1 gene coding for two of four malignant brain tumor domains and the sterile alpha motif domain is controlled by the NUMA1 promoter. The data support a neoplastic genesis of aneurysmal benign fibrous histiocytoma and indicate a pathogenetic role for LAMTOR1-PRKCD and NUMA1-SFMBT1. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. The hypothalamic photoreceptors regulating seasonal reproduction in birds: a prime role for VA opsin.

    Science.gov (United States)

    García-Fernández, José M; Cernuda-Cernuda, Rafael; Davies, Wayne I L; Rodgers, Jessica; Turton, Michael; Peirson, Stuart N; Follett, Brian K; Halford, Stephanie; Hughes, Steven; Hankins, Mark W; Foster, Russell G

    2015-04-01

    Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼ 492 nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

  20. Simple Eyes, Extraocular Photoreceptors and Opsins in the American Horseshoe Crab.

    Science.gov (United States)

    Battelle, Barbara-Anne

    2016-11-01

    The eyes and photoreceptors of the American horseshoe crab Limulus polyphemus have been studied since the 1930s, and this work has been critical for understanding basic mechanisms of vision. One of the attractions of Limulus as a preparation for studies of vision is that it has three different types of eyes-a pair of later compound, image-forming eyes and two types of simple eyes, a pair of median ocelli, and three pair of larval eyes. Each eye type is tractable for experimentation. Limulus also has extraocular photoreceptors in its segmental ganglia and tail. The current contribution focuses on photoreceptors in Limulus larval eyes and ocelli and its extraocular photoreceptors with the goal of summarizing what is currently known and not known about their physiology and function and the opsins they express. The Limulus genome encodes a surprisingly large number of opsins (18), and studies of their expression pattern have raised new questions about the role of opsin co-expression, the functions of peropsins expressed outside of eyes, and the physiological relevance of opsins with apparently very low expression levels. Studies of opsin expression in Limulus lead one to wonder whether photoreceptors yet to be discovered might be present throughout its central nervous system. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

  1. Gene identification and analysis: an application of neural network-based information fusion

    Energy Technology Data Exchange (ETDEWEB)

    Matis, S.; Xu, Y.; Shah, M.B.; Mural, R.J.; Einstein, J.R.; Uberbacher, E.C.

    1996-10-01

    Identifying genes within large regions of uncharacterized DNA is a difficult undertaking and is currently the focus of many research efforts. We describe a gene localization and modeling system called GRAIL. GRAIL is a multiple sensor-neural network based system. It localizes genes in anonymous DNA sequence by recognizing gene features related to protein-coding slice sites, and then combines the recognized features using a neural network system. Localized coding regions are then optimally parsed into a gene mode. RNA polymerase II promoters can also be predicted. Through years of extensive testing, GRAIL consistently localizes about 90 percent of coding portions of test genes with a false positive rate of about 10 percent. A number of genes for major genetic diseases have been located through the use of GRAIL, and over 1000 research laboratories worldwide use GRAIL on regular bases for localization of genes on their newly sequenced DNA.

  2. Opsin expression in Limulus eyes: a UV opsin is expressed in each eye type and co-expressed with a visible light-sensitive opsin in ventral larval eyes.

    Science.gov (United States)

    Battelle, Barbara-Anne; Kempler, Karen E; Harrison, Alexandra; Dugger, Donald R; Payne, Richard

    2014-09-01

    The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors.

  3. TALE-PvuII fusion proteins--novel tools for gene targeting.

    Directory of Open Access Journals (Sweden)

    Mert Yanik

    Full Text Available Zinc finger nucleases (ZFNs consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs, in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site, but not isolated TALE or PvuII recognition sites (unaddressed sites, even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  4. TALE-PvuII fusion proteins--novel tools for gene targeting.

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  5. Cysteine-rich secretory protein-3 (CRISP3 is strongly up-regulated in prostate carcinomas with the TMPRSS2-ERG fusion gene.

    Directory of Open Access Journals (Sweden)

    Franclim R Ribeiro

    Full Text Available A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16 and without (n = 8 TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (r(s = 0.65, p<0.001 and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene.

  6. Maps of cone opsin input to mouse V1 and higher visual areas.

    Science.gov (United States)

    Rhim, Issac; Coello-Reyes, Gabriela; Ko, Hee-Kyoung; Nauhaus, Ian

    2017-04-01

    Studies in the mouse retina have characterized the spatial distribution of an anisotropic ganglion cell and photoreceptor mosaic, which provides a solid foundation to study how the cortex pools from afferent parallel color channels. In particular, the mouse's retinal mosaic exhibits a gradient of wavelength sensitivity along its dorsoventral axis. Cones at the ventral extreme mainly express S opsin, which is sensitive to ultraviolet (UV) wavelengths. Then, moving toward the retina's dorsal extreme, there is a transition to M-opsin dominance. Here, we tested the hypothesis that the retina's opsin gradient is recapitulated in cortical visual areas as a functional map of wavelength sensitivity. We first identified visual areas in each mouse by mapping retinotopy with intrinsic signal imaging (ISI). Next, we measured ISI responses to stimuli along different directions of the S- and M-color plane to quantify the magnitude of S and M input to each location of the retinotopic maps in five visual cortical areas (V1, AL, LM, PM, and RL). The results illustrate a significant change in the S:M-opsin input ratio along the axis of vertical retinotopy that is consistent with the gradient along the dorsoventral axis of the retina. In particular, V1 populations encoding the upper visual field responded to S-opsin contrast with 6.1-fold greater amplitude than to M-opsin contrast. V1 neurons encoding lower fields responded with 4.6-fold greater amplitude to M- than S-opsin contrast. The maps in V1 and higher visual areas (HVAs) underscore the significance of a wavelength sensitivity gradient for guiding the mouse's behavior.NEW & NOTEWORTHY Two elements of this study are particularly novel. For one, it is the first to quantify cone inputs to mouse visual cortex; we have measured cone input in five visual areas. Next, it is the first study to identify a feature map in the mouse visual cortex that is based on well-characterized anisotropy of cones in the retina; we have identified

  7. CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma

    DEFF Research Database (Denmark)

    Engelholm, Lars H; Riaz, Anjum; Serra, Denise

    2017-01-01

    ) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8...... to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. METHODS: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein......, as observed in human FL-HCC. CONCLUSIONS: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics...

  8. Fusion protein gene nucleotide sequence similarities, shared antigenic sites and phylogenetic analysis suggest that phocid distemper virus 2 and canine distemper virus belong to the same virus entity.

    NARCIS (Netherlands)

    I.K.G. Visser (Ilona); R.W.J. van der Heijden (Roger); M.W.G. van de Bildt (Marco); M.J.H. Kenter (Marcel); C. Örvell; A.D.M.E. Osterhaus (Albert)

    1993-01-01

    textabstractNucleotide sequencing of the fusion protein (F) gene of phocid distemper virus-2 (PDV-2), recently isolated from Baikal seals (Phoca sibirica), revealed an open reading frame (nucleotides 84 to 2075) with two potential in-frame ATG translation initiation codons. We suggest that the secon

  9. pBaSysBioll : an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

    NARCIS (Netherlands)

    Botella, Eric; Fogg, Mark; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette; Denham, Emma. L.; Le Chat, Ludovic; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; van Dijl, Jan Maarten; Aymerich, Stephane; Wilkinson, Anthony J.; Devine, Kevin M.

    Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type

  10. pBaSysBioll : an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

    NARCIS (Netherlands)

    Botella, Eric; Fogg, Mark; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette; Denham, Emma. L.; Le Chat, Ludovic; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; van Dijl, Jan Maarten; Aymerich, Stephane; Wilkinson, Anthony J.; Devine, Kevin M.

    2010-01-01

    Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type

  11. Mammary Analogue Secretory Carcinoma of Salivary Glands: Molecular Analysis of 25 ETV6 Gene Rearranged Tumors With Lack of Detection of Classical ETV6-NTRK3 Fusion Transcript by Standard RT-PCR: Report of 4 Cases Harboring ETV6-X Gene Fusion.

    Science.gov (United States)

    Skálová, Alena; Vanecek, Tomas; Simpson, Roderick H W; Laco, Jan; Majewska, Hanna; Baneckova, Martina; Steiner, Petr; Michal, Michal

    2016-01-01

    ETV6 gene abnormalities are well described in tumor pathology. Many fusion partners of ETV6 have been reported in a variety of epithelial and hematological malignancies. In salivary gland tumor pathology, however, the ETV6-NTRK3 translocation is specific for mammary analogue secretory carcinoma (MASC), and has not been documented in any other salivary tumor type. The present study comprised a clinical and molecular analysis of 25 cases morphologically and immunohistochemically typical of MASC. They all also displayed the ETV6 rearrangement as visualized by fluorescent in situ hybridization but lacked the classical ETV6-NTRK3 fusion transcript by standard reverse-transcriptase-polymerase chain reaction. In 4 cases, the classical fusion transcript was found by more sensitive, nested reverse-transcription-polymerase chain reaction. Five other cases harbored atypical fusion transcripts as detected by both standard and nested reverse-transcription-polymerase chain reaction. In addition, fluorescent in situ hybridization with an NTRK3 break-apart probe was also performed; rearrangement of NTRK3 gene was detected in 16 of 25 cases. In 3 other cases, the tissue was not analyzable, and in 2 further cases analysis could not be performed because of a lack of appropriate tissue material. Finally, in the 4 remaining cases whose profile was NTRK3 split-negative and ETV6 split-positive, unknown (non-NTRK) genes appeared to fuse with ETV6 (ETV6-X fusion). In looking for possible fusion partners, analysis of rearrangement of other kinase genes known to fuse with ETV6 was also performed, but without positive results. Although numbers were small, correlating the clinico-pathologic features of the 4 ETV6-X fusion tumors and 5 MASC cases with atypical fusion transcripts raises the possibility of that they may behave more aggressively.

  12. In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the in-vitro activation of cytotoxic T lymphocytes (CTLs) by fusion of mouse hepatocellular carcinoma (HCC) cells and lymphotactin gene-modified dendritic cells (DCs).METHODS: Lymphotactin gene modified DCs (DCLptn) were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α). DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTLs by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay.RESULTS: Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-γ and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn+H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn + H22, DC/H22 groups.CONCLUSION: Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

  13. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    Directory of Open Access Journals (Sweden)

    Chiemi Noguchi

    Full Text Available Amplification of the dihydrofolate reductase gene (Dhfr by methotrexate (Mtx exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR and a matrix attachment region (MAR, which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  14. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    Science.gov (United States)

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  15. Genome-wide repression of eRNA and target gene loci by the ETV6-RUNX1 fusion in acute leukemia.

    Science.gov (United States)

    Teppo, Susanna; Laukkanen, Saara; Liuksiala, Thomas; Nordlund, Jessica; Oittinen, Mikko; Teittinen, Kaisa; Grönroos, Toni; St-Onge, Pascal; Sinnett, Daniel; Syvänen, Ann-Christine; Nykter, Matti; Viiri, Keijo; Heinäniemi, Merja; Lohi, Olli

    2016-11-01

    Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19(+)/CD20(+)-lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis. © 2016 Teppo et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Obesity and Prostate Cancer Risk According to Tumor TMPRSS2:ERG Gene Fusion Status

    National Research Council Canada - National Science Library

    Egbers, Lieke; Luedeke, Manuel; Rinckleb, Antje; Kolb, Suzanne; Wright, Jonathan L; Maier, Christiane; Neuhouser, Marian L; Stanford, Janet L

    2015-01-01

    ...) with the erythroblast transformation-specific (ETS)-related gene (ERG), is found in approximately 50% of prostate cancers and may characterize distinct molecular subtypes of prostate cancer with different etiologies...

  17. Temporal Resolution of ChR2 and Chronos in an Optogenetic-based Auditory Brainstem Implant Model: Implications for the Development and Application of Auditory Opsins

    Science.gov (United States)

    Hight, A. E.; Kozin, Elliott D.; Darrow, Keith; Lehmann, Ashton; Boyden, Edward; Brown, M. Christian; Lee, Daniel J.

    2015-01-01

    The contemporary auditory brainstem implant (ABI) performance is limited by reliance on electrical stimulation with its accompanying channel cross talk and current spread to non-auditory neurons. A new generation ABI based on optogenetic-technology may ameliorate limitations fundamental to electrical neurostimulation. The most widely studied opsin is channelrhodopsin-2 (ChR2); however, its relatively slow kinetic properties may prevent the encoding of auditory information at high stimulation rates. In the present study, we compare the temporal resolution of light-evoked responses of a recently developed fast opsin, Chronos, to ChR2 in a murine ABI model. Viral mediated gene transfer via a posterolateral craniotomy was used to express Chronos or ChR2 in the mouse nucleus (CN). Following a four to six week incubation period, blue light (473 nm) was delivered via an optical fiber placed directly on the surface of the infected CN, and neural activity was recorded in the contralateral inferior colliculus (IC). Both ChR2 and Chronos evoked sustained responses to all stimuli, even at high driven rates. In addition, optical stimulation evoked excitatory responses throughout the tonotopic axis of the IC. Synchrony of the light-evoked response to stimulus rates of 14–448 pulses/s was higher in Chronos compared to ChR2 mice (p<0.05 at 56, 168, and 224 pulses/s). Our results demonstrate that Chronos has the ability to drive the auditory system at higher stimulation rates than ChR2 and may be a more ideal opsin for manipulation of auditory pathways in future optogenetic-based neuroprostheses. PMID:25598479

  18. NAB2-STAT6 gene fusion and STAT6 immunoexpression in extrathoracic solitary fibrous tumors: the association between fusion variants and locations.

    Science.gov (United States)

    Chuang, I-Chieh; Liao, Kuan-Cho; Huang, Hsuan-Ying; Kao, Yu-Chien; Li, Chien-Feng; Huang, Shih-Chiang; Tsai, Jen-Wei; Chen, Ko-Chin; Lan, Jui; Lin, Po-Chun

    2016-05-01

    Solitary fibrous tumor (SFT) is a rare mesenchymal neoplasm harboring NAB2-STAT6 fusion, which drives STAT6 nuclear relocation. For extrathoracic SFTs, the clinical relevance of this molecular hallmark remains obscure. We assessed STAT6 immunoexpression for 61 extrathoracic SFTs exclusive of the meninges and head and neck, and 25 had analyzable RNAs to distinguish fusion variants by RT-PCR. The immunohistochemical and molecular findings were correlated with clincopathological features and disease-free survival (DFS). Twenty-eight males and 33 females had SFTs in the body cavities (n = 31), extremities (n = 17), and trunk (n = 13), categorized into 53 non-malignant and 8 malignant tumors. The vast majority (n = 57, 93%) exhibited distinctive STAT6 nuclear expression, including malignant ones. The common fusion variants were NAB2ex6-STAT6ex16/17 in 13 SFTs and NAB2ex4-STAT6ex2 in 8, while miscellaneous variants were detected only in 4 SFTs in the limbs and trunk but not in any body cavity-based cases (P = 0.026). The worse DFS was univariately associated with malignant histology (P = 0.04) but unrelated to tumor size, location, or fusion variant. Conclusively, extrathoracic SFTs mostly harbor NAB2ex6-STAT6ex16/17, followed by NAB2ex4-STAT6ex2. Miscellaneous variants are significantly rare in SFTs within the body cavities. The clinical aggressiveness of extrathoraic SFTs is associated with malignant histology but unrelated to the NAB2-STAT6 fusion variants.

  19. RNA-seq analysis of prostate cancer in the Chinese population identifies recurrent gene fusions,cancer-associated long noncoding RNAs and aberrant alternative splicings

    Institute of Scientific and Technical Information of China (English)

    Shancheng Ren; Weidong Xu; Chao Chen; Fubo Wang; Xinwu Guo; Ji Lu; Jun Yang; Min Wei; Zhijian Tian; Yinghui Guan; Liang Tang; Zhiyu Peng; Chuanliang Xu; Linhui Wang; Xu Gao; Wei Tian; Jian Wang; Huanming Yang; Jun Wang; Yinghao Sun; Jian-Hua Mao; Yongwei Yu; Changjun Yin; Xin Gao; Zilian Cui; Jibin Zhang; Kang Yi

    2012-01-01

    There are remarkable disparities among patients of different races with prostate cancer; however,the mechanism underlying this difference remains unclear.Here,we present a comprehensive landscape of the transcriptome profiles of 14 primary prostate cancers and their paired normal counterparts from the Chinese population using RNA-seq,revealing tremendous diversity across prostate cancer transcriptomes with respect to gene fusions,long noneoding RNAs (long ncRNA),alternative splicing and somatic mutations.Three of the 14 tumors (21.4%) harbored a TMPRSS2-ERG fusion,and the low prevalence of this fusion in Chinese patients was further confirmed in an additional tumor set (10/54=18.5%).Notably,two novel gene fusions,CTAGE5-KHDRBS3 (20/54=37%) and USP9Y-TTTY15(19/54=35.2%),occurred frequently in our patient cohort.Further systematic transcriptional profiling identified numerous long ncRNAs that were differentially expressed in the tumors.An analysis of the correlation between expression of long ncRNA and genes suggested that long ncRNAs may have functions beyond transcriptional regulation.This study yielded new insights into the pathogenesis of prostate cancer in the Chinese population.

  20. Secretory breast carcinomas with ETV6-NTRK3 fusion gene belong to the basal-like carcinoma spectrum.

    Science.gov (United States)

    Laé, Marick; Fréneaux, Paul; Sastre-Garau, Xavier; Chouchane, Olfa; Sigal-Zafrani, Brigitte; Vincent-Salomon, Anne

    2009-02-01

    Secretory breast carcinomas (carcinoma. A series of six secretory breast carcinomas were identified in our files. The ETV6 rearrangement was confirmed in all cases by fluorescence in situ hybridization. Immunophenotype was assessed with anti-ER, PR, ERBB2, KIT, EGFR, E-cadherin, vimentin, PS100, smooth muscle actin, basal (CK5/6 and 14), luminal cytokeratins (CK8/18) and p63 antibodies. In situ and invasive components shared the same immunoprofile and were ER, PR, ERBB2 negative with expression of basal cytokeratins. ETV6 gene alterations were present in both in situ and invasive components, highlighting their genetic similarities. The immunoprofile data (triple-negative with expression of basal markers) showed that secretory breast carcinomas with ETV6-NTRK3 fusion gene belong to the phenotypic basal-like spectrum of breast carcinomas. These results support the hypothesis that secretory breast carcinomas have immunohistochemical and genetic features that distinguish them from other basal-like tumors of the breast.

  1. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion.

    Science.gov (United States)

    Nikolaidis, Nikolas; Doran, Nicole; Cosgrove, Daniel J

    2014-02-01

    Horizontal gene transfer (HGT) has been described as a common mechanism of transferring genetic material between prokaryotes, whereas genetic transfers from eukaryotes to prokaryotes have been rarely documented. Here we report a rare case of HGT in which plant expansin genes that code for plant cell-wall loosening proteins were transferred from plants to bacteria, fungi, and amoebozoa. In several cases, the species in which the expansin gene was found is either in intimate association with plants or is a known plant pathogen. Our analyses suggest that at least two independent genetic transfers occurred from plants to bacteria and fungi. These events were followed by multiple HGT events within bacteria and fungi. We have also observed that in bacteria expansin genes have been independently fused to DNA fragments that code for an endoglucanase domain or for a carbohydrate binding module, pointing to functional convergence at the molecular level. Furthermore, the functional similarities between microbial expansins and their plant xenologs suggest that these proteins mediate microbial-plant interactions by altering the plant cell wall and therefore may provide adaptive advantages to these species. The evolution of these nonplant expansins represents a unique case in which bacteria and fungi have found innovative and adaptive ways to interact with and infect plants by acquiring genes from their host. This evolutionary paradigm suggests that despite their low frequency such HGT events may have significantly contributed to the evolution of prokaryotic and eukaryotic species.

  2. Identification of SETD2-NF1 fusion gene in a pediatric spindle cell tumor with the chromosomal translocation t(3;17)(p21;q12).

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Lobmaier, Ingvild; Bjerkehagen, Bodil; Heim, Sverre

    2017-06-01

    Spindle cell tumors are clinically heterogeneous but morphologically similar neoplasms. The term refers to the tumor cells' long and slender microscopic appearance. Distinct subgroups of spindle cell tumors are characterized by chromosomal translocations and also fusion genes. Other spindle cell tumors exist that have not yet been found to have characteristic, let alone pathognomonic, genetic or pathogenetic features. Continuous examination of spindle cell tumors is likely to reveal other subgroups that may, in the future, be seen to correspond to meaningful clinical differences and may even be therapeutically decisive. We analyzed genetically a pediatric spindle cell tumor. Karyotyping showed the tumor cells to carry a t(3;17)(p21;q12) chromosomal translocation whereas RNA sequencing identified a SETD2-NF1 fusion gene caused by the translocation. RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript. Interphase FISH analysis confirmed the existence of the chimeric gene and showed that there was no reciprocal fusion. The fusion transcript codes for a protein in which the last 114 amino acids of SETD2, i.e., the entire Set2 Rpb1 interacting (SRI) domain of SETD2, are replaced by 30 amino acids encoded by the NF1 sequence. The result would be similar to that seen with truncating SETD2 mutations in leukemias. Absence of the SRI domain would result in inability to recruit SETD2 to its target gene locus through binding to the phosphor-C-terminal repeat domain of elongating RNA polymerase II and may affect H3K36 methylation. Alternatively, loss of one of two functional SETD2 alleles might be the crucial tumorigenic factor.

  3. Bioinformatic analysis of patient-derived ASPS gene expressions and ASPL-TFE3 fusion transcript levels identify potential therapeutic targets.

    Directory of Open Access Journals (Sweden)

    David G Covell

    Full Text Available Gene expression data, collected from ASPS tumors of seven different patients and from one immortalized ASPS cell line (ASPS-1, was analyzed jointly with patient ASPL-TFE3 (t(X;17(p11;q25 fusion transcript data to identify disease-specific pathways and their component genes. Data analysis of the pooled patient and ASPS-1 gene expression data, using conventional clustering methods, revealed a relatively small set of pathways and genes characterizing the biology of ASPS. These results could be largely recapitulated using only the gene expression data collected from patient tumor samples. The concordance between expression measures derived from ASPS-1 and both pooled and individual patient tumor data provided a rationale for extending the analysis to include patient ASPL-TFE3 fusion transcript data. A novel linear model was exploited to link gene expressions to fusion transcript data and used to identify a small set of ASPS-specific pathways and their gene expression. Cellular pathways that appear aberrantly regulated in response to the t(X;17(p11;q25 translocation include the cell cycle and cell adhesion. The identification of pathways and gene subsets characteristic of ASPS support current therapeutic strategies that target the FLT1 and MET, while also proposing additional targeting of genes found in pathways involved in the cell cycle (CHK1, cell adhesion (ARHGD1A, cell division (CDC6, control of meiosis (RAD51L3 and mitosis (BIRC5, and chemokine-related protein tyrosine kinase activity (CCL4.

  4. Enforced effect of tk-MCP-1 fusion gene in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Hong Shuhui

    2012-09-01

    Full Text Available Abstract Objective The efficiency of HSV-tk/GCV system is not high because of insufficient gene transfer and incompletely initiative of host antineoplastic potency. The present study was designed to assess the antitumor efficacy of tk-MCP-1 on ovarian cancer in vitro and vivo. Methods A novel bicistronic expression system can help to improve the expression level of a gene in a stable manner. pLXSN/tk-MCP-1 co-expressing tk and MCP-1 genes was constructed using a pLXSN retroviral vector and an internal ribosome entry site sequence by restriction enzyme. Western blot was performed to determine tk and MCP-1 expression in the infected SKOV3. The GCV-sensitively tumoricidal activities of SKOV3/tk-MCP-1 with or without monocytes were compared to those of SKOV3 expressing HSV-tk or MCP-1. We investigated the growth of subcutaneous tumors in SCID mice immuno-reconstituted, and evaluated the antitumor effect of MCP-1 in conjunction with suicide gene. Results The significant GCV-sensitively tumoricidal activity of pLXSN/tk-MCP-1 was observed when compared with those of pLXSN/tk, pLXSN/MCP-1 and pLXSN/neo, especially when monocytes were added. The growth of subcutaneous tumors in SCID mice immuno-reconstituted was markedly suppressed by co-delivery of HSV-tk and MCP-1 genes, and the enhanced antitumor effect was associated with the recruitment of monocytes. Conclusion These results demonstrated pLXSN/tk-MCP-1 presented an enhanced antitumor effects on ovarian cancer by orchestration of immune responses.

  5. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion.

    Science.gov (United States)

    Paquette, Stéphane G; Banner, David; Chi, Le Thi Bao; Leόn, Alberto J; Xu, Luoling; Ran, Longsi; Huang, Stephen S H; Farooqui, Amber; Kelvin, David J; Kelvin, Alyson A

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus-epithelial cell interaction.

  6. Fusion of the TBL1XR1 and HMGA1 genes in splenic hemangioma with t(3;6)(q26;p21).

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Bjerkehagen, Bodil; Lobmaier, Ingvild; Heim, Sverre

    2016-03-01

    RNA-sequencing of a splenic hemangioma with the karyotype 45~47,XX,t(3;6)(q26;p21) showed that this translocation generated a chimeric TBL1XR1-HMGA1 gene. This is the first time that this tumor has been subjected to genetic analysis, but the finding of an acquired clonal chromosome abnormality in cells cultured from the lesion and the presence of the TBL1XR1-HMGA1 fusion in them strongly favor the conclusion that splenic hemangiomas are of a neoplastic nature. Genomic PCR confirmed the presence of the TBL1XR1-HMGA1 fusion gene, and RT-PCR together with Sanger sequencing verified the presence of the fusion transcripts. The molecular consequences of the t(3;6) would be substantial. The cells carrying the translocation would retain only one functional copy of the wild-type TBL1XR1 gene while the other, rearranged allele could produce a putative truncated form of TBL1XR1 protein containing the LiSH and F-box-like domains. In the TBL1XR1-HMGA1 fusion transcript, furthermore, untranslated exons of HMGA1 are replaced by the first 5 exons of the TBL1XR1 gene. The result is that the entire coding region of HMGA1 comes under the control of the TBL1XR1 promoter, bringing about dysregulation of HMGA1. This is reminiscent of similar pathogenetic mechanisms involving high mobility genes in benign connective tissue tumors such as lipomas and leiomyomas.

  7. Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina.

    Directory of Open Access Journals (Sweden)

    Paula S Nieto

    Full Text Available The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+ mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR, encephalopsin/panopsin (Opn3, neuropsin (Opn5 and cone opsin (Opn1mw but not melanopsin (Opn4 or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL. Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+ levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+ mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

  8. ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer

    Science.gov (United States)

    2011-08-01

    facilitates DNA end processing and rejoin- ing in a multistep procedure that requires the XRCC4/ DNA Ligase IV complex. In fact, XRCC4 and DNA Ligase IV are...Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr. Biol. 8, 1395–1398. Bennett, E.J., and Harper, J.W. (2008...1998). Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177. Gallagher, D.J., Gaudet, M.M

  9. Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans

    OpenAIRE

    Ahmed, Zubair M.; Masmoudi, Saber; Kalay, Ersan; Belyantseva, Inna A.; Mosrati, Mohamed Ali; Collin, Rob W. J.; Riazuddin, Saima; Hmani-Aifa, Mounira; Venselaar, Hanka; Kawar, Mayya N; Abdelaziz, Tlili; van der Zwaag, Bert; Khan, Shahid Y.; Ayadi, Leila; Riazuddin, S. Amer

    2008-01-01

    Many proteins necessary for sound transduction have been discovered through positional cloning of genes that cause deafness 1–3 . In this study, we report that mutations of LRTOMT are associated with profound non-syndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, that are detected by Western blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, nove...

  10. Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes.

    Science.gov (United States)

    Battelle, Barbara-Anne; Kempler, Karen E; Saraf, Spencer R; Marten, Catherine E; Dugger, Donald R; Speiser, Daniel I; Oakley, Todd H

    2015-02-01

    The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.

  11. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  12. Renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion: imaging findings in 21 patients

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiao; Zhou, Hao; Duan, Na; Liu, Yongkang; Wang, Zhongqiu [Affiliated Hospital of Nanjing University of Chinese Medicine, Department of Radiology, Nanjing (China); Zhu, Qingqiang [Medical School of Yangzhou University, Department of Medical Imaging, Subei People' s Hospital, Yangzhou (China); Li, Baoxin [Gulou Hospital, Department of Radiology, Nanjing (China); Cui, Wenjing [Affiliated Hospital of Nanjing University of Chinese Medicine, Department of Radiology, Nanjing (China); Nanjing University Medical School, Department of Radiology, Jinling Hospital, Nanjing (China); Kundra, Vikas [The University of Texas, M.D. Anderson Cancer Center, Department of Radiology, Houston, TX (United States)

    2017-02-15

    To characterize imaging features of renal cell carcinoma (RCC) associated with Xp11.2 translocation/TFE gene fusion. Twenty-one patients with Xp11.2/TFE RCC were retrospectively evaluated. Tumour location, size, density, cystic or solid appearance, calcification, capsule sign, enhancement pattern and metastases were assessed. Fourteen women and seven men were identified with 12 being 25 years old or younger. Tumours were solitary and cystic-solid (76.2 %) masses with a capsule (76.2 %); 90.5 % were located in the medulla. Calcifications and lymph node metastases were each observed in 24 %. On unenhanced CT, tumour attenuation was greater than in normal renal parenchyma (85.7 %). Tumour enhancement was less than in normal renal cortex on all enhanced phases, greater than in normal renal medulla on cortical and medullary phases, but less than in normal renal medulla on delayed phase. On MR, the tumours were isointense on T1WI, heterogeneously hypointense on T2WI and slightly hyperintense on diffusion-weighted imaging. Xp11.2/TFE RCC usually occurs in young women. It is a cystic-solid, hyperdense mass with a capsule. It arises from the renal medulla with enhancement less than in the cortex but greater than in the medulla in all phases except the delayed phase, when it is lower than in the medulla. (orig.)

  13. Mutations in the Drosophila pushover gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion

    Energy Technology Data Exchange (ETDEWEB)

    Richards, S.; Hillman, T.; Stern, M. [Rice Univ., Houston, TX (United States)

    1996-04-01

    We describe the identification of a gene called pushover (push), which affects both behavior and synaptic transmission at the neuromuscular junction. Adults carrying either of two mutations in push exhibit sluggishness, uncoordination, a defective escape response, and male sterility. Larvae defective in push exhibit increased release of transmitter at the neuromuscular junction. In particular, the frequency of spontaneous transmitter release and the amount of transmitter release evoked by nerve stimulation are each increased two- to threefold in push mutants at the lowest external [(Ca{sup 2+})] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following quinidine application, push mutants, but not wild-type, display repetitive firing of the motor axon, leading to repetitive muscle postsynaptic potentials. The push gene thus might affect both neuronal excitability and the transmitter release process. Complementation tests and recombinational mapping suggest that the push mutations are allelic to a previously identified P-element-induced mutation, which also causes behavorial abnormalities and male sterility. 43 refs., 5 figs., 1 tab.

  14. The molecular pathology examination analysis of BRAF gene mutation and RET fusion gene in thyroid cancer%甲状腺癌中BARF基因突变和RET融合基因的分子病理检测分析

    Institute of Scientific and Technical Information of China (English)

    吴永芳; 许春伟; 宋业颖; 班怡; 张博; 李晓兵

    2015-01-01

    Objective: To investigate the mutations of BRAF gene and RET fusion gene in thyroid cancer. Methods:Taqman-ARMS was used to test in 106 cases of thyroid cancer with BRAF gene mutation and RET fusion gene. Results:hTe total mutation rate of BRAF gene was 66.98%(71/106) in thyroid cancer, and they were all V600E mutation. BRAF gene mutation with age (0.05). Conclusion:BRAF gene mutation has a higher mutation rate in thyroid cancer, especially in young, capsule invasion or metastasis, andⅠorⅡstage. Fusion partner CCDC6-or NCOA4-is common RET fusion gene in thyroid cancer.%目的:探讨甲状腺癌中BRAF基因突变和RET融合基因各亚型情况。方法:应用Taqman-ARMS方法检测106例甲状腺癌中BRAF基因突变和RET融合基因情况。结果:甲状腺癌中BRAF基因总突变率为66.98%(71/106),均为V600E突变,BRAF基因突变在年龄(0.05)。结论:甲状腺癌患者中BRAF基因存在较高的突变率,低龄、被膜未浸润或转移及Ⅰ或Ⅱ期甲状腺癌多见,RET融合基因中融合伙伴CCDC6-或NCOA4-较为常见。

  15. Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling

    Directory of Open Access Journals (Sweden)

    Steven R. Ahrendt

    2017-04-01

    Full Text Available Background Opsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins are a subfamily of G-protein coupled receptors (GPCRs, a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores. Methods We used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if the sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus. Results Our results indicate that the Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins. Discussion This represents the first test of structure/function relationship of a type 2 rhodopsin

  16. De Novo Adult Transcriptomes of Two European Brittle Stars: Spotlight on Opsin-Based Photoreception.

    Directory of Open Access Journals (Sweden)

    Jérôme Delroisse

    Full Text Available Next generation sequencing (NGS technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeqTM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric and mammal (ciliary classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic

  17. The Neurospora Transcription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion

    Directory of Open Access Journals (Sweden)

    Rigzin Dekhang

    2017-01-01

    Full Text Available Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa. A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in Δadv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism.

  18. Genes encoding Cher-TPR fusion proteins are predominantly found in gene clusters encoding chemosensory pathways with alternative cellular functions.

    Science.gov (United States)

    Muñoz-Martínez, Francisco; García-Fontana, Cristina; Rico-Jiménez, Miriam; Alfonso, Carlos; Krell, Tino

    2012-01-01

    Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be

  19. Frequency of the ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and MLL-AFF1 fusion genes in Guatemalan pediatric acute lymphoblastic leukemia patients and their ethnic associations.

    Science.gov (United States)

    Carranza, Claudia; Granados, Lilian; Morales, Oneida; Jo, Wendy; Villagran, Swuanny; Tinti, Damaris; Villegas, Mauricio; Antillón, Federico; Torselli, Silvana; Silva, Gabriel

    2013-06-01

    Fusion genes involved in acute lymphoblastic leukemia (ALL) occur mostly due to genetic and environmental factors, and only a limited number of studies have reported any ethnic influence. This study assesses whether an ethnic influence has an effect on the frequency of any of the four fusion genes: BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and MLL-AFF1 found in ALL. To study this ethnic influence, mononuclear cells were obtained from bone marrow samples from 143 patients with ALL. We performed RNA extraction and reverse transcription, then assessed the quality of the cDNA by amplifying the ABL1 control gene, and finally evaluated the presence of the four transcripts by multiplex polymerase chain reaction. We found 10 patients who had the BCR-ABL1 fusion gene (7%); 3 patients (2%) were TCF3-PBX1 positive; and 6 patients (4.5%) were ETV6-RUNX1 positive. The incidence of this last fusion gene is quite low when compared to the values reported in most countries. The low incidence of the ETV6-RUNX1 fusion gene found in Guatemala matches the incidence rates that have been reported in Spain and Indian Romani. Since it is known that an ethnic resemblance exists among these three populations, as shown by ancestral marker studies, the ALL data suggests an ethnic influence on the occurrence and frequency of this particular fusion gene.

  20. Intraparenchymal mesenchymal chondrosarcoma of the frontal lobe--a case report and molecular detection of specific gene fusions from archival FFPE sample.

    Science.gov (United States)

    Sajjad, Emir Ahmed; Sikora, Katarzyna; Paciejewski, Tomasz; Garbicz, Filip; Paskal, Wiktor; Szacht, Milena; Grajkowska, Wieslawa; Włodarski, Pawel Krzysztof

    2015-01-01

    Mesenchymal chondrosarcoma is a rare tumor of cartilaginous origin characterized by its bimorphic pattern composed of highly undifferentiated small round cells separated by islands of well-differentiated hyaline cartilage. It exhibits higher malignancy and earlier occurrence in comparison to classic chondrosarcomas. Recently identified HEY1-NCOA2 and IRF2BP2-CDX1 gene fusions confirm their distinct molecular origin and pose a promising diagnostic marker. The majority of cases arise from craniofacial bones. In this study, we present a rare case of mesenchymal chondrosarcoma encompassed within the brain parenchyma of the frontal lobe without any dural or bone attachment. We demonstrate histopathological findings and confirm the HEY1-NCOA2 gene fusion in a formalin-fixed paraffin-embedded archival sample using simple reverse transcription polymerase chain reaction (RT-PCR) method. IRF2BP2-CDX1 gene fusion was absent in the analyzed sample. The clinical follow-up is also presented with a review of treatment modalities for this entity.

  1. Atrophic dermatofibrosarcoma protuberans with the fusion gene COL1A1-PDGFB detected by RT-PCR using only a single primer pair.

    Science.gov (United States)

    Xu, Wen-Jun; Wang, Ju-Sheng

    2015-01-01

    Dermatofibrosarcoma protuberans (DFSPs) is an uncommon dermal tumor of intermediate to low-grade malignancy. A few patients have clinically persistent plaques that might be atrophic, and they are difficult to be diagnosed clinically. With the development of cytogenetic and molecular biology techniques, the detection of fusion transcripts of the collagen type 1a1 (COL1A1) and platelet-derived growth factor-BB (PDGFB) genes has been recognized as a reliable and valuable molecular tool for the diagnosis of DFSPs. We reported a 24-year-old woman who had a 2 years history of atrophic DFSPs, and detected the gene fusion between COL1A1 to PDGFB by one-step method of RT-PCR using only a single primer pair. The gene fusion detected by this rapid and efficient one-step method in our patient appears to be the first report of atrophic DFSPs, and we detected a novel COL1A1 breakpoint between exon 2 and exon 3.

  2. Genetic interaction between Tmprss2-ERG gene fusion and Nkx3.1-loss does not enhance prostate tumorigenesis in mouse models.

    Science.gov (United States)

    Linn, Douglas E; Bronson, Roderick T; Li, Zhe

    2015-01-01

    Gene fusions involving ETS family transcription factors (mainly TMPRSS2-ERG and TMPRSS2-ETV1 fusions) have been found in ~50% of human prostate cancer cases. Although expression of TMPRSS2-ERG or TMPRSS2-ETV1 fusion alone is insufficient to initiate prostate tumorigenesis, they appear to sensitize prostate epithelial cells for cooperation with additional oncogenic mutations to drive frank prostate adenocarcinoma. To search for such ETS-cooperating oncogenic events, we focused on a well-studied prostate tumor suppressor NKX3.1, as loss of NKX3.1 is another common genetic alteration in human prostate cancer. Previous studies have shown that deletions at 8p21 (harboring NKX3.1) and 21q22 (resulting in TMPRSS2-ERG fusion) were both present in a subtype of prostate cancer cases, and that ERG can lead to epigenetic silencing of NKX3.1 in prostate cancer cells, whereas NKX3.1 can in turn negatively regulate TMPRSS2-ERG fusion expression via suppression of the TMPRSS2 promoter activity. We recently generated knockin mouse models for TMPRSS2-ERG and TMPRSS2-ETV1 fusions, utilizing the endogenous Tmprss2 promoter. We crossed these knockin models to an Nkx3.1 knockout mouse model. In Tmprss2-ERG;Nkx3.1+/- (or -/-) male mice, although we observed a slight but significant upregulation of Tmprss2-ERG fusion expression upon Nkx3.1 loss, we did not detect any significant cooperation between these two genetic events to enhance prostate tumorigenesis in vivo. Furthermore, retrospective analysis of a previously published human prostate cancer dataset revealed that within ERG-overexpressing prostate cancer cases, NKX3.1 loss or deletion did not predict biochemical relapse after radical prostatectomy. Collectively, these data suggest that although TMPRSS2-ERG fusion and loss of NKX3.1 are among the most common mutational events found in prostate cancer, and although each of them can sensitize prostate epithelial cells for cooperating with other oncogenic events, these two events

  3. Genetic interaction between Tmprss2-ERG gene fusion and Nkx3.1-loss does not enhance prostate tumorigenesis in mouse models.

    Directory of Open Access Journals (Sweden)

    Douglas E Linn

    Full Text Available Gene fusions involving ETS family transcription factors (mainly TMPRSS2-ERG and TMPRSS2-ETV1 fusions have been found in ~50% of human prostate cancer cases. Although expression of TMPRSS2-ERG or TMPRSS2-ETV1 fusion alone is insufficient to initiate prostate tumorigenesis, they appear to sensitize prostate epithelial cells for cooperation with additional oncogenic mutations to drive frank prostate adenocarcinoma. To search for such ETS-cooperating oncogenic events, we focused on a well-studied prostate tumor suppressor NKX3.1, as loss of NKX3.1 is another common genetic alteration in human prostate cancer. Previous studies have shown that deletions at 8p21 (harboring NKX3.1 and 21q22 (resulting in TMPRSS2-ERG fusion were both present in a subtype of prostate cancer cases, and that ERG can lead to epigenetic silencing of NKX3.1 in prostate cancer cells, whereas NKX3.1 can in turn negatively regulate TMPRSS2-ERG fusion expression via suppression of the TMPRSS2 promoter activity. We recently generated knockin mouse models for TMPRSS2-ERG and TMPRSS2-ETV1 fusions, utilizing the endogenous Tmprss2 promoter. We crossed these knockin models to an Nkx3.1 knockout mouse model. In Tmprss2-ERG;Nkx3.1+/- (or -/- male mice, although we observed a slight but significant upregulation of Tmprss2-ERG fusion expression upon Nkx3.1 loss, we did not detect any significant cooperation between these two genetic events to enhance prostate tumorigenesis in vivo. Furthermore, retrospective analysis of a previously published human prostate cancer dataset revealed that within ERG-overexpressing prostate cancer cases, NKX3.1 loss or deletion did not predict biochemical relapse after radical prostatectomy. Collectively, these data suggest that although TMPRSS2-ERG fusion and loss of NKX3.1 are among the most common mutational events found in prostate cancer, and although each of them can sensitize prostate epithelial cells for cooperating with other oncogenic events, these

  4. PARP inhibition sensitizes to low dose-rate radiation TMPRSS2-ERG fusion gene-expressing and PTEN-deficient prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Payel Chatterjee

    Full Text Available Exposure to genotoxic agents, such as irradiation produces DNA damage, the toxicity of which is augmented when the DNA repair is impaired. Poly (ADP-ribose polymerase (PARP inhibitors were found to be "synthetic lethal" in cells deficient in BRCA1 and BRCA2 that impair homologous recombination. However, since many tumors, including prostate cancer (PCa rarely have on such mutations, there is considerable interest in finding alternative determinants of PARP inhibitor sensitivity. We evaluated the effectiveness of radiation in combination with the PARP inhibitor, rucaparib in PCa cells. The combination index for clonogenic survival following radiation and rucaparib treatments revealed synergistic interactions in a panel of PCa cell lines, being strongest for LNCaP and VCaP cells that express ETS gene fusion proteins. These findings correlated with synergistic interactions for senescence activation, as indicated by β--galactosidase staining. Absence of PTEN and presence of ETS gene fusion thus facilitated activation of senescence, which contributed to decreased clonogenic survival. Increased radiosensitivity in the presence of rucaparib was associated with persistent DNA breaks, as determined by χ-H2AX, p53BP1, and Rad51 foci. VCaP cells, which harbor the TMPRSS2-ERG gene fusion and PC3 cells that stably express a similar construct (fusion III showed enhanced sensitivity towards rucaparib, which, in turn, increased the radiation response to a similar extent as the DNA-PKcs inhibitor NU7441. Rucaparib radiosensitized PCa cells, with a clear benefit of low dose-rate radiation (LDR administered over a longer period of time that caused enhanced DNA damage. LDR mimicking brachytherapy, which is used successfully in the clinic, was most effective when combined with rucaparib by inducing persistent DNA damage and senescence, leading to decreased clonogenic survival. This combination was most effective in the presence of the TMPRSS2-ERG and in the

  5. PARP inhibition sensitizes to low dose-rate radiation TMPRSS2-ERG fusion gene-expressing and PTEN-deficient prostate cancer cells.

    Science.gov (United States)

    Chatterjee, Payel; Choudhary, Gaurav S; Sharma, Arishya; Singh, Kamini; Heston, Warren D; Ciezki, Jay; Klein, Eric A; Almasan, Alexandru

    2013-01-01

    Exposure to genotoxic agents, such as irradiation produces DNA damage, the toxicity of which is augmented when the DNA repair is impaired. Poly (ADP-ribose) polymerase (PARP) inhibitors were found to be "synthetic lethal" in cells deficient in BRCA1 and BRCA2 that impair homologous recombination. However, since many tumors, including prostate cancer (PCa) rarely have on such mutations, there is considerable interest in finding alternative determinants of PARP inhibitor sensitivity. We evaluated the effectiveness of radiation in combination with the PARP inhibitor, rucaparib in PCa cells. The combination index for clonogenic survival following radiation and rucaparib treatments revealed synergistic interactions in a panel of PCa cell lines, being strongest for LNCaP and VCaP cells that express ETS gene fusion proteins. These findings correlated with synergistic interactions for senescence activation, as indicated by β--galactosidase staining. Absence of PTEN and presence of ETS gene fusion thus facilitated activation of senescence, which contributed to decreased clonogenic survival. Increased radiosensitivity in the presence of rucaparib was associated with persistent DNA breaks, as determined by χ-H2AX, p53BP1, and Rad51 foci. VCaP cells, which harbor the TMPRSS2-ERG gene fusion and PC3 cells that stably express a similar construct (fusion III) showed enhanced sensitivity towards rucaparib, which, in turn, increased the radiation response to a similar extent as the DNA-PKcs inhibitor NU7441. Rucaparib radiosensitized PCa cells, with a clear benefit of low dose-rate radiation (LDR) administered over a longer period of time that caused enhanced DNA damage. LDR mimicking brachytherapy, which is used successfully in the clinic, was most effective when combined with rucaparib by inducing persistent DNA damage and senescence, leading to decreased clonogenic survival. This combination was most effective in the presence of the TMPRSS2-ERG and in the absence of PTEN

  6. Nonvisual Opsins and the Regulation of Peripheral Clocks by Light and Hormones.

    Science.gov (United States)

    Poletini, Maristela O; Ramos, Bruno C; Moraes, Maria Nathalia; Castrucci, Ana Maria L

    2015-01-01

    The molecular clock machinery is conserved throughout evolution. However, how environmental cues are perceived has evolved in such a way that peripheral clocks in mammals require a variety of signals, including hormones. On the other hand, in nonmammalian cells able to directly detect light, light seems to play a major role in the synchronization of the clock. The interaction between perception of circadian light by nonvisual opsins and hormones will be discussed under the perspective of clock synchronization at the molecular level.

  7. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Paquette, Stéphane G. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Banner, David [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Chi, Le Thi Bao [Department of Microbiology, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Carlo Urbani Centre, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Leon, Alberto J. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); Xu, Luoling; Ran, Longsi [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Huang, Stephen S.H. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Farooqui, Amber [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); and others

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

  8. Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene.

    Science.gov (United States)

    Aström, A K; Voz, M L; Kas, K; Röijer, E; Wedell, B; Mandahl, N; Van de Ven, W; Mark, J; Stenman, G

    1999-02-15

    We have previously shown (K. Kas et al, Nat. Genet., 15: 170-174, 1997) that the developmentally regulated zinc finger gene pleomorphic adenoma gene 1 (PLAG1) is the target gene in 8q12 in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between PLAG1 and the constitutively expressed gene for beta-catenin (CTNNB1), leading to activation of PLAG1 expression and reduced expression of CTNNB1. Here we have studied the expression of PLAG1 by Northern blot analysis in 47 primary benign and malignant human tumors with or without cytogenetic abnormalities of 8q12. Overexpression of PLAG1 was found in 23 tumors (49%). Thirteen of 17 pleomorphic adenomas with a normal karyotype and 5 of 10 with 12q13-15 abnormalities overexpressed PLAG1, which demonstrates that PLAG1 activation is a frequent event in adenomas irrespective of karyotype. In contrast, PLAG1 was overexpressed in only 2 of 11 malignant salivary gland tumors analyzed, which suggests that, at least in salivary gland tumors, PLAG1 activation preferentially occurs in benign tumors. PLAG1 over-expression was also found in three of nine mesenchymal tumors, i.e., in two uterine leiomyomas and one leiomyosarcoma. RNase protection, rapid amplification of 5'-cDNA ends (5'-RACE), and reverse transcription-PCR analyses of five adenomas with a normal karyotype revealed fusion transcripts in three tumors. Nucleotide sequence analysis of these showed that they contained fusions between PLAG1 and CTNNB1 (one case) or PLAG1 and a novel fusion partner gene, i.e., the gene encoding the transcription elongation factor SII (two cases). The fusions occurred in the 5' noncoding region of PLAG1, leading to exchange of regulatory control elements and, as a consequence, activation of PLAG1 gene expression. Because all of the cases had grossly normal karyotypes, the rearrangements must result from cryptic rearrangements. The results suggest that in addition to

  9. Two novel alkane hydroxylase-rubredoxin fusion genes isolated from a Dietzia bacterium and the functions of fused rubredoxin domains in long-chain n-alkane degradation.

    Science.gov (United States)

    Nie, Yong; Liang, Jieliang; Fang, Hui; Tang, Yue-Qin; Wu, Xiao-Lei

    2011-10-01

    Two alkane hydroxylase-rubredoxin fusion gene homologs (alkW1 and alkW2) were cloned from a Dietzia strain, designated DQ12-45-1b, which can grow on crude oil and n-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1- and AlkG2-type Rds. Expression of the alkW1 gene in DQ12-45-1b was induced when cells were grown on C(8) to C(32) n-alkanes as sole carbon sources, but expression of the alkW2 gene was not detected. Functional heterologous expression in an alkB deletion mutant of Pseudomonas fluorescens KOB2Δ1 suggested the alkW1 could restore the growth of KOB2Δ1 on C(14) and C(16) n-alkanes and induce faster growth on C(18) to C(32) n-alkanes than alkW1ΔRd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2Δ1 itself. In addition, the artificial fusion of AlkB from the Gram-negative P. fluorescens CHA0 and the Rds from both Gram-negative P. fluorescens CHA0 and Gram-positive Dietzia sp. DQ12-45-1b significantly increased the degradation of C(32) alkane compared to that seen with AlkB itself. In conclusion, the alkW1 gene cloned from Dietzia species encoded an alkane hydroxylase which increased growth on and degradation of n-alkanes up to C(32) in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chain n-alkanes (such as C(32)) in the Gram-negative bacterium.

  10. Cardiac optogenetic pacing in drosophila melanogaster using red-shifted opsins (Conference Presentation)

    Science.gov (United States)

    Men, Jing; Li, Airong; Jerwick, Jason; Tanzi, Rudolph E.; Zhou, Chao

    2017-02-01

    Electrical pacing is the current gold standard for investigation of mammalian cardiac electrical conduction systems as well as for treatment of certain cardiac pathologies. However, this method requires an invasive surgical procedure to implant the pacing electrodes. Recently, optogenetic pacing has been developed as an alternative, non-invasive method for heartbeat pacing in animals. It induces heartbeats by shining pulsed light on transgene-generated microbial opsins which in turn activate light gated ion channels in animal hearts. However, commonly used opsins, such as channelrhodopsin-2 (ChR2), require short light wavelength stimulation (475 nm), which is strongly absorbed and scattered by tissue. Here, we expressed recently engineered red-shifted opsins, ReaChR and CsChrimson, in the heart of a well-developed animal model, Drosophila melanogaster, for the first time. Optogenetic pacing was successfully conducted in both ReaChR and CsChrimson flies at their larval, pupal, and adult stages using 617 nm excitation light pulse, enabling a much deeper tissue penetration compared to blue stimulation light. A customized high speed and ultrahigh resolution OCM system was used to non-invasively monitor the heartbeat pacing in Drosophila. Compared to previous studies on optogenetic pacing of Drosophila, higher penetration depth of optogenetic excitation light was achieved in opaque late pupal flies. Lower stimulating power density is needed for excitation at each developmental stage of both groups, which improves the safety of this technique for heart rhythm studies.

  11. Atomistic design of microbial opsin-based blue-shifted optogenetics tools

    Science.gov (United States)

    Kato, Hideaki E.; Kamiya, Motoshi; Sugo, Seiya; Ito, Jumpei; Taniguchi, Reiya; Orito, Ayaka; Hirata, Kunio; Inutsuka, Ayumu; Yamanaka, Akihiro; Maturana, Andrés D.; Ishitani, Ryuichiro; Sudo, Yuki; Hayashi, Shigehiko; Nureki, Osamu

    2015-05-01

    Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity. Molecular engineering has been utilized to create colour variants for the functional augmentation of optogenetics tools, but was limited by the complexity of the protein-chromophore interactions. Here we report the development of blue-shifted colour variants by rational design at atomic resolution, achieved through accurate hybrid molecular simulations, electrophysiology and X-ray crystallography. The molecular simulation models and the crystal structure reveal the precisely designed conformational changes of the chromophore induced by combinatory mutations that shrink its π-conjugated system which, together with electrostatic tuning, produce large blue shifts of the absorption spectra by maximally 100 nm, while maintaining photosensitive ion transport activities. The design principle we elaborate is applicable to other microbial opsins, and clarifies the underlying molecular mechanism of the blue-shifted action spectra of microbial opsins recently isolated from natural sources.

  12. Horizontal transmission of malignancy: in-vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology.

    Directory of Open Access Journals (Sweden)

    David M Goldenberg

    Full Text Available We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5-6 years GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH, lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b suggests that this uniform population may be the clonal initiating (malignant cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.

  13. Preliminary comparing the toxicities of the hybrid cry1Acs fused with different heterogenous genes provided guidance for the fusion expression of Cry proteins.

    Science.gov (United States)

    Tang, Ying; Tong, Jinying; Zhang, Yunlei; Wang, Lei; Hu, Shengbiao; Li, Wenping; Lv, Yuan

    2012-01-01

    In order to provide guidance for selecting suitable heterogenous gene that can efficiently enhance toxicity or broaden insecticidal spectrum of Cry1Ac through fusion expression, two hybrid cry1Acs fused with chitinase-encoding gene tchiB and neurotoxin gene hwtx-1 respectively were constructed and their toxicities were compared. A Bacillus thuringiensis strain harboring the cry1Ac gene in vector pHT315 was used as control. Bioassay revealed that LC(50) (after 72 h) of Cry1Ac protoxin was 41.01 μg mL(-1), while the hybrid cry1Acs fused with tchiB and hwtx-1 were 4.89 and 23.14 μg mL(-1), which were 8.23- and 1.77-fold higher than Cry1Ac protoxin in terms of relative toxicity respectively. Both fusion crystals had a higher toxicity than the original Cry1Ac protein and the toxicity of hybrid cry1Acs fused with hwtx-1 experienced a more significant increase than that fused with tchiB.

  14. Osteoclast Fusion

    DEFF Research Database (Denmark)

    Marie Julie Møller, Anaïs; Delaissé, Jean-Marie; Søe, Kent

    2017-01-01

    suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed...... on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono-nucleated pre-osteoclasts, syncytin-1 promotes fusion of two multi-nucleated osteoclasts, but also reduces the number of fusions between mono-nucleated pre-osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through......Investigations addressing the molecular keys of osteoclast fusion are primarily based on end-point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi-nucleated cells, the number of nuclei per...

  15. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Science.gov (United States)

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  16. Acute promyelocytic leukaemia in patients originating in Latin America is associated with an increased frequency of the bcr1 subtype of the PML/RARalpha fusion gene.

    Science.gov (United States)

    Douer, Dan; Santillana, Sergio; Ramezani, Laleh; Samanez, Cesar; Slovak, Marilyn L; Lee, Ming S; Watkins, Kristy; Williams, Tony; Vallejos, Carlos

    2003-08-01

    The PML/RARalpha fusion gene in acute promyelocytic leukaemia (APL) has three subtypes based on the breakpoint site of the PML gene: long (bcr1), short (bcr3) and variable (bcr2) subtypes. The PML/RARalpha fusion protein is involved in the pathogenesis of APL and the breakpoint site of the PML gene might be associated with aetiological factor(s). Because APL is over-represented in patients that originate in Latin America (Latinos), we evaluated whether the distribution of the PML/RARalpha fusion mRNA in this population is different to that reported in non-Latinos. Among 52 APL patients (28 from Mexico and Central America diagnosed in Los Angeles and 24 from Peru, South America), bcr1, bcr2 and bcr3 expression was 75%, 10% and 15% respectively. However, bcr1 breakpoints were significantly higher compared with non-Latino patients (340/654, 52%) reported in four studies. Often bcr1 and bcr2 are reported together; 862 (60%) of 1429 non-Latino APL patients reported in nine studies were either bcr1 or bcr2, compared with 44 (85%) in our 52 Latino patients. This difference was also statistically significant when our patients were compared to each of the individual studies from USA and Europe, but not for a small series from China and Japan. These results suggest that the overrepresentation of APL among Latin American patients can be accounted for by an increase of a single subtype--bcr1, and the breakage sites in the PML gene may not be random but possibly influenced by genetic and/or environmental factor(s).

  17. Fusion of the FUS and CREB3L2 genes in a supernumerary ring chromosome in low-grade fibromyxoid sarcoma.

    Science.gov (United States)

    Bartuma, Hammurabi; Möller, Emely; Collin, Anna; Domanski, Henryk A; Von Steyern, Fredrik Vult; Mandahl, Nils; Mertens, Fredrik

    2010-06-01

    Low-grade fibromyxoid sarcoma (LGFMS) is a rare, low-grade malignant soft tissue tumor that is often mistaken for either benign or more malignant tumor types. Commonly, this tumor affects young adults and typically arises in the deep proximal extremities or trunk with frequent recurrences and can metastasize to the lungs many years later. Most cases have a recurrent balanced translocation involving chromosomes 7 and 16, t(7;16)(q32-34;p11), which leads to the fusion of the FUS and CREB3L2 genes. However, supernumerary ring chromosomes have been identified in a subset of FUS/CREB3L2-positive LGFMS, but it has not yet been formally demonstrated that such ring chromosomes harbor the FUS/CREB3L2 fusion gene. Here, we report the genetic findings of a supernumerary ring chromosome from an LGFMS from a 77-year-old man. Chromosome banding analysis revealed a supernumerary ring chromosome, and further studies with fluorescence in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the ring contained material from chromosomes 7 and 16, that the FUS gene was present in two rearranged copies, and that it expressed the FUS/CREB3L2 fusion gene. Moreover, an assessment of previously reported cases showed that tumors with ring chromosomes relapsed more often than tumors with a balanced t(7;16), suggesting that ring formation in LGFMS is correlated with tumor progression. Copyright 2010 Elsevier Inc. All rights reserved.

  18. Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

    Science.gov (United States)

    Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

    2016-12-20

    Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

  19. Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

    Science.gov (United States)

    Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

    2008-10-15

    In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.

  20. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  1. CDKN2D-WDFY2 is a cancer-specific fusion gene recurrent in high-grade serous ovarian carcinoma.

    Directory of Open Access Journals (Sweden)

    Kalpana Kannan

    2014-03-01

    Full Text Available Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian

  2. CDKN2D-WDFY2 Is a Cancer-Specific Fusion Gene Recurrent in High-Grade Serous Ovarian Carcinoma

    Science.gov (United States)

    Rajapakshe, Kimal; Hawkins, Shannon M.; Matzuk, Martin M.; Milosavljevic, Aleksandar; Yen, Laising

    2014-01-01

    Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas. PMID

  3. The evolution of the histone methyltransferase gene Su(var3-9 in metazoans includes a fusion with and a re-fission from a functionally unrelated gene

    Directory of Open Access Journals (Sweden)

    Patties Ina

    2006-03-01

    Full Text Available Abstract Background In eukaryotes, histone H3 lysine 9 (H3K9 methylation is a common mechanism involved in gene silencing and the establishment of heterochromatin. The loci of the major heterochromatic H3K9 methyltransferase Su(var3-9 and the functionally unrelated γ subunit of the translation initiation factor eIF2 are fused in Drosophila melanogaster. Here we examined the phylogenetic distribution of this unusual gene fusion and the molecular evolution of the H3K9 HMTase Su(var3-9. Results We show that the gene fusion had taken place in the ancestral line of winged insects and silverfishs (Dicondylia about 400 million years ago. We cloned Su(var3-9 genes from a collembolan and a spider where both genes ancestrally exist as independent transcription units. In contrast, we found a Su(var3-9-specific exon inside the conserved intron position 81-1 of the eIF2γ gene structure in species of eight different insect orders. Intriguinly, in the pea aphid Acyrthosiphon pisum, we detected only sequence remains of this Su(var3-9 exon in the eIF2γ intron, along with an eIF2γ-independent Su(var3-9 gene. This reveals an evolutionary re-fission of both genes in aphids. Su(var3-9 chromo domains are similar to HP1 chromo domains, which points to a potential binding activity to methylated K9 of histone H3. SET domain comparisons suggest a weaker methyltransferase activity of Su(var3-9 in comparison to other H3K9 HMTases. Astonishingly, 11 of 19 previously described, deleterious amino acid substitutions found in Drosophila Su(var3-9 are seemingly compensable through accompanying substitutions during evolution. Conclusion Examination of the Su(var3-9 evolution revealed strong evidence for the establishment of the Su(var3-9/eIF2γ gene fusion in an ancestor of dicondylic insects and a re-fission of this fusion during the evolution of aphids. Our comparison of 65 selected chromo domains and 93 selected SET domains from Su(var3-9 and related proteins offers

  4. Fusion rings and fusion ideals

    DEFF Research Database (Denmark)

    Andersen, Troels Bak

    by the so-called fusion ideals. The fusion rings of Wess-Zumino-Witten models have been widely studied and are well understood in terms of precise combinatorial descriptions and explicit generating sets of the fusion ideals. They also appear in another, more general, setting via tilting modules for quantum...

  5. Recurrence of lung adenocarcinoma after an interval of 15 years revealed by demonstration of the same type of EML4-ALK fusion gene.

    Science.gov (United States)

    Tsukamoto, Yoshitane; Kanamori, Kiyonobu; Watanabe, Takahiro; Mikami, Koji; Ieki, Ryuji; Nakano, Takashi; Kajimoto, Kazuyoshi; Hirota, Seiichi

    2014-12-01

    We carried out an experiment on a 58-year-old man with multiple left lung tumors and swelling of multiple lymph nodes. For clinical staging and therapeutic purposes, bronchoalveolar lavage (BAL) cytology and lung biopsy were performed. The biopsy specimen revealed the left lower lung mass to be immunohistochemically ALK (anaplastic lymphoma kinase)-positive adenocarcinoma. Using the BAL specimen from the left lower lung, EML4 (echinoderm microtubule-associated protein-like 4)-ALK variant 1 fusion gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). His past history showed that he had undergone an operation for lung adenocarcinoma of the right lower lobe 15 years before, and the pathological specimen at that time revealed that the lung adenocarcinoma with pleural invasion and single metastasis of mediastinal lymph node showed a mucinous cribriform pattern and/or signet-ring cell pattern. The typical histology led us to examine the ALK rearrangement in the primary lung cancer and mediastinal metastatic tumor. Immunohistochemistry (IHC) for ALK was positive, and ALK break apart fluorescence in situ hybridization (FISH) showed a positive result. Moreover, RT-PCR using formalin-fixed, paraffin-embedded tissue from the right lung cancer also demonstrated EML4-ALK variant 1 fusion gene. Although there is a possibility that the left lung cancer is de novo one with multiple metastases, detection of the same fusion gene of the very rare EML4-ALK variant 1 in both tumors suggests that the left cancer is a recurrence of the right lung cancer after an interval of 15 years. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaochar, Salma; Shanks, Lisa; Weinert, Ted

    2010-12-14

    Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.

  7. Fusion neutronics

    CERN Document Server

    Wu, Yican

    2017-01-01

    This book provides a systematic and comprehensive introduction to fusion neutronics, covering all key topics from the fundamental theories and methodologies, as well as a wide range of fusion system designs and experiments. It is the first-ever book focusing on the subject of fusion neutronics research. Compared with other nuclear devices such as fission reactors and accelerators, fusion systems are normally characterized by their complex geometry and nuclear physics, which entail new challenges for neutronics such as complicated modeling, deep penetration, low simulation efficiency, multi-physics coupling, etc. The book focuses on the neutronics characteristics of fusion systems and introduces a series of theories and methodologies that were developed to address the challenges of fusion neutronics, and which have since been widely applied all over the world. Further, it introduces readers to neutronics design’s unique principles and procedures, experimental methodologies and technologies for fusion systems...

  8. Temsirolimus in the treatment of renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion proteins: a case report and review of literature

    Directory of Open Access Journals (Sweden)

    James Brown

    2009-12-01

    Full Text Available Xp11.2 translocation renal cell carcinomas (TRCCs are a rare family of tumors newly recognized by the World Health Organization (WHO in 2004. These tumors result in the fusion of partner genes to the TFE3 gene located on Xp11.2. They are most common in the pediatric population, but have been recently implicated in adult renal cell carcinoma (RCC presenting at an early age. TFE3-mediated direct transcriptional upregulation of the Met tyrosine kinase receptor triggers dramatic activation of downstream signaling pathways including the protein kinase B (Akt/phosphatidylinositol-3 kinase (PI3K and mammalian target of rapamycin (mTOR pathways. Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR kinase, a component of intracellular signaling pathways involved in the growth and proliferation of malignant cells. Here we present a case of a 22-year old female who has been treated with temsirolimus for her Xp11.2/TFE3 gene fusion RCC.

  9. Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct ItB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacterpylori(Hpylori) strain Y06 and the ItB gene from Escherichiacoli(E coli) strain 44851 were linked into ItB-ureB fusiongene by PCR. The fusion gene sequence was analyzedafter T-A cloning. A prokaryotic recombinant expressionvector pET32a inserted with ItB-ureB fusion gene (pET32aItB-ureB) was constructed. Expression of the recombinantLTB-UreB protein (rLTB-UreB)in E. coliBL21DE3 inducedby isopropylthio-β-D-galactoside (IPTG) at differentconcentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTBUreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant proteinon inducing specific antibody in rabbits. GM1-ELISA wasused to demonstrate the adjuvanticity of rLTB-UreB. Immunoreaction of rLTB-UreB to the UreB antibody positivesera from 125 gastric patients was determined by using ELISA. RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ItB-ureB fusion gene were 100%. IPTG withdifferent dosages of 0.1-1.0 mmol/L could efficiently inducepET32a-ItB-ureB-E.coli BL21DE3 to express the rLTB-UreB. The output of the target recombinant protein expressed by pET32a-ureB-E. coli BL21DE3 was approximately 35%of the total bacterial proteins. rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori andanti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from

  10. Molecular Characterization of TMPRSS2-ERG Gene Fusion in the NCI-H660 Prostate Cancer Cell Line: A New Perspective for an Old Model

    Directory of Open Access Journals (Sweden)

    Kirsten D. Mertz

    2007-03-01

    Full Text Available Recent studies have established that a significant fraction of prostate cancers harbor a signature gene fusion between the 5' region of androgen-regulated TMPRSS2 and an ETS family transcription factor, most commonly ERG. Studies on the molecular mechanisms and functional consequences of this important chromosomal rearrangement are currently limited to the VCaP cell line derived from a vertebral bone metastasis of a hormone-refractory prostate tumor. Here we report on the NCI-H660 cell line, derived from a metastasic site of an extrapulmonary small cell carcinoma arising from the prostate. NCI-H660 harbors TMPRSS2-ERG fusion with a homozygous intronic deletion between TMPRSS2 and ERG. We demonstrate this by real-time quantitative polymerase chain reaction, a two-stage dual-color interphase fluorescence in situ hybridization (FISH assay testing for TMPRSS2 and ERG break-aparts, and single-nucleotide polymorphism oligonucleotide arrays. The deletion is consistent with the common intronic deletion found on chromosome 21q22.2-3 in human prostate cancer samples. We demonstrate the physical juxtaposition of TMPRSS2 and ERG on the DNA level by fiber FISH. The androgen receptor-negative NCI-H660 cell line expresses ERG in an androgen-independent fashion. This in vitro model system has the potential to provide important pathobiologic insights into TMPRSS2-ERG fusion prostate cancer.

  11. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule

    Directory of Open Access Journals (Sweden)

    Navarro Xavier

    2011-03-01

    Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is one of the most devastating neurodegenerative diseases. Neurotrophic factors have been widely tested to counteract neurodegenerative conditions, despite their unspecific neuronal access. The non-toxic C-terminal fragment of the tetanus toxin (TTC heavy chain has been studied not only as a carrier molecule to the CNS but also as a neuroprotective agent. Because the neurotrophic effects of BDNF have been demonstrated in vitro and in vivo, the question addressed in this work is whether a fusion molecule of BDNF-TTC may have a synergistic effect and enhance the neuroprotective properties of TTC alone in a mouse model of ALS. Methods Recombinant plasmid constructs (pCMV-TTC and pCMV-BDNF-TTC were injected into the quadriceps femoris and triceps brachialis muscles of SOD1G93A transgenic mice at 8 weeks of age. The hanging wire and rotarod tests were performed to assess motor coordination, strength and balance. Electrophysiological tests, morphological assays of spinal cord sections of L2 and L4 segments, and gene and protein expression analyses were performed. The Kaplan-Meier survival analysis test was used for comparisons of survival. Multiple comparisons of data were analyzed using a one-way analysis of variance (ANOVA. Results Treatment with the fusion-molecule BDNF-TTC and with TTC alone significantly delayed the onset of symptoms and functional deficits of SOD1G93A mice. Muscle innervation was partially preserved with these treatments, and the number of surviving motoneurons in L2 spinal cord segment was increased particularly by the fusion protein induction. Inhibition of pro-apoptotic protein targets (caspase-3 and Bax and significant phosphorylation of Akt and ERK were also found in the spinal cord of treated mice. Conclusions Significant improvements in behavioral and electrophysiological results, motoneuron survival and anti-apoptotic/survival-activated pathways were observed with

  12. Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.

    Science.gov (United States)

    Sequeira, Ana Filipa; Turchetto, Jeremy; Saez, Natalie J; Peysson, Fanny; Ramond, Laurie; Duhoo, Yoan; Blémont, Marilyne; Fernandes, Vânia O; Gama, Luís T; Ferreira, Luís M A; Guerreiro, Catarina I P I; Gilles, Nicolas; Darbon, Hervé; Fontes, Carlos M G A; Vincentelli, Renaud

    2017-01-17

    Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.

  13. Translocation t(1;11(p32;q23 with MLL-EPS15 fusion gene formation in acute leukemias: a review and 6 new case reports. Approaches to minimal residual disease monitoring

    Directory of Open Access Journals (Sweden)

    G. A. Tsaur

    2014-07-01

    Full Text Available We performed clinical and laboratory characterization of patients with rare translocation t(1;11(p32;q23 leading to MLL-EPS15 fusion gene formation. Study cohort consisted of 33 primary acute leukemia (AL cases including 6 newly diagnosed and 27 patients previously described in literature. Among study group patients t(1;11(p32;q23 was found most frequently in infant AL cases (median age 8 months. In acute lymphoblastic leukemia (ALL male/female ratio was 1:3, in acute myeloid leukemia (AML it was 1:1. Additional cytogenetic aberrations in 38 % of patients were revealed. The most frequent breakpoint position in EPS15 gene was intron 1. Four different types of MLLEPS15 fusion gene transcripts were detected. Primers-probe-plasmid combination for MLL-EPS15 fusion gene transcript monitoring by realtime quantitative polymerase chain reaction (RQ-PCR was developed and successfully applied. In 3 patients RQ-PCR was done on genomic DNA for absolute quantification of MLL-EPS15 fusion gene. High qualitative concordance rate (92 % was noted between minimal residual disease data obtained in cDNA and genomic DNA for MLL-EPS15 fusion detection.

  14. Translocation t(1;11(p32;q23 with MLL-EPS15 fusion gene formation in acute leukemias: a review and 6 new case reports. Approaches to minimal residual disease monitoring

    Directory of Open Access Journals (Sweden)

    G. A. Tsaur

    2013-01-01

    Full Text Available We performed clinical and laboratory characterization of patients with rare translocation t(1;11(p32;q23 leading to MLL-EPS15 fusion gene formation. Study cohort consisted of 33 primary acute leukemia (AL cases including 6 newly diagnosed and 27 patients previously described in literature. Among study group patients t(1;11(p32;q23 was found most frequently in infant AL cases (median age 8 months. In acute lymphoblastic leukemia (ALL male/female ratio was 1:3, in acute myeloid leukemia (AML it was 1:1. Additional cytogenetic aberrations in 38 % of patients were revealed. The most frequent breakpoint position in EPS15 gene was intron 1. Four different types of MLLEPS15 fusion gene transcripts were detected. Primers-probe-plasmid combination for MLL-EPS15 fusion gene transcript monitoring by realtime quantitative polymerase chain reaction (RQ-PCR was developed and successfully applied. In 3 patients RQ-PCR was done on genomic DNA for absolute quantification of MLL-EPS15 fusion gene. High qualitative concordance rate (92 % was noted between minimal residual disease data obtained in cDNA and genomic DNA for MLL-EPS15 fusion detection.

  15. Transformation of Arabidopsis by Rice OsWRKY78:: GFP Fusion Gene and Subcellular Localization of OsWRKY78 Protein

    Institute of Scientific and Technical Information of China (English)

    Shunzhi LIU; Mei ZHANG; Xin TANG; Xiaolan WANG

    2012-01-01

    [Objective] The study was to understand the subcellular localization of OsWRKY78 protein in plants. [Method] Primers specific for OsWRKY78 gene were designed according to the OsWRKY78 full length sequence in Genbank. The gene was cloned by RT-PCR method. The gene was then recombined into a plasmid ex- pression vector carrying green fluorescent protein (GFP) gene, pBinGFP. The re- combinant was confirmed by PCR and enzyme digestion. The recombinant plasmid pBinGFP-OsWRKY was transformed into Arabidopsis through Agrobacterium tume- faciens strain GV3101 and transgenic plants were obtained. [Result] Measured by fluorescence microscopy, the expression of OsWRKY78 and GFP fusion protein in root tip cells was localized in the nucleus. [Conclusion] This study laid the foundation for further investigating the function of OsWRKY78 gene and its role in related sig- nal transduction and provided theoretical basis for exploring the relation between OsWRKY78 gene and brown planthoppers.

  16. Fusion of the HMGA2 and C9orf92 genes in myolipoma with t(9;12)(p22;q14).

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Agostini, Antonio; Lobmaier, Ingvild; Bjerkehagen, Bodil; Heim, Sverre

    2016-02-09

    Myolipoma of soft tissue is an extremely rare benign tumor composed of mature adipose tissue and smooth muscle cells. It is found predominantly in women. The cytogenetic and molecular genetic features of myolipomas remain largely unexplored. Here we present the first cytogenetically analyzed myolipoma. Cytogenetic and molecular genetic analyses were done on a myolipoma. G-banding analysis of short-term cultured cells from the myolipoma yielded a karyotype with a single clonal chromosome abnormality: 46,XX,t(9;12)(p22;q14). Fluorescence in situ hybridization experiments demonstrated that HMGA2 (in 12q14) was rearranged. Molecular genetic analysis showed that the translocation resulted in fusion of HMGA2 with the C9orf92 gene (from 9p22). The HMGA2-C9orf92 fusion transcript would code for a putative protein containing amino acid residues 1-94 of HMGA2 and 6 amino acid residues from the out-of-frame fusion with exon 4 of C9orf92. The pattern of HMGA2 rearrangement in the present case of myolipoma is similar to what is found in other benign connective tissue tumor types, including lipomas, i.e., disruption of the HMGA2 locus leaves intact exons which encode the AT-hook domains but separates them from the 3´-terminal part of the gene. Whether any genetic features differentiate myolipomas from regular lipomas with HMGA2-involvement is a question that cannot be answered until more cases of the former tumor type are subjected to genetic analysis.

  17. Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector

    Indian Academy of Sciences (India)

    T Swathi Anuradha; S K Jami; R S Datla; P B Kirti

    2006-06-01

    We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-dayold seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in -glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T0 and corresponding T1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

  18. [Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity].

    Science.gov (United States)

    Tian, Wei-Wei; Qiao, Zhen-Hua; Yang, Lin-Hua; Wang, Hong-Wei; Tang, Yan-Hong; Bian, Si-Cheng

    2011-04-01

    This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.

  19. Renal Cell Carcinoma Associated with Xp11.2 Translocation/TFE3 Gene Fusion: A Rare Case Report with Review of the Literature

    Directory of Open Access Journals (Sweden)

    Puneet Ahluwalia

    2013-01-01

    Full Text Available Introduction. The recently recognized renal cell carcinomas associated with Xp11.2 translocations are rare tumors predominantly reported in children. Chromosome Xp11.2 translocation results in gene fusion related to transcription factor E3 (TFE3 that plays an important role in proliferation and survival. Case Report. Herein, we present two cases of a TFE3 translocation-associated RCC in young female adults, one detected incidentally and the other one presenting with gross hematuria. Tumor is characterized by immunohistochemistry and a literature review with optimal treatment regimen is presented. Discussion. Xp11.2 translocation RCCs in adult patients are associated with advanced stages, large tumors, and extracapsular disease and usually have an aggressive clinical course. Conclusion. In TFE3 RCC, the genetic background may not only contribute to tumorigenesis, but also determine the response to chemotherapy and targeted therapy. Therefore it is necessary to diagnose this tumor entity accurately. Because of the small number of TFE3 gene fusion-related renal tumors described in the literature, the exact biologic behavior and impact of current treatment modalities remain to be uncertain.

  20. Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

    Science.gov (United States)

    Firouzamandi, Masoumeh; Moeini, Hassan; Hosseini, Davood; Bejo, Mohd Hair; Omar, Abdul Rahman; Mehrbod, Parvaneh; Ideris, Aini

    2016-03-01

    The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

  1. Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

    CSIR Research Space (South Africa)

    Du Plessis, A

    2006-10-01

    Full Text Available N-terminal sequencing gave rise to homology to flagellin protein, product of the hag gene. protein, product of the hag gene. Gene was cloned by using degenerate primers and inverse PCR. The gene sequence as well as the up- and down- stream regions...

  2. 小地老虎UV视蛋白基因的克隆及序列分析%Molecular Cloning and Sequence Analysis of UV Opsin in Agrotis ypsilon

    Institute of Scientific and Technical Information of China (English)

    闫硕; 张青文; 熊晓菲; 韩娜娜; 王琼; 张璟; 刘小侠

    2012-01-01

    视蛋白是感光物质的重要组成成分,是动物感知周围光环境的重要途径之一.本文以小地老虎(Agrotis ypsilon)3日龄成虫为材料,利用RT-PCR和RACE末端扩增技术克隆得到小地老虎UV视蛋白基因的cDNA序列.序列分析表明,小地老虎视蛋白基因的cDNA序列1 632 bp,包括一个1 140 bp的完整开放阅读框架,编码379个氨基酸,理论蛋白分子量(Mw) 41.50 ku,等电点(pI) 7.56.GenBank登录号为JN185654.UV视蛋白包括7个跨膜拓扑结构和一个G蛋白偶联受体家族,第107位赖氨酸与UV视蛋白的紫外敏感性有重要关系.同源性分析显示,小地老虎UV视蛋白基因与其他昆虫的UV视蛋白基因具有较高同源性.本研究对深入探究UV视蛋白在动物夜行生活中的作用具有重要意义.%Opsin is the main content of photoactive substances, and it is important for the light perception of animals. Total RNA was isolated from the third day adult of the Agrotis ypsilon. The cDNA sequence was cloned by RT-PCR and rapid amplification of cDNA ends (RACE). UV opsin cDNA sequence is 1 632 bp in length, containing an open reading frame of 1 140 bp, which encodes a polypeptide of 379 amino acids residues. The predicted molecular weight of the amino acid is about 41. 50 ku,with an isoelectric point of 7. 56. GenBank accession number; JN185654. The analysis of phylogenetic relationship showed that the cloned gene had high amino acid sequences homology with other insect UV opsins. The molecular cloning of UV opsin gene may play an important role in the study of nocturnal insect vision in the night.

  3. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bone marrow mesenchymal stemcells(MSCs)are pluripotential stemcells that have the capacitytodifferentiate into chondrocytes and osteoblasts[1].Ithas been well documented that bone morphogeneticproteins(BMPs),a group of proteins belonging tothe TGF-βsuperfamily,can induce bone for mationbothin vivoandin vitroas well as promote osteo-blastic differentiation of MSC[2].HeterologousBMP2is successfully transferred to MSCs and genetherapy is employed based on repairing bony andcartilage defects,spinal fusion[3-5]....

  4. Genetic variability available through cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Smith, H.H.; Mastrangelo-Hough, I.A.

    1977-01-01

    Results are reported for the following studies: plant hybridization through protoplast fusion using species of Nicotiana and Petunia; chromosome instability studies on culture-induced chromosome changes and chromosome elimination; chloroplast distribution in parasexual hybrids; chromosomal introgression following fusion; plant-animal fusion; and microcell-mediated chromosome transfer and chromosome-mediated gene transfer. (HLW)

  5. The biological mechanisms and behavioral functions of opsin-based light detection by the skin

    Directory of Open Access Journals (Sweden)

    Jennifer L Kelley

    2016-08-01

    Full Text Available Light detection not only forms the basis of vision (via visual retinal photoreceptors, but can also occur in other parts of the body, including many non-rod/non-cone ocular cells, the pineal complex, the deep brain, and the skin. Indeed, many of the photopigments (an opsin linked to a light-sensitive 11-cis retinal chromophore that mediate color vision in the eyes of vertebrates are also present in the skin of animals such as reptiles, amphibians, crustaceans and fishes (with related photoreceptive molecules present in cephalopods, providing a localized mechanism for light detection across the surface of the body. This form of non-visual photosensitivity may be particularly important for animals that can change their coloration by altering the dispersion of pigments within the chromatophores (pigment containing cells of the skin. Thus, skin coloration may be directly color matched or tuned to both the luminance and spectral properties of the local background environment, thereby facilitating behavioral functions such as camouflage, thermoregulation, and social signaling. This review examines the diversity and sensitivity of opsin-based photopigments present in the skin and considers their putative functional roles in mediating animal behavior. Furthermore, it discusses the potential underlying biochemical and molecular pathways that link shifts in environmental light to both photopigment expression and chromatophore photoresponses. Although photoreception that occurs independently of image formation remains poorly understood, this review highlights the important role of non-visual light detection in facilitating the multiple functions of animal coloration.

  6. The evolution of irradiance detection: melanopsin and the non-visual opsins.

    Science.gov (United States)

    Peirson, Stuart N; Halford, Stephanie; Foster, Russell G

    2009-10-12

    Circadian rhythms are endogenous 24 h cycles that persist in the absence of external time cues. These rhythms provide an internal representation of day length and optimize physiology and behaviour to the varying demands of the solar cycle. These clocks require daily adjustment to local time and the primary time cue (zeitgeber) used by most vertebrates is the daily change in the amount of environmental light (irradiance) at dawn and dusk, a process termed photoentrainment. Attempts to understand the photoreceptor mechanisms mediating non-image-forming responses to light, such as photoentrainment, have resulted in the discovery of a remarkable array of different photoreceptors and photopigment families, all of which appear to use a basic opsin/vitamin A-based photopigment biochemistry. In non-mammalian vertebrates, specialized photoreceptors are located within the pineal complex, deep brain and dermal melanophores. There is also strong evidence in fish and amphibians for the direct photic regulation of circadian clocks in multiple tissues. By contrast, mammals possess only ocular photoreceptors. However, in addition to the image-forming rods and cones of the retina, there exists a third photoreceptor system based on a subset of melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). In this review, we discuss the range of vertebrate photoreceptors and their opsin photopigments, describe the melanopsin/pRGC system in some detail and then finally consider the molecular evolution and sensory ecology of these non-image-forming photoreceptor systems.

  7. Gene : CBRC-ACAR-01-0922 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available ve opsin (Blue photoreceptor pigment) (RH2 opsin) gb|AAB35062.1| RH2 opsin [Anolis carolinensis] gb|AAD32620.1| RH2 opsin [Anolis car...olinensis] 0.0 100% MNGTEGINFYVPLSNKTGLVRSPFEYPQYYLAEPWKYKVVCCYIFFLIFTGLPINILTLLVTF

  8. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Chen; Jie, Leng; Yongqi, Wang [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Weiming, Xiao [Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Juqun, Xi [Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225009 (China); Yanbing, Ding [Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Li, Qian [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Xingyuan, Pan [Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009 (China); Mingchun, Ji [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Weijuan, Gong, E-mail: wjgong@yzu.edu.cn [Department of Immunology, School of Medicine, Yangzhou University, Yangzhou, 225009 (China); Department of Gastroenterology, The Second Clinical Medical College, Yangzhou University, Yangzhou, 225009 (China); Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225009 (China); Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009 (China); Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009 (China)

    2015-07-31

    Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8{sup +} T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. - Highlights: • Generation of a nanoparticle for delivery of dsNKG2D-IL-15 gene. • Characterization of the gene nanoparticle. • Antitumor activity mediated by the gene nanoparticle.

  9. High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli.

    Science.gov (United States)

    Zhang, Jiaxin; Movahedi, Ali; Wei, Zhiheng; Sang, Ming; Wu, Xiaolong; Wang, Mengyang; Wei, Hui; Pan, Huixin; Yin, Tongming; Zhuge, Qiang

    2016-09-15

    The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve the expression level of ABP-dHC-cecropin A in E. coli, tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors (n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins (n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A.

  10. Vertebrate cone opsins enable sustained and highly sensitive rapid control of Gi/o signaling in anxiety circuitry.

    Science.gov (United States)

    Masseck, Olivia A; Spoida, Katharina; Dalkara, Deniz; Maejima, Takashi; Rubelowski, Johanna M; Wallhorn, Lutz; Deneris, Evan S; Herlitze, Stefan

    2014-03-19

    G protein-coupled receptors (GPCRs) coupling to Gi/o signaling pathways are involved in the control of important physiological functions, which are difficult to investigate because of the limitation of tools to control the signaling pathway with precise kinetics and specificity. We established two vertebrate cone opsins, short- and long-wavelength opsin, for long-lasting and repetitive activation of Gi/o signaling pathways in vitro and in vivo. We demonstrate for both opsins the repetitive fast, membrane-delimited, ultra light-sensitive, and wavelength-dependent activation of the Gi/o pathway in HEK cells. We also show repetitive control of Gi/o pathway activation in 5-HT1A receptor domains in the dorsal raphe nucleus (DRN) in brain slices and in vivo, which is sufficient to modulate anxiety behavior in mice. Thus, vertebrate cone opsins represent a class of tools for understanding the role of Gi/o-coupled GPCRs in health and disease. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Reproducible and sustained regulation of Gαs signalling using a metazoan opsin as an optogenetic tool.

    Directory of Open Access Journals (Sweden)

    Helena J Bailes

    Full Text Available Originally developed to regulate neuronal excitability, optogenetics is increasingly also used to control other cellular processes with unprecedented spatiotemporal resolution. Optogenetic modulation of all major G-protein signalling pathways (Gq, Gi and Gs has been achieved using variants of mammalian rod opsin. We show here that the light response driven by such rod opsin-based tools dissipates under repeated exposure, consistent with the known bleaching characteristics of this photopigment. We continue to show that replacing rod opsin with a bleach resistant opsin from Carybdea rastonii, the box jellyfish, (JellyOp overcomes this limitation. Visible light induced high amplitude, reversible, and reproducible increases in cAMP in mammalian cells expressing JellyOp. While single flashes produced a brief cAMP spike, repeated stimulation could sustain elevated levels for 10s of minutes. JellyOp was more photosensitive than currently available optogenetic tools, responding to white light at irradiances ≥1 µW/cm(2. We conclude that JellyOp is a promising new tool for mimicking the activity of Gs-coupled G protein coupled receptors with fine spatiotemporal resolution.

  12. Expression of γ-aminobutyric acid ρ1 and ρ1Δ450 as gene fusions with the green fluorescent protein

    Science.gov (United States)

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2001-01-01

    The functional characteristics and cellular localization of the γaminobutyric acid (GABA) ρ1 receptor and its nonfunctional isoform ρ1Δ450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with ρ1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type ρ1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, ρ1Δ450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing ρ1Δ450-GFP was distributed similarly to that of ρ1-GFP. Mammalian cells transfected with the ρ1-GFP or ρ1Δ450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that ρ1Δ450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, ρ1- and ρ1Δ450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors. PMID:11172056

  13. Variable light environments induce plastic spectral tuning by regional opsin coexpression in the African cichlid fish, Metriaclima zebra.

    Science.gov (United States)

    Dalton, Brian E; Lu, Jessica; Leips, Jeff; Cronin, Thomas W; Carleton, Karen L

    2015-08-01

    Critical behaviours such as predation and mate choice often depend on vision. Visual systems are sensitive to the spectrum of light in their environment, which can vary extensively both within and among habitats. Evolutionary changes in spectral sensitivity contribute to divergence and speciation. Spectral sensitivity of the retina is primarily determined by visual pigments, which are opsin proteins bound to a chromophore. We recently discovered that photoreceptors in different regions of the retina, which view objects against distinct environmental backgrounds, coexpress different pairs of opsins in an African cichlid fish, Metriaclima zebra. This coexpression tunes the sensitivity of the retinal regions to the corresponding backgrounds and may aid in detection of dark objects, such as predators. Although intraretinal regionalization of spectral sensitivity in many animals correlates with their light environments, it is unknown whether variation in the light environment induces developmentally plastic alterations of intraretinal sensitivity regions. Here, we demonstrate with fluorescent in situ hybridization and qPCR that the spectrum and angle of environmental light both influence the development of spectral sensitivity regions by altering the distribution and level of opsins across the retina. Normally, M. zebra coexpresses LWS opsin with RH2Aα opsin in double cones of the ventral but not the dorsal retina. However, when illuminated from below throughout development, adult M. zebra coexpressed LWS and RH2Aα in double cones both dorsally and ventrally. Thus, environmental background spectra alter the spectral sensitivity pattern that develops across the retina, potentially influencing behaviours and related evolutionary processes such as courtship and speciation.

  14. Variable light environments induce plastic spectral tuning by regional opsin coexpression in the African cichlid fish, Metriaclima zebra

    Science.gov (United States)

    Dalton, Brian E.; Lu, Jessica; Leips, Jeff; Cronin, Thomas W.; Carleton, Karen L.

    2015-01-01

    Critical behaviors such as predation and mate choice often depend on vision. Visual systems are sensitive to the spectrum of light in their environment, which can vary extensively both within and among habitats. Evolutionary changes in spectral sensitivity contribute to divergence and speciation. Spectral sensitivity of the retina is primarily determined by visual pigments, which are opsin proteins bound to a chromophore. We recently discovered that photoreceptors in different regions of the retina, which view objects against distinct environmental backgrounds, coexpress different pairs of opsins in an African cichlid fish, Metriaclima zebra. This coexpression tunes the sensitivity of the retinal regions to the corresponding backgrounds and may aid detection of dark objects, such as predators. Although intraretinal regionalization of spectral sensitivity in many animals correlates with their light environments, it is unknown whether variation in the light environment induces developmentally plastic alterations of intraretinal sensitivity regions. Here, we demonstrate with fluorescent in situ hybridization and qPCR that the spectrum and angle of environmental light both influence the development of spectral sensitivity regions by altering the distribution and level of opsins across the retina. Normally M. zebra coexpresses LWS opsin with RH2Aα opsin in double cones of the ventral but not the dorsal retina. However, when illuminated from below throughout development, adult M. zebra coexpressed LWS and RH2Aα in double cones both dorsally and ventrally. Thus, environmental background spectra alter the spectral sensitivity pattern that develops across the retina, potentially influencing behaviors and related evolutionary processes such as courtship and speciation. PMID:26175094

  15. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity.

    Science.gov (United States)

    Yan, Chen; Jie, Leng; Yongqi, Wang; Weiming, Xiao; Juqun, Xi; Yanbing, Ding; Li, Qian; Xingyuan, Pan; Mingchun, Ji; Weijuan, Gong

    2015-07-31

    Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8(+) T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy.

  16. Therapeutic efficacy of a tuberculosis DNA vaccine encoding heat shock protein 65 of Mycobacterium tuberculosis and the human interleukin 2 fusion gene.

    Science.gov (United States)

    Changhong, Shi; Hai, Zhang; Limei, Wang; Jiaze, An; Li, Xi; Tingfen, Zhang; Zhikai, Xu; Yong, Zhao

    2009-01-01

    Use of therapeutic DNA vaccines is a promising strategy against tuberculosis (TB), however, their immunogenicity still needs to be improved. In this study, a plasmid DNA vaccine expressing heat shock protein 65 (HSP65) and the human interleukin 2 (IL-2) fusion gene was constructed. Immune responses induced by the vaccine in the mice and protection against Mycobacterium tuberculosis (MTB) were investigated, along with the therapeutic effect of the DNA vaccine on tuberculosis in mice. Administration of the HSP65-IL-2-DNA vaccine enhanced Th1-type cellular responses by producing greater amounts of interferon-gamma (IFN-gamma) and IL-2 with a higher titer of antigen-specific anti-Hsp65 IgG2a. Compared with the Bacille Calmette-Guérin (BCG) vaccine, the DNA vaccine was able to evoke both CD4 and CD8 T-cell responses, with an especially high percentage of CD8 T-cells. The DNA vaccine was also able to induce high antigen-specific cytotoxicity activity against target cells. When the mice were challenged with virulent MTB H37Rv, a dramatic decrease in the numbers of MTB colony forming units in the spleen and lungs was observed in the mice immunized with HSP65-IL-2-DNA (P<0.05). Meanwhile, the bacterial numbers in TB infected mice treated with the DNA vaccine were also significantly reduced. The protective and therapeutic effects of the HSP65-IL-2-DNA vaccine in the spleen and lungs were superior to that of the HSP65-DNA vaccine (P<0.05). These results suggest that the DNA vaccine expression of IL-2 and the HSP65 fusion gene enhances the immunogenicity and protective as well as therapeutic effects of the HSP65-DNA vaccine against TB in mice by improving the Th1-type response.

  17. Cold fusion

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Suk Yong; Sung, Ki Woong; Kang, Joo Sang; Lee, Jong Jik [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1995-02-01

    So called `cold fusion phenomena` are not confirmed yet. Excess heat generation is very delicate one. Neutron generation is most reliable results, however, the records are erratic and the same results could not be repeated. So there is no reason to exclude the malfunction of testing instruments. The same arguments arise in recording {sup 4}He, {sup 3}He, {sup 3}H, which are not rich in quantity basically. An experiment where plenty of {sup 4}He were recorded is attached in appendix. The problem is that we are trying to search cold fusion which is permitted by nature or not. The famous tunneling effect in quantum mechanics will answer it, however, the most fusion rate is known to be negligible. The focus of this project is on the theme that how to increase that negligible fusion rate. 6 figs, 4 tabs, 1512 refs. (Author).

  18. Spinal Fusion

    Science.gov (United States)

    ... results in predictable healing. Autograft is currently the “gold standard” source of bone for a fusion. The ... pump. With this technique, the patient presses a button that delivers a predetermined amount of narcotic pain ...

  19. Phylogenetic characterization of the fusion genes of the Newcastle disease viruses isolated in Fars province poultry farms during 2009-2011.

    Science.gov (United States)

    Mehrabanpour, Mohammad Javad; Khoobyar, Setareh; Rahimian, Abdollah; Nazari, Mohammad Bagher; Keshtkar, Mohammad Reza

    2014-01-01

    Despite routine vaccination programs against Newcastle disease (ND), sporadic cases have occasionally occurred that remain a constant threat to commercial poultry. Ten isolates of Newcastle disease viruses (NDV) from infected broiler chicken cases were obtained from various locations in Fars province during 2009-2011 and genetically analyzed using reverse transcription polymerase chain reaction (RT- PCR) with primers specific to the viral fusion (F) protein- gene. The viruses were confirmed as NDV by hemagglutination inhibition assay and RT- PCR. The isolates based on the sequence and phylogenetic analyses of partial F gene were genotypically analyzed by RT PCR. In the present investigation, the pathogenicity of NDV strains was determined by internationally recognized test mean death time (MDT). Analysis based on F gene showed that characterized isolates possess three different types of protease cleavage site motifs and appear to show maximum identities with isolates in the region. The subsequent phylogenetic analysis was implemented using MEGA and the phylogenetic tree. The results of RT-PCR and MDT showed that 10 isolates were positive for NDV, (60% velogenic, 30% mesogenic and 10% lentogenic). The results of the phylogenetic analysis showed that 10 NDV isolates from Iran belong to the class II, genotype III viruses. This information is fundamental to improve the efficacy of controlling strategies and vaccine development for NDV.

  20. Spatial localization of genes determined by intranuclear DNA fragmentation with the fusion proteins lamin KRED and histone KRED und visible light.

    Science.gov (United States)

    Waldeck, Waldemar; Mueller, Gabriele; Glatting, Karl-Heinz; Hotz-Wagenblatt, Agnes; Diessl, Nicolle; Chotewutmonti, Sasithorn; Langowski, Jörg; Semmler, Wolfhard; Wiessler, Manfred; Braun, Klaus

    2013-01-01

    The highly organized DNA architecture inside of the nuclei of cells is accepted in the scientific world. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. Small chromosomes are localized in a central nuclear position whereas the large chromosomes are peripherally positioned. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and also histone H2A, both combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light. The extent of DNA damage was strongly dependent on its localization inside of nuclei. The ROS activity allowed to gain information about the location of genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and some diseases was found.

  1. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  2. DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV

    Institute of Scientific and Technical Information of China (English)

    Jing Jin; Jian-Ying Yang; Jing Liu; Yu-Ying Kong; Yuan Wang; Guang-Di Li

    2002-01-01

    AIM: Both Hepatitis B virus (HBV) and Hepatitis C virus(HCV) are major causative agents of transfusion-associatedand community-acquired hepatitis worldwide. Developmentof a HCV vaccine as well as more effective HBV vaccines isan urgent task. DNA immunization provides a promisingapproach to elicit protective humoral and cellular immuneresponses against viral infection. The aim of this study is toachieve immune responses against both HCV and HBV by DNAimmunization with fusion constructs comprising various HCVE2 gene fragments fused to HBsAg gane of HBV.METHODS: C57BL/6 mice were immunized with plasmid DNAexpressing five fragments of HCV E2 fused to the gene forHBsAg respectively. After one primary and one boostingimmunizations, antibodies against HCV E2 and HBsAg weretested and subtyped in ELISA. Splenic cytokine expressionof IFN-γ and IL-10 was analyzed using an RT-PCR assay.Post-immune mouse antisera also were tested for theirability to capture HCV viruses in the serum of a hepatitis Cpatient in vitro.RESUTLTS: After immunization, antibodies against bothHBsAg and HCV E2 were detected in mouse sera, withIgG2a being the dominant immunoglobulin sub-class. High-level expression of INF-γ was deuetected in cultured splenic cells.Mouse antisera against three of the five fusion constructs wereable to capture HCV viruses in an in vitro assay.CONCLUSION: The results indicate that these fusionconstructs could efficiently elicit humoral and Th1 dominantcellular immune responses against both HBV S and HCV E2antigens in DNA-immunized mice. They thus could serve ascandidates for a bivalent vaccine against HBV and HCVinfection. In addition, the capacity of mouse antisera againstthree of the five fusion constnucts to capture HCV virusses invitro suggested that neutralizing epitopes may be present inother regions of E2 besides the hypervariable region 1.

  3. An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

    Science.gov (United States)

    Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

    2016-01-01

    The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

  4. Allele-specific analysis of cell fusion-mediated pluripotent reprograming reveals distinct and predictive susceptibilities of human X-linked genes to reactivation.

    Science.gov (United States)

    Cantone, Irene; Dharmalingam, Gopuraja; Chan, Yi-Wah; Kohler, Anne-Celine; Lenhard, Boris; Merkenschlager, Matthias; Fisher, Amanda G

    2017-01-25

    Inactivation of one X chromosome is established early in female mammalian development and can be reversed in vivo and in vitro when pluripotency factors are re-expressed. The extent of reactivation along the inactive X chromosome (Xi) and the determinants of locus susceptibility are, however, poorly understood. Here we use cell fusion-mediated pluripotent reprograming to study human Xi reactivation and allele-specific single nucleotide polymorphisms (SNPs) to identify reactivated loci. We show that a subset of human Xi genes is rapidly reactivated upon re-expression of the pluripotency network. These genes lie within the most evolutionary recent segments of the human X chromosome that are depleted of LINE1 and enriched for SINE elements, predicted to impair XIST spreading. Interestingly, this cadre of genes displays stochastic Xi expression in human fibroblasts ahead of reprograming. This stochastic variability is evident between clones, by RNA-sequencing, and at the single-cell level, by RNA-FISH, and is not attributable to differences in repressive histone H3K9me3 or H3K27me3 levels. Treatment with the DNA demethylating agent 5-deoxy-azacytidine does not increase Xi expression ahead of reprograming, but instead reveals a second cadre of genes that only become susceptible to reactivation upon induction of pluripotency. Collectively, these data not only underscore the multiple pathways that contribute to maintaining silencing along the human Xi chromosome but also suggest that transcriptional stochasticity among human cells could be useful for predicting and engineering epigenetic strategies to achieve locus-specific or domain-specific human Xi gene reactivation.

  5. Fusion of the Tumor-Suppressor Gene CHEK2 and the Gene for the Regulatory Subunit B of Protein Phosphatase 2 PPP2R2A in Childhood Teratoma

    Directory of Open Access Journals (Sweden)

    Yuesheng Jin

    2006-05-01

    Full Text Available We characterized the molecular genetic consequences of a balanced chromosome translocation t(8;22(p21; q12, which occurred as the sole cytogenetic aberration in short-term cultured cells from an intrathoracic mature teratoma in a 15-year-old girl. Fluorescence in situ hybridization and reverse transcription- polymerase chain reaction disclosed that t(8;22 resulted in the fusion of the genes PPP2R2A and CHEK2, with an inserted fragment belonging to class I endogenous retrovirus-related sequences at the junction. Sequencing of the two genes did not reveal any additional mutation. None of the three detected PPP2R2A/CHEK2 fusion transcripts resulted in an in-frame PPP2R2A/CHEK2 chimerical open reading frame; however, in all of them, the known open reading frame of CHEK2 was preserved. Thus, promoter swapping leading to deregulated CHEK2 expression would be the most likely oncogenic mechanism. Whereas inactivating mutations of CHEK2 previously have been described in a variety of sporadic tumors and in inherited cancer-predisposing syndromes, PPP2R2A, encoding a regulatory subunit of the multimeric enzyme phosphatase 2, has not been directly implicated in tumorigenesis. Our findings suggest that deregulation of CHEK2 and/or PPP2R2A is of pathogenetic importance in at least a subset of germ cell tumors.

  6. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    Energy Technology Data Exchange (ETDEWEB)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang, E-mail: wolfgang.marwan@ovgu.de

    2013-05-24

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  7. Biochemical Measurements of Free Opsin in Macular Degeneration Eyes: Examining the 11-CIS Retinal Deficiency Hypothesis of Delayed Dark Adaptation (An American Ophthalmological Society Thesis).

    Science.gov (United States)

    Hanneken, Anne; Neikirk, Thomas; Johnson, Jennifer; Kono, Masahiro

    2017-08-01

    To test the hypothesis that delayed dark adaptation in patients with macular degeneration is due to an excess of free unliganded opsin (apo-opsin) and a deficiency of the visual chromophore, 11-cis retinal, in rod outer segments. A total of 50 human autopsy eyes were harvested from donors with and without macular degeneration within 2-24 hrs. postmortem. Protocols were developed which permitted dark adaptation of normal human eyes after death and enucleation. Biochemical methods of purifying rod outer segments were optimized and the concentration of rhodopsin and apo-opsin was measured with UV-visible scanning spectroscopy. The presence of apo-opsin was calculated by measuring the difference in the rhodopsin absorption spectra before and after the addition of 11-cis retinal. A total of 20 normal eyes and 16 eyes from donors with early, intermediate and advanced stages of macular degeneration were included in the final analysis. Dark adaptation was achieved by harvesting whole globes in low light, transferring into dark (light-proof) canisters and dissecting the globes using infrared light and image converters for visualization. Apo-opsin was readily detected in positive controls after the addition of 11-cis retinal. Normal autopsy eyes showed no evidence of apo-opsin. Eyes with macular degeneration also showed no evidence of apo-opsin, regardless of the severity of disease. Methods have been developed to study dark adaptation in human autopsy eyes. Eyes with age-related macular degeneration do not show a deficiency of 11-cis retinal or an excess of apo-opsin within rod outer segments.

  8. Structural and functional studies of FKHR-PAX3, a reciprocal fusion gene of the t(2;13 chromosomal translocation in alveolar rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Qiande Hu

    Full Text Available Alveolar rhabdomyosarcoma (ARMS is an aggressive pediatric cancer of skeletal muscle. More than 70% of ARMS tumors carry balanced t(2;13 chromosomal translocation that leads to the production of two novel fusion genes, PAX3-FKHR and FKHR-PAX3. While the PAX3-FKHR gene has been intensely studied, the reciprocal FKHR-PAX3 gene has rarely been described. We report here the cloning and functional characterization of the FKHR-PAX3 gene as the first step towards a better understanding of its potential impact on ARMS biology. From RH30 ARMS cells, we detected and isolated three versions of FKHR-PAX3 cDNAs whose C-terminal sequences corresponded to PAX3c, PAX3d, and PAX3e isoforms. Unlike the nuclear-specific localization of PAX3-FKHR, the reciprocal FKHR-PAX3 proteins stayed predominantly in the cytoplasm. FKHR-PAX3 potently inhibited myogenesis in both non-transformed myoblast cells and ARMS cells. We showed that FKHR-PAX3 was not a classic oncogene but could act as a facilitator in oncogenic pathways by stabilizing PAX3-FKHR expression, enhancing cell proliferation, clonogenicity, anchorage-independent growth, and matrix adhesion in vitro, and accelerating the onset of tumor formation in xenograft mouse model in vivo. In addition to these pro-oncogenic behaviors, FKHR-PAX3 also negatively affected cell migration and invasion in vitro and lung metastasis in vivo. Taken together, these functional characteristics suggested that FKHR-PAX3 might have a critical role in the early stage of ARMS development.

  9. Trophoblast fusion.

    Science.gov (United States)

    Huppertz, Berthold; Gauster, Martin

    2011-01-01

    The villous trophoblast of the human placenta is the epithelial cover of the fetal chorionic villi floating in maternal blood. This epithelial cover is organized in two distinct layers, the multinucleated syncytiotrophoblast directly facing maternal blood and a second layer of mononucleated cytotrophoblasts. During pregnancy single cytotrophoblasts continuously fuse with the overlying syncytiotrophoblast to preserve this end-differentiated layer until delivery. Syncytial fusion continuously supplies the syncytiotrophoblast with compounds of fusing cytotrophoblasts such as proteins, nucleic acids and lipids as well as organelles. At the same time the input of cytotrophoblastic components is counterbalanced by a continuous release of apoptotic material from the syncytiotrophoblast into maternal blood. Fusion is an essential step in maintaining the syncytiotrophoblast. Trophoblast fusion was shown to be dependant on and regulated by multiple factors such as fusion proteins, proteases and cytoskeletal proteins as well as cytokines, hormones and transcription factors. In this chapter we focus on factors that may be involved in the fusion process of trophoblast directly or that may prepare the cytotrophoblast to fuse.

  10. An ancient history of gene duplications, fusions and losses in the evolution of APOBEC3 mutators in mammals

    Directory of Open Access Journals (Sweden)

    Münk Carsten

    2012-05-01

    Full Text Available Abstract Background The APOBEC3 (A3 genes play a key role in innate antiviral defense in mammals by introducing directed mutations in the DNA. The human genome encodes for seven A3 genes, with multiple splice alternatives. Different A3 proteins display different substrate specificity, but the very basic question on how discerning self from non-self still remains unresolved. Further, the expression of A3 activity/ies shapes the way both viral and host genomes evolve. Results We present here a detailed temporal analysis of the origin and expansion of the A3 repertoire in mammals. Our data support an evolutionary scenario where the genome of the mammalian ancestor encoded for at least one ancestral A3 gene, and where the genome of the ancestor of placental mammals (and possibly of the ancestor of all mammals already encoded for an A3Z1-A3Z2-A3Z3 arrangement. Duplication events of the A3 genes have occurred independently in different lineages: humans, cats and horses. In all of them, gene duplication has resulted in changes in enzyme activity and/or substrate specificity, in a paradigmatic example of convergent adaptive evolution at the genomic level. Finally, our results show that evolutionary rates for the three A3Z1, A3Z2 and A3Z3 motifs have significantly decreased in the last 100 Mya. The analysis constitutes a textbook example of the evolution of a gene locus by duplication and sub/neofunctionalization in the context of virus-host arms race. Conclusions Our results provide a time framework for identifying ancestral and derived genomic arrangements in the APOBEC loci, and to date the expansion of this gene family for different lineages through time, as a response to changes in viral/retroviral/retrotransposon pressure.

  11. Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry.

    Science.gov (United States)

    Wu, Fang; Wang, Peng; Young, Leah C; Lai, Raymond; Li, Liang

    2009-02-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

  12. A new transcriptional variant and small azurophilic granules in an acute promyelocytic leukemia case with NPM1/RARA fusion gene.

    Science.gov (United States)

    Kikuma, Tomoe; Nakamachi, Yuji; Noguchi, Yoriko; Okazaki, Yoko; Shimomura, Daisuke; Yakushijin, Kimikazu; Yamamoto, Katsuya; Matsuoka, Hiroshi; Minami, Hironobu; Itoh, Tomoo; Kawano, Seiji

    2015-12-01

    We report here the first case of NPM1/RARA-positive acute promyelocytic leukemia (APL) preceded by myeloid sarcoma (MS) in the vertebra. A 52-year-old man was diagnosed with MS, as the tumor cells were positive for myeloperoxidase and CD68 but negative for CD163. After treatment with steroids and radiation, the size of the tumor was markedly reduced and peripheral blood count was normal. Bone marrow examination showed 89.2% consisted of unclassified promyelocytes characterized by round nuclei and abundant small azurophilic granules but no Auer rods. The results of chromosome analysis showed 46,XY,t(5;17)(q35;q12). Reverse-transcription polymerase chain reaction amplified the NPM1/RARA fusion transcripts derived from a combination of NPM1 exon 4 and RARA exon 5, or of NPM1 exon 1 and RARA exon 5; the latter of these has not been reported previously. Electron microscopic examination of the promyelocyte nuclei showed they were oval with mild nuclear chromatin condensation and small- to medium-sized nucleoli. Hematological and molecular complete remission was attained after induction therapy including all-trans retinoic acid. As MS was also diagnosed in two of the seven other reported cases of APL with NPM1/RARA, MS may occur more frequently in APL with NPM1/RARA than APL with PML/RARA.

  13. Crizotinib Treatment in a Lung Adenocarcinoma Harboring ALK Fusion Gene with Bone Marrow Metastasis: Case Report and Literature Review

    Directory of Open Access Journals (Sweden)

    Xiaoyan LI

    2015-02-01

    Full Text Available Background and objective Distant metastasis was common in lung cancer, especially patients with bone marrow metastasis had poor prognosis and there were few effective methods. Crizotinib had been confirmed to be used in anaplastic lymphoma kinase (ALK positive lung adenocarcinoma, but the efficacy in lung cancer with bone marrow metastasis was unknown. In the present study, we reported one case of ALK-positive lung adenocarcinoma with bone marrow matastasis given crizotinib treatment, the safety and efficacy was summarized. Methods ALK fusion was tested by fluorescence in situ hybridization (FISH. Crizotinib was given with dose of 250 mg, bid. Objective response was evaluated by Response Evaluation Criteriation in Solid Tumours (RECIST v1.1 and bone marrow response was evaluated by bone marrow puncture and biopsy. Adeverse events were evaluated according to Common Terminology Criteria for Adverse Events (CTC AE v4.0. Results The patient achieved partial response (PR after 6 weeks of crizotinib, especially the objective response of bone marrow metastasis was complete response (CR. The patient stopped crizotinib because of pneumonia. The progression free survival (PFS and overall survival (OS was 20 weeks and 22 weeks respectively. Conclusion Crizotinib could be an effective method for ALK-positive lung cancer with bone marrow metastasis and showed good tolerance.

  14. Membrane fusion during poxvirus entry.

    Science.gov (United States)

    Moss, Bernard

    2016-12-01

    Poxviruses comprise a large family of enveloped DNA viruses that infect vertebrates and invertebrates. Poxviruses, unlike most DNA viruses, replicate in the cytoplasm and encode enzymes and other proteins that enable entry, gene expression, genome replication, virion assembly and resistance to host defenses. Entry of vaccinia virus, the prototype member of the family, can occur at the plasma membrane or following endocytosis. Whereas many viruses encode one or two proteins for attachment and membrane fusion, vaccinia virus encodes four proteins for attachment and eleven more for membrane fusion and core entry. The entry-fusion proteins are conserved in all poxviruses and form a complex, known as the Entry Fusion Complex (EFC), which is embedded in the membrane of the mature virion. An additional membrane that encloses the mature virion and is discarded prior to entry is present on an extracellular form of the virus. The EFC is held together by multiple interactions that depend on nine of the eleven proteins. The entry process can be divided into attachment, hemifusion and core entry. All eleven EFC proteins are required for core entry and at least eight for hemifusion. To mediate fusion the virus particle is activated by low pH, which removes one or more fusion repressors that interact with EFC components. Additional EFC-interacting fusion repressors insert into cell membranes and prevent secondary infection. The absence of detailed structural information, except for two attachment proteins and one EFC protein, is delaying efforts to determine the fusion mechanism.

  15. Fusion Machinery

    DEFF Research Database (Denmark)

    Sørensen, Jakob Balslev; Milosevic, Ira

    2015-01-01

    the vesicular SNARE VAMP2/synaptobrevin-2 and the target (plasma membrane) SNAREs SNAP25 and syntaxin-1 results in fusion and release of neurotransmitter, synchronized to the electrical activity of the cell by calcium influx and binding to synaptotagmin. Formation of the SNARE complex is tightly regulated...... and appears to start with syntaxin-1 bound to an SM (Sec1/Munc18-like) protein. Proteins of the Munc13-family are responsible for opening up syntaxin and allowing sequential binding of SNAP-25 and VAMP2/synaptobrevin-2. N- to C-terminal “zippering” of the SNARE domains leads to membrane fusion...

  16. Heterogeneous breakpoints on the immunoglobulin genes are involved in fusion with the 5' region of BCL2 in B-cell tumors.

    Science.gov (United States)

    Yonetani, N; Ueda, C; Akasaka, T; Nishikori, M; Uchiyama, T; Ohno, H

    2001-09-01

    The 5' flanking region of the BCL2 gene (5'-BCL2) is a breakpoint cluster of rearrangements with immunoglobulin genes (IGs). In contrast to t(14;18)(q32;q21) affecting the 3' region of BCL2, 5'-BCL2 can fuse to not only the heavy chain gene (IGH), but also two light chain gene (IGL) loci. We report here cloning and sequencing of a total of eleven 5'-BCL2 / IGs junctional areas of B-cell tumors, which were amplified by long-distance polymerase chain reaction-based assays. The breakpoints on 5'-BCL2 were distributed from 378 to 2312 bp upstream of the translational initiation site and, reflecting the alteration of regulatory sequences of BCL2, 5'-BCL2 / IGs-positive cells showed markedly higher levels of BCL2 expression than those of t(14;18)-positive cells. In contrast, the breakpoints on the IGs were variable. Two 5'-BCL2 / IGH and two 5'-BCL2 / IGLkappa junctions occurred 5' of the joining (J) segments, suggesting operation of an erroneous variable (V) / diversity (D) / J and V / J rearrangement mechanism. However, two other 5'-BCL2 / IGH junctions affected switch regions, and the kappa-deleting element, which is located 24 kb downstream of the constant region of IGLkappa, followed the 5'-BCL2 in another case. One 5'-BCL2 / IGLkappa and two 5'-BCL2 / IGLlambda junctions involved intronic regions where the normal recombination process does not occur. In the remaining one case, the 5'-BCL2 fused 3' of a Vlambda gene that was upstream of another Vlambda / Jlambda complex carrying a non-producing configuration, indicating that the receptor editing mechanism was likely involved in this rearrangement. Our study revealed heterogeneous anatomy of the 5'-BCL2 / IGs fusion gene leading to transcriptional activation of BCL2, and suggested that the mechanisms underlying the formation of this particular oncogene / IGs recombination are not identical to those of t(14;18).

  17. Multiple horizontal gene transfer events and domain fusions have created novel regulatory and metabolic networks in the oomycete genome.

    Directory of Open Access Journals (Sweden)

    Paul Francis Morris

    Full Text Available Complex enzymes with multiple catalytic activities are hypothesized to have evolved from more primitive precursors. Global analysis of the Phytophthora sojae genome using conservative criteria for evaluation of complex proteins identified 273 novel multifunctional proteins that were also conserved in P. ramorum. Each of these proteins contains combinations of protein motifs that are not present in bacterial, plant, animal, or fungal genomes. A subset of these proteins were also identified in the two diatom genomes, but the majority of these proteins have formed after the split between diatoms and oomycetes. Documentation of multiple cases of domain fusions that are common to both oomycetes and diatom genomes lends additional support for the hypothesis that oomycetes and diatoms are monophyletic. Bifunctional proteins that catalyze two steps in a metabolic pathway can be used to infer the interaction of orthologous proteins that exist as separate entities in other genomes. We postulated that the novel multifunctional proteins of oomycetes could function as potential Rosetta Stones to identify interacting proteins of conserved metabolic and regulatory networks in other eukaryotic genomes. However ortholog analysis of each domain within our set of 273 multifunctional proteins against 39 sequenced bacterial and eukaryotic genomes, identified only 18 candidate Rosetta Stone proteins. Thus the majority of multifunctional proteins are not Rosetta Stones, but they may nonetheless be useful in identifying novel metabolic and regulatory networks in oomycetes. Phylogenetic analysis of all the enzymes in three pathways with one or more novel multifunctional proteins was conducted to determine the probable origins of individual enzymes. These analyses revealed multiple examples of horizontal transfer from both bacterial genomes and the photosynthetic endosymbiont in the ancestral genome of Stramenopiles. The complexity of the phylogenetic origins of these

  18. Repair of Staphylococcus aureus-infected wound with gene-modified C3H10T1/2 cells expressing BPI-BD3 fusion antibiotic peptide

    Directory of Open Access Journals (Sweden)

    Xin-ran ZHANG

    2015-10-01

    Full Text Available Objective To study the antibacterial and tissue reparative effect of BPI-BD3 gene-modified mesenchymal stem cells in a mouse model of wound infection. Methods C3H10T1/2 cells were transfected with recombinant adenovirus vector pAdxsi-BPI-BD3, the expression of BPI-BD3 fusion protein was verified by RT-PCR and Western blotting. Excision wound with a diameter of 1cm was inoculated with Staphylococcus aureuswas made on the back of 30 mice. The mice were randomly divided into 3 groups (10 each. Mice in group T were injected with BPI-BD3 gene-modified C3H10T1/2 cells through caudal vein, those in group C were injected with unmodified C3H10T1/2 cells, and in group N were injected with PBS as control. The wound repair result was evaluated by estimation of the percentage of remaining wound area and the amount of wound bacteria under the scar, followed by observation of pathological changes. Inflammatory reactions of the wounds were assessed accordingly. Results The amount of bacteria under the scar was less in group T than in the other two groups (P<0.05. It was also found that the wound healing process was faster in group T than in group C and group N. Pathological observation showed that the inflammatory reaction in group T was also significantly milder than in the other two groups. Conclusion BPI-BD3 gene-modified mesenchymal stem cells may enhance wound repair by controlling infection and promoting tissue regeneration, thus it may be promising in clinical application. DOI: 10.11855/j.issn.0577-7402.2015.09.07

  19. Using electroretinograms and multi-model inference to identify spectral classes of photoreceptors and relative opsin expression levels

    Directory of Open Access Journals (Sweden)

    Nicolas Lessios

    2017-07-01

    Full Text Available Understanding how individual photoreceptor cells factor in the spectral sensitivity of a visual system is essential to explain how they contribute to the visual ecology of the animal in question. Existing methods that model the absorption of visual pigments use templates which correspond closely to data from thin cross-sections of photoreceptor cells. However, few modeling approaches use a single framework to incorporate physical parameters of real photoreceptors, which can be fused, and can form vertical tiers. Akaike’s information criterion (AICc was used here to select absorptance models of multiple classes of photoreceptor cells that maximize information, given visual system spectral sensitivity data obtained using extracellular electroretinograms and structural parameters obtained by histological methods. This framework was first used to select among alternative hypotheses of photoreceptor number. It identified spectral classes from a range of dark-adapted visual systems which have between one and four spectral photoreceptor classes. These were the velvet worm, Principapillatus hitoyensis, the branchiopod water flea, Daphnia magna, normal humans, and humans with enhanced S-cone syndrome, a condition in which S-cone frequency is increased due to mutations in a transcription factor that controls photoreceptor expression. Data from the Asian swallowtail, Papilio xuthus, which has at least five main spectral photoreceptor classes in its compound eyes, were included to illustrate potential effects of model over-simplification on multi-model inference. The multi-model framework was then used with parameters of spectral photoreceptor classes and the structural photoreceptor array kept constant. The goal was to map relative opsin expression to visual pigment concentration. It identified relative opsin expression differences for two populations of the bluefin killifish, Lucania goodei. The modeling approach presented here will be useful in

  20. Development of novel prime-boost strategies based on a tri-gene fusion recombinant L. tarentolae vaccine against experimental murine visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Noushin Saljoughian

    Full Text Available Visceral leishmaniasis (VL is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L. tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB(-CTE as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB(-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB(-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.

  1. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants.

    Science.gov (United States)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

    2013-05-24

    Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Attenuated human parainfluenza virus type 1 (HPIV1) expressing the respiratory syncytial virus (RSV) fusion F glycoprotein from an added gene: effects of pre-fusion stabilization and packaging of RSV F.

    Science.gov (United States)

    Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D; Graham, Barney S; Collins, Peter L; Munir, Shirin

    2017-08-23

    Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the pre-fusion (pre-F) conformation by previously-described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane (TM) and cytoplasmic tail (CT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.Importance. RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a

  3. Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

    Science.gov (United States)

    Nikonov, Sergei S; Daniele, Lauren L; Zhu, Xuemei; Craft, Cheryl M; Swaroop, Anand; Pugh, Edward N

    2005-03-01

    normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

  4. Magnetic fusion; La fusion magnetique

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-07-01

    This document is a detailed lecture on thermonuclear fusion. The basic physics principles are recalled and the technological choices that have led to tokamaks or stellarators are exposed. Different aspects concerning thermonuclear reactors such as safety, economy and feasibility are discussed. Tore-supra is described in details as well as the ITER project.

  5. Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueping [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Wang, Li [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Department of Medical Laboratory, Chongqing Emergency Medical Center (Chongqing The Fourth Hospital), Chongqing, 400016 (China); Sheng, Shangchun [The No.2 Peoples' Hospital of Yibin, Sichuan, 644000 (China); Wang, Teng; Yang, Juan [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Xie, Guoming, E-mail: guomingxie@cqmu.edu.cn [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Feng, Wenli, E-mail: fengwlcqmu@sina.com [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China)

    2015-08-19

    This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application. - Highlights: • A transducer hairpin was designed to improve the versatility of DNA circuit. • GS/PANI/AuNPs were introduced to the DNA circuit for further signal amplification. • The established biosensor displayed high sensitivity and good specificity.

  6. Genetic diversity, seasonality and transmission network of human metapneumovirus: identification of a unique sub-lineage of the fusion and attachment genes

    Science.gov (United States)

    Chow, Wei Zhen; Chan, Yoke Fun; Oong, Xiang Yong; Ng, Liang Jie; Nor’E, Siti Sarah; Ng, Kim Tien; Chan, Kok Gan; Hanafi, Nik Sherina; Pang, Yong Kek; Kamarulzaman, Adeeba; Tee, Kok Keng

    2016-01-01

    Human metapneumovirus (HMPV) is an important viral respiratory pathogen worldwide. Current knowledge regarding the genetic diversity, seasonality and transmission dynamics of HMPV among adults and children living in tropical climate remains limited. HMPV prevailed at 2.2% (n = 86/3,935) among individuals presented with acute respiratory tract infections in Kuala Lumpur, Malaysia between 2012 and 2014. Seasonal peaks were observed during the northeast monsoon season (November–April) and correlated with higher relative humidity and number of rainy days (P < 0.05). Phylogenetic analysis of the fusion and attachment genes identified the co-circulation of three known HMPV sub-lineages, A2b and B1 (30.2% each, 26/86) and B2 (20.9%, 18/86), with genotype shift from sub-lineage B1 to A2b observed in 2013. Interestingly, a previously unrecognized sub-lineage of A2 was identified in 18.6% (16/86) of the population. Using a custom script for network construction based on the TN93 pairwise genetic distance, we identified up to nine HMPV transmission clusters circulating as multiple sub-epidemics. Although no apparent major outbreak was observed, the increased frequency of transmission clusters (dyads) during seasonal peaks suggests the potential roles of transmission clusters in driving the spread of HMPV. Our findings provide essential information for therapeutic research, prevention strategies, and disease outbreak monitoring of HMPV. PMID:27279080

  7. Protocol standardization for detection of TMPRSS2 fusions: ERG and gene expression of EZH2, SPINK-1 and NKX3.1 in prostate cancer (PCa

    Directory of Open Access Journals (Sweden)

    Yenifer Yamile Segura Moreno

    2016-09-01

    Full Text Available At present doesn't exist tool to differentiate patients with prostate cancer (PCa of poor prognosis of those with indolent disease that only require a controlled monitoring of the disease. Because of the coexistence of different premalignant and malignant foci in CaP, the understanding of the carcinogenesis process requires a better understanding. Currently, the morphological heterogeneity in PCa is evaluated with Gleason score, which is closely related to the prognosis of the disease, but this is insufficient so it is currently to work on identifying molecular alterations to identify subtypes that can establish more precisely the patient's prognosis. This preliminary study aimed to standardization of the method of quantification in prostatic samples of FFPE of expression of transcripts of possible biomarkers, such as the oncogenes, SPINK-1 y EZH2, the tumour suppressor, NKX3.1, together with the determination of the presence/absence of gene fusion, TMPRSS2:ERG, being that these transcripts are involved in apparent exclusive events of the natural evolution of PCa, that support the possibility of a molecular classification for this disease.

  8. Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

    Directory of Open Access Journals (Sweden)

    Zhongshan Wang

    2011-01-01

    Full Text Available The cloning, expression and purification of the glutathione (sulfur import system ATP-binding protein (gsiA was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3 was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3 provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.

  9. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

    Science.gov (United States)

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

    2015-12-01

    Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

  10. RT-PCR ANALYSIS OF E2A-PBX1, TEL-AML1, BCR-ABL AND MLL-AF4 FUSION GENE TRANSCRIPTS IN B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    Iuliu-Cristian Ivanov

    2013-11-01

    Full Text Available Acute lymphoblastic leukemia represents a heterogeneous group of hematological malignancies, defined by clonal proliferation of lymphoid cells. Immunophenotyping by flow cytometry and molecular analysis for the detection of genetic anomalies are clinical standard procedures for diagnosis, sub-classification and post-therapeutic evaluation. Samples from 105 patients diagnosed with acute lymphoblastic leukemia were immunophenotyped at diagnosis and were investigated by molecular analysis in order to identify the occurrence of four fusion genes: MLL-AF4, TEL-AML-1, BCR-ABL-p190, E2A-PBX-1. There were no associations found between the immunophenotype and the presence of any fusion genes evaluated. Both methods in combination remain a prerequisite for an improved subclassification of hematological malignancies, therapeutic decision, and evaluation of treatment response.

  11. The significance of TMPRSS2-ETS fusion gene in urine in diagnosis of prostate cancer%尿沉淀细胞中TMPRSS2-ETS融合基因对诊断前列腺癌的意义

    Institute of Scientific and Technical Information of China (English)

    丁奕星; 齐隽; 马妍慧; 沈立松

    2013-01-01

    目的:检测尿沉淀细胞中TMPRSS2-ETS融合基因对诊断前列腺癌的意义.方法:将2010~2011年我院收治的经病理检查证实为前列腺癌者归为前列腺癌组,经病理检查证实为BPH者归为BPH组.收集两组患者前列腺按摩后首次尿液标本,用FISH检测其TMPRSS2-ERG、TMPRSS2-ETV1和TMPRSS2-ETV4融合基因的表达.结果:纳入本次研究的前列腺癌组和BPH组分别为51例和20例,TMPRSS2-ERG(+)的病例分别为26例(50.98%)和4例(20%),差异有显著统计学意义(P<0.05).其敏感度为50.98%,特异度为80%,假阳性率为20%,假阴性率为49.02%,阳性似然比为2.549,阴性似然比为0.613,Youden指数为0.3098.两组中均未发现有TMPRSS2-ETV1或TMPRSS2-ETV4融合基因阳性患者.结论:用FISH方法检测尿沉淀细胞TMPRSS2 ERG融合基因有助于区分前列腺癌与BPH.TMPRSS2-ETV1和TMPRSS2-ETV4融合基因在前列腺癌中出现率较低,故不推荐用于前列腺癌的诊断检测.%Objective:To investigate the significance of detecting TMRPSS2-ETS fusion gene in urine in diagnosis of prostate cancer. Methods: The patients who was treated in the department of urology in our hospital from 2010-2011 were divided into 2 groups. The prostate cancer group including the patients who was diagnosed as prostate cancer by pathology while the benign prostate hyperplasia group including the patients who was diagnosed as benign prostate hyperplasia by pathology. The first voiding urine after prostate massage of the patients in 2 groups was collected and was detected the expression of TMPRSS2-ERG, TMPRSS2-ETV1 and TMPRSS2-ETV4 fusion gene by FISH. Results: There are 51 cases in the prostate cancer group while TMPRSSW-ERG fusion gene in 26 cases(50. 98%)was positive and 20 cases in the benign prostate hyperplasia group while the fusion gene in 4 cases(20%)was positive, which has showing significant difference statistically(P<0. 05). The sensitivity of detection of TMPRSS2-ERG fusion

  12. Silencing of the PiAvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element.

    Science.gov (United States)

    Vetukuri, Ramesh R; Tian, Zhendong; Avrova, Anna O; Savenkov, Eugene I; Dixelius, Christina; Whisson, Stephen C

    2011-12-01

    Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5'-infSINEm::PiAvr3a-3' or 5'-PiAvr3a::SINEm-3' chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements.

  13. Tame Fusion

    Institute of Scientific and Technical Information of China (English)

    S.D. Scott

    2003-01-01

    The first section of this paper covers preliminaries. Essentially, the next four cover units. It is shown that a compatible nearring with DCCR is Nnilpotent if and only if every maximal right N-subgroup is a right ideal. The last five sections relate to fusion (I.e., N-groups minimal for being generated by Nsubgroups, where each is N-isomorphic to a given N-group). Right N-subgroups of a tame nearring N with DCCR, minimal for not annihilating a minimal ideal from the left, are self monogenic and N-isomorphic. That this holds for any collection of minimal ideals is significant. Here, the right N-subgroup involved is a 'fusion product' of the 'components'.

  14. Carpal Fusion

    OpenAIRE

    2012-01-01

    Carpal fusion may be seen in hereditary and nonhereditary conditions such as acrocallosal syndrome,acromegaly, Apert syndrome, arthrogryposis, Carpenter syndrome, chromosomal abnormalities, ectrodactyly-ectodermal dysplasia-cleft (EEC) syndrome, the F form of acropectorovertebral dysgenesis or the F syndrome, fetal alcohol syndrome, Holt-Oram syndrome, Leopard syndrome, multiple synostosis syndrome, oligosyndactyly syndrome, Pfeiffer-like syndrome, scleroderma, split hand and foot malformatio...

  15. Fusion rules of equivariantizations of fusion categories

    OpenAIRE

    2012-01-01

    We determine the fusion rules of the equivariantization of a fusion category $\\mathcal{C}$ under the action of a finite group $G$ in terms of the fusion rules of $\\mathcal{C}$ and group-theoretical data associated to the group action. As an application we obtain a formula for the fusion rules in an equivariantization of a pointed fusion category in terms of group-theoretical data. This entails a description of the fusion rules in any braided group-theoretical fusion category.

  16. Fusion rules of equivariantizations of fusion categories

    OpenAIRE

    Burciu, Sebastian; Natale, Sonia

    2012-01-01

    We determine the fusion rules of the equivariantization of a fusion category $\\mathcal{C}$ under the action of a finite group $G$ in terms of the fusion rules of $\\mathcal{C}$ and group-theoretical data associated to the group action. As an application we obtain a formula for the fusion rules in an equivariantization of a pointed fusion category in terms of group-theoretical data. This entails a description of the fusion rules in any braided group-theoretical fusion category.

  17. Unintended Changes in Genetically Modified Rice Expressing the Lysine-Rich Fusion Protein Gene Revealed by a Proteomics Approach

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xiang-xiang; TANG Tang; LIU Fu-xia; LU Chang-li; HU Xiao-lan; JI Li-lian; LIU Qiao-quan

    2013-01-01

    Development of new technologies for evaluating genetically modiifed (GM) crops has revealed that there are unintended insertions and expression changes in GM crops. Proifling techniques are non-targeted approaches and are capable of detecting more unintended changes in GM crops. Here, we report the application of a comparative proteomic approach to investigate the protein proifle differences between a GM rice line, which has a lysine-rich protein gene, and its non-transgenic parental line. Proteome analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrum analysis of the seeds identiifed 22 differentially expressed protein spots. Apart from a number of glutelins that were detected as targeted proteins in the GM line, the majority of the other changed proteins were involved in carbohydrate metabolism, protein synthesis and stress responses. These results indicated that the altered proteins were not associated with plant allergens or toxicity.

  18. Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto's thyroiditis

    DEFF Research Database (Denmark)

    Wirtschafter, A; Schmidt, R; Rosen, D

    1997-01-01

    -polymerase chain reaction (RT-PCR) assay, we found messenger RNA (mRNA) expression for the RET/PTC1 and RET/PTC3 oncogenes in 95% of the Hashimoto's patients studied. All Hashimoto's patients presenting without histopathologic evidence of papillary thyroid cancer showed molecular genetic evidence of cancer......Hashimoto's thyroiditis is an inflammatory disease of the thyroid gland with autoimmune etiology. Patients afflicted with Hashimoto's have a higher risk of thyroid malignancies such as papillary thyroid carcinoma. In the present study, we investigated the frequency of papillary thyroid carcinoma...... specific genes in patients diagnosed with Hashimoto's disease. The newly identified oncogenes RET/PTC1 and RET/PTC3 provide useful and specific markers of the early stages of papillary carcinoma as they are highly specific for malignant cells. Using a sensitive and specific reverse transcriptase...

  19. Response of the Gypsy Moth, Lymantria dispar to Transgenic Poplar, Populus simonii x P. nigra, Expressing Fusion Protein Gene of the Spider Insecticidal Peptide and Bt-toxin C-peptide

    OpenAIRE

    Cao, Chuan-Wang; Liu, Gui-Feng; Wang, Zhi-Ying; Yan, Shan-Chun; Ma, Ling; Yang, Chuan-Ping

    2010-01-01

    The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω?-ACTX-Ar1 sequence coding for an ω?-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The...

  20. FISH技术在儿童ALL常见融合基因检测中的应用价值%Value of fluorescence in situ hybridization in Identification of fusion gene in pediatric cases with acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    徐雪琴; 林小玲; 谢番妮; 郑昭科; 唐少华

    2013-01-01

    Objective:To discuss the role of interphase fluorescence in situ hybridization (I-FISH) in TELAML1 and BCR/ABL fusion gene detection in pediatric cases with acute lymphoblastic leukemia.Methods:Dual color I-FISH and conventional cytogenetic analysis (CCA) were combined to detect t(12;21) TEL/AML1 and t(9;22) BCR/ABL fusion gene in bone marrow mononuclear cells from 35 newly and 4 oddly pediatric ALL patients.Results:Twelve cases were found the TEL/AML1 fusion gene by FISH (30.8%).But no dubious t (12; 21) gene was detected by CCA.Four cases were found the BCR/ABL fusion gene by FISH (10.3%).Only 1 case was found dubious t (9;22) gene by CCA,the incidence was 2.5%.TEL/AML1 fusion gene was more than BCR/ABL fusion gene in pediatric ALL patients and showed lower peripheral tumor load in diagnosis.Conclusion:Dual color IFISH is more sensitive and specific than conventional,cytogenetic analysis (CCA)in the identification of TELAML1 and BCP/ABL fusion gene.So it is playing a significant role in diagnosis and therapy of pediatric cases with acute lymphoblastic leukemia.%目的:探讨双色间期荧光原位杂交(I-FISH)技术在儿童急性淋巴细胞白血病TEL/AML1和BCR/ABL融合基因检测中的应用价值.方法:联合双色间期荧光原位杂交(I-FISH)技术和常规染色体核型分析技术(CCA)对35例儿童初治ALL和4例儿童复发ALL的骨髓有核细胞进行t(12;21) TEL/AML1和t(9;22)BCR/ABL融合基因进行检测.结果:12例患几经FISH检测发现TEL/AML1融合基因,占总病例的30.8%;而CCA检测均未发现有可疑t(12;21).4例患儿经FISH检测发现BCR/ABL融合基因,占总病例的10.3%;而CCA检测发现1例可疑t(9;22),占总病例的2.5%.TEL/AML1在儿童ALL中阳性率较BCR/ABL高,该融合基因阳性的患儿病情较轻.结论:FISH技术较常规染色体核型分析技术特异性强、敏感度高,可以有效检测出TEL/AML1和BCR/ABL融合基因,从而为儿童ALL的诊断和个体化治疗方案提供重要的依据.

  1. Quantitative PCR detection of NPM/ALK fusion gene and CD30 gene expression in patients with anaplastic large cell lymphoma--residual disease monitoring and a correlation with the disease status.

    Science.gov (United States)

    Kalinova, Marketa; Krskova, Lenka; Brizova, Helena; Kabickova, Edita; Kepak, Tomas; Kodet, Roman

    2008-01-01

    Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of malignant lymphoproliferative diseases with a consistent expression of the cytokine receptor CD30. ALCL is frequently associated with a NPM/ALK fusion gene which is found in up to 75% of pediatric ALCLs. Real-time quantitative RT-PCR (RQ-RT-PCR) of NPM/ALK and CD30 gene expression was employed to analyze minimal residual disease (MRD) in 10 patients with NPM/ALK positive ALCL in 79 follow-up bone marrow (BM) and/or peripheral blood (PB) samples. In all BM samples from relapses and/or closely before a relapse, BM samples revealed NPM/ALK and CD30 positivity in at least one of the iliac BM trephines. Five out of nine relapses were preceded or were accompanied by minimally half log increased NPM/ALK levels in the BM. We found that RQ-RT-PCR of the CD30 expression is not suitable for MRD detection--only two relapses were accompanied by an increase of the CD30 level above a level which was detected in BM/PB samples from healthy individuals. RQ-RT-PCR of NPM/ALK expression is a promising and rapid approach for monitoring MRD.

  2. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1.

    OpenAIRE

    May, W A; Lessnick, S L; Braun, B S; Klemsz, M; Lewis, B. C.; Lunsford, L B; Hromas, R; Denny, C T

    1993-01-01

    EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewing's sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-1 has been shown to be a potent transforming gene, suggesting that it plays an important role in the genesis of these human tumors. We now demonstrate that EWS/FLI-1 has the characteristics of an aberrant transcription factor. Subcellular fractionation experiments localized the EWS/FLI-1 protein to the nucleus of primitive neuro...

  3. Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.

    Science.gov (United States)

    Lee, Jong Hyun; Jeong, Yeong Ran; Kim, Yeon-Gu; Lee, Gyun Min

    2017-03-07

    To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After three days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. This article is protected

  4. LDL receptor-GFP fusion proteins: new tools for the characterization of disease-causing mutations in the LDL receptor gene

    DEFF Research Database (Denmark)

    Holst, Henrik Uffe; Dagnæs-Hansen, Frederik; Corydon, Thomas Juhl;

    2001-01-01

    The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of a LDL receptor mutation (W556S) found in FH patients and characterized as transport defective....... In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-locallisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum....

  5. FUSION WORLD

    Institute of Scientific and Technical Information of China (English)

    Caroline; 黄颖(翻译)

    2009-01-01

    Fusion World”科技展示体验中心是英国设计公司MET Studio为新加坡科技研究局(A*Star)的科学工程委员会(SERC)所设计的,位于启汇城的办公地点,用于展示该委员会的精选技术作品,以吸引潜在的客户和启汇城内的学生购买群体。

  6. The TMPRSS2-ERG Gene Fusion Blocks XRCC4-Mediated Nonhomologous End-Joining Repair and Radiosensitizes Prostate Cancer Cells to PARP Inhibition.

    Science.gov (United States)

    Chatterjee, Payel; Choudhary, Gaurav S; Alswillah, Turkeyah; Xiong, Xiahui; Heston, Warren D; Magi-Galluzzi, Cristina; Zhang, Junran; Klein, Eric A; Almasan, Alexandru

    2015-08-01

    Exposure to genotoxic agents, such as ionizing radiation (IR), produces DNA damage, leading to DNA double-strand breaks (DSB); IR toxicity is augmented when the DNA repair is impaired. We reported that radiosensitization by a PARP inhibitor (PARPi) was highly prominent in prostate cancer cells expressing the TMPRSS2-ERG gene fusion protein. Here, we show that TMPRSS2-ERG blocks nonhomologous end-joining (NHEJ) DNA repair by inhibiting DNA-PKcs. VCaP cells, which harbor TMPRSS2-ERG and PC3 cells that stably express it, displayed γH2AX and 53BP1 foci constitutively, indicating persistent DNA damage that was absent if TMPRSS2-ERG was depleted by siRNA in VCaP cells. The extent of DNA damage was enhanced and associated with TMPRSS2-ERG's ability to inhibit DNA-PKcs function, as indicated by its own phosphorylation (Thr2609, Ser2056) and that of its substrate, Ser1778-53BP1. DNA-PKcs deficiency caused by TMPRSS2-ERG destabilized critical NHEJ components on chromatin. Thus, XRCC4 was not recruited to chromatin, with retention of other NHEJ core factors being reduced. DNA-PKcs autophosphorylation was restored to the level of parental cells when TMPRSS2-ERG was depleted by siRNA. Following IR, TMPRSS2-ERG-expressing PC3 cells had elevated Rad51 foci and homologous recombination (HR) activity, indicating that HR compensated for defective NHEJ in these cells, hence addressing why TMPRSS2-ERG alone did not lead to radiosensitization. However, the presence of TMPRSS2-ERG, by inhibiting NHEJ DNA repair, enhanced PARPi-mediated radiosensitization. IR in combination with PARPi resulted in enhanced DNA damage in TMPRSS2-ERG-expressing cells. Therefore, by inhibiting NHEJ, TMPRSS2-ERG provides a synthetic lethal interaction with PARPi in prostate cancer patients expressing TMPRSS2-ERG. ©2015 American Association for Cancer Research.

  7. Carpal Fusion

    Directory of Open Access Journals (Sweden)

    Jalal Jalalshokouhi*

    2012-05-01

    Full Text Available Carpal fusion may be seen in hereditary and nonhereditary conditions such as acrocallosal syndrome,acromegaly, Apert syndrome, arthrogryposis, Carpenter syndrome, chromosomal abnormalities, ectrodactyly-ectodermal dysplasia-cleft (EEC syndrome, the F form of acropectorovertebral dysgenesis or the F syndrome, fetal alcohol syndrome, Holt-Oram syndrome, Leopard syndrome, multiple synostosis syndrome, oligosyndactyly syndrome, Pfeiffer-like syndrome, scleroderma, split hand and foot malformation, Stickler syndrome, thalidomide embryopathy, Turner syndrome and many other conditions as mentioned in Rubinstein-Taybi's book. Sometimes there is no known causative disease.Diagnosis is usually made by plain X-ray during studying a syndrome or congenital disease or could be an incidental finding like our patients. Hand bone anomalies are more common in syndromes or other congenital or non-hereditary conditions, but polydactyly, syndactyly or oligodactyly and carpal fusions are interesting. X-ray is the modality of choice, but MRI and X-ray CT with multiplanar reconstructions may be used for diagnosis.

  8. Gene : CBRC-TGUT-22-0004 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-22-0004 Novel 19 A Opsins OPSP_COLLI 1e-177 85% sp|P51476|OPSP_COLLI Pinopsin (Pineal... opsin) (P-opsin) (Pineal gland-specific opsin) gb|AAB40945.1| pineal gland-specific opsin 1e-17

  9. Catalysed fusion

    CERN Document Server

    Farley, Francis

    2012-01-01

    A sizzling romance and a romp with subatomic particles at CERN. Love, discovery and adventure in the city where nations meet and beams collide. Life in a large laboratory. As always, the challenges are the same. Who leads? Who follows? Who succeeds? Who gets the credit? Who gets the women or the men? Young Jeremy arrives in CERN and joins the quest for green energy. Coping with baffling jargon and manifold dangers, he is distracted by radioactive rats, lovely ladies and an unscrupulous rival. Full of doubts and hesitations, he falls for a dazzling Danish girl, who leads him astray. His brilliant idea leads to a discovery and a new route to cold fusion. But his personal life is scrambled. Does it bring fame or failure? Tragedy or triumph?

  10. Detection of EML4-ALK fusion gene in patient with lung adenocarcinoma%检测EML4-ALK融合基因对肺腺癌患者的临床意义

    Institute of Scientific and Technical Information of China (English)

    金涛; 朱理; 赵琼; 方维嘉; 曾蕾; 彭玲; 王一青

    2013-01-01

      目的探讨检测EML4-ALK融合基因对肺腺癌患者的临床意义。方法收集2012年7至12月手术或活检肺腺癌组织标本37例,所有标本均经病理证实为腺癌,用RT-PCR方法检测EML4-ALK融合基因的表达,设计9种探针检测EML4-ALK融合基因最常见的9种类型,并随访阳性患者的治疗结果。结果37例腺癌标本中,1例检出EML4-ALK融合基因表达阳性,并经测序法证实为1型变异,检出率2.70%。为女性患者,左上肺肿块,T1N0M0,行左肺上叶肺癌根治术,术后给予克唑替尼,随访6个月无复发。结论使用RT-PCR结合测序是一种快速、简便、容易推广的EML4-ALK融合基因检测技术,可以在肺腺癌患者检测中推广使用。%Objective To detect echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma ki-nase (ALK) fusion gene in patients with non-smal cel lung cancer (NSCLC). Methods Biopsy or surgical specimens were col-lected from 37 patients with pathological y confirmed adenocarcinoma from July 2012 to Dec 2012. The expression of ELM4-ALK fusion gene was detected by RT-PCR with primers designed for 9 subtypes of the fusion gene, and the detected gene was veri-fied by sequencing. Results The expression of ELM4-ALK fusion gene was detected and verified in the surgical specimen from a 36y female patient with T1N0M0 lung adenocarcinoma with a detection rate of 2.70%. The radical resection of upper lobe of the left lung was performed and XALKORI was given one month after the surgery. No metastasis was found after 6-months fol-low-up. Conclusion The detection rate for ELM4-ALK fusion gene is similar as reported in patients with lung adenocarcinoma, and the RT-PCR combined with sequencing is a sensitive and effective method for detection of EML4-ALK fusion gene in surgi-cal or biopsy specimens of patients with lung adenocarninoma.

  11. Possible involvement of cone opsins in distinct photoresponses of intrinsically photosensitive dermal chromatophores in tilapia Oreochromis niloticus.

    Directory of Open Access Journals (Sweden)

    Shyh-Chi Chen

    Full Text Available Dermal specialized pigment cells (chromatophores are thought to be one type of extraretinal photoreceptors responsible for a wide variety of sensory tasks, including adjusting body coloration. Unlike the well-studied image-forming function in retinal photoreceptors, direct evidence characterizing the mechanism of chromatophore photoresponses is less understood, particularly at the molecular and cellular levels. In the present study, cone opsin expression was detected in tilapia caudal fin where photosensitive chromatophores exist. Single-cell RT-PCR revealed co-existence of different cone opsins within melanophores and erythrophores. By stimulating cells with six wavelengths ranging from 380 to 580 nm, we found melanophores and erythrophores showed distinct photoresponses. After exposed to light, regardless of wavelength presentation, melanophores dispersed and maintained cell shape in an expansion stage by shuttling pigment granules. Conversely, erythrophores aggregated or dispersed pigment granules when exposed to short- or middle/long-wavelength light, respectively. These results suggest that diverse molecular mechanisms and light-detecting strategies may be employed by different types of tilapia chromatophores, which are instrumental in pigment pattern formation.

  12. Correcting mitochondrial fusion by manipulating mitofusin conformations

    Science.gov (United States)

    Franco, Antonietta; Kitsis, Richard N.; Fleischer, Julie A.; Gavathiotis, Evripidis; Kornfeld, Opher S.; Gong, Guohua; Biris, Nikolaos; Benz, Ann; Qvit, Nir; Donnelly, Sara K; Chen, Yun; Mennerick, Steven; Hodgson, Louis; Mochly-Rosen, Daria; Dorn, Gerald W

    2017-01-01

    Summary Mitochondria are dynamic organelles, remodeling and exchanging contents during cyclic fusion and fission. Genetic mutations of mitofusin (Mfn) 2 interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition, Charcot Marie Tooth disease type 2A (CMT2A). It has not been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is incompletely understood. Here we show that mitofusins adopt either a fusion-constrained or fusion-permissive molecular conformation directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated by targeting these conformational transitions. Based on this model we engineered a cell-permeant minipeptide to destabilize fusion-constrained mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons harboring CMT2A gene defects. The relationship between mitofusin conformational plasticity and mitochondrial dynamism uncovers a central mechanism regulating mitochondrial fusion whose manipulation can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics. PMID:27775718

  13. 急性早幼粒细胞白血病PML/RARα融合基因检测的临床意义%Clinical significance of detecting PML/RARα fusion gene in acute promyelocytic leukemia

    Institute of Scientific and Technical Information of China (English)

    刘小宁; 李元媛; 甘宜敏; 陈凤丽; 衡春; 于亮

    2016-01-01

    目的:探讨荧光原位杂交技术(FISH)检测PML/RARα融合基因在急性早幼粒细胞白血病(APL)诊断中的应用价值。方法应用常规细胞遗传学分析28例APL患者的染色体核型,同时用FISH法检测PML/RARα融合基因。结果28例初诊的APL患者254例常规核型分析检出t(15;17)(q22;q12);2例患者核型正常;另1例未检出t(15;17),核型分析结果为涉及15和17号染色体的复杂异常;FISH检测所有病例均存在PML/RARα融合基因。结论 FISH法行PML/RARα融合基因检测特异性和敏感性好,是诊断APL的可靠方法。%Objective To explore the application value of detecting PML/RARα fusion gene in the diagnosis of acute promye-locytic leukemia(APL) with fluorescence in situ hybridization(FISH). Methods The chromosome karyotype of 28 cases of APL pa-tients was analyzed with the conventional cytogenetic analysis method and the PML/RARα fusion gene was detected with FISH method. Results Among 28 first diagnosed APL patients,the t (15;17)(q22;q12) was detected in 24 25 patients with the conven-tional karyotype analysis;Two patients were detected to be normal karyotype;And the t (15;17) was not detected in one case. The karyotype analysis results showed the complex abnormalities involving in the chromosome 15 and 17;The PML/RARα fusion gene was detected to exist in all cases with FISH detection. Conclusion The PML/RARα fusion gene detection with FISH method is a reliable way for the diagnosis of APL with fine specificity and sensitivity.

  14. 原发性胃淋巴瘤凋亡抑制蛋白2-黏膜相关淋巴瘤转位基因1融合基因检测的意义%The clinical value of apoptosis protein 2-mucosa associated lymphoid tissue lymphoma translocation gene 1 fusion gene detection in tissue of primary gastric lymphoma

    Institute of Scientific and Technical Information of China (English)

    许国强; 陈妙辉; 陈洪潭; 单国栋; 胡凤玲; 杨铭

    2011-01-01

    Objective To explore the feasibility of inhibitor of apoptosis protein 2-mucosa associated lymphoid tissue lymphoma translocation gene 1 (API2-MALT1) fusion gene detection in endoscopic biopsy tissue of primary gastric lymphoma (PGL), and to study the expression of this fusion gene in PGL and its clinical values in diagnosis and treatment of PGL.Methods A total of 32 suspicious PGL patients underwent endoscopic ultrasonography (EUS) examination and mucosal biopsy.The biopsy specimens were conducted histopathology examination, immunohistochemistry detection and real time RT-PCR for API2-MALT1 fusion gene expression.The expression of API2MALT1 fusion gene in clearly diagnosed PGL and its relation with PGL diagnosis, classification and treatment were analyzed and summarized.Results A total of 14 cases of 32 suspicious PGL patients were diagnosed as PGL by histopathology and immunohistochemistry examination, which including 11 cases of gastric mucosa-associated lymphoid tissue (MALT) lymphoma and 3 cases of gastric diffuse large B-cell lymphoma (DLBCL).There were 5 API2-MALT1 fusion gene positive cases, which were all MALT lymphoma, about 5 out of total 11 MALT lymphoma patients.API2-MALT1 fusion gene expression was negative in all 3 gastric DLBCL cases.The depth of lesion invasion and lymph nodes metastasis were more severe in API2-MALT1 fusion gene negative group than those of positive group.There were 2 of 5 API2-MALT1 fusion gene positive cases were Hp positive, and 5 of 9 negative cases were Hp positive.The anti-Hp therapy in 5 API2-MALT1 fusion gene positive cases was ineffective,however, chemotherapy was effective.In negative group, 2 of 5 Hp positive cases was complete remission and 4 Hp negative cases were ineffective with anti-Hp therapy.Concltsions API2-MALT1 fusion gene is common genetic abnormality in gastric MALT lymphoma.The detection of this fusion gene expression in endoscopic biopsy specimen by real time RT-PCR is clinically practical, and the

  15. An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli.

    Science.gov (United States)

    Macián, F; Pérez-Roger, I; Armengod, M E

    1994-07-22

    We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.

  16. Photoreceptors for a light biotransducer: a comparative study of the electrical responses of two (type-1)-opsins

    CERN Document Server

    Alfinito, E; Reggiani, L; Lee, K

    2013-01-01

    The increasing interest in photoactivated proteins as natural replacement of standard inorganic materials in photocells drives to the compared analysis of bacteriorhodopsin and proteorhodopsin, two widely diffused proteins belonging to the family of \\textit{type-1} opsins. These proteins share similar behaviours but exhibit relevant differences in the sequential chain of the amino acids constituting their tertiary structure. The use of an impedance network analogue to model the protein main features provides a microscopic interpretation of a set of experiments on their photoconductance properties. In particular, this model links the protein electrical responses to the tertiary structure and to the interactions among neighbouring amino acids. The same model is also used to predict the small-signal response in terms of the Nyquist plot. Interesting enough, these rhodopsins are found to behave like a wide gap semiconductor with intrinsic conductivities of the order of $10^{-7}$ S/cm.

  17. Cold nuclear fusion

    National Research Council Canada - National Science Library

    Huang Zhenqiang Huang Yuxiang

    2013-01-01

    ...... And with a magnetic moment of light nuclei controlled cold nuclear collide fusion, belongs to the nuclear energy research and development in the field of applied technology "cold nuclear collide fusion...

  18. Cold fusion research

    Energy Technology Data Exchange (ETDEWEB)

    None

    1989-11-01

    I am pleased to forward to you the Final Report of the Cold Fusion Panel. This report reviews the current status of cold fusion and includes major chapters on Calorimetry and Excess Heat, Fusion Products and Materials Characterization. In addition, the report makes a number of conclusions and recommendations, as requested by the Secretary of Energy.

  19. The recurrent chromosomal translocation t(12;18)(q14~15;q12~21) causes the fusion gene HMGA2-SETBP1 and HMGA2 expression in lipoma and osteochondrolipoma.

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Bjerkehagen, Bodil; Lobmaier, Ingvild; Heim, Sverre

    2015-09-01

    Lipomas are the most common soft tissue tumors in adults. They often carry chromosome aberrations involving 12q13~15 leading to rearrangements of the HMGA2 gene in 12q14.3, with breakpoints occurring within or outside of the gene. Here, we present eleven lipomas and one osteochondrolipoma with a novel recurrent chromosome aberration, t(12;18)(q14~15;q12~21). Molecular studies on eight of the tumors showed that full-length HMGA2 transcript was expressed in three and a chimeric HMGA2 transcript in five of them. In three lipomas and in the osteochondrolipoma, exons 1-3 of HMGA2 were fused to a sequence of SETBP1 on 18q12.3 or an intragenic sequence from 18q12.3 circa 10 kbp distal to SETBP1. In another lipoma, exons 1-4 of HMGA2 were fused to an intronic sequence of GRIP1 which maps to chromosome band 12q14.3, distal to HMGA2. The ensuing HMGA2 fusion transcripts code for putative proteins which contain amino acid residues of HMGA2 corresponding to exons 1-3 (or exons 1-4 in one case) followed by amino acid residues corresponding to the fused sequences. Thus, the pattern is similar to the rearrangements of HMGA2 found in other lipomas, i.e., disruption of the HMGA2 locus leaves intact exons 1-3 which encode the AT-hooks domains and separates them from the 3'-terminal part of the gene. The fact that the examined osteochondrolipoma had a t(12;18) and a HMGA2-SETBP1 fusion identical to the findings in the much more common ordinary lipomas, underscores the close developmental relationship between the two tumor types.

  20. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

    2015-05-15

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

  1. Protein-protein fusion catalyzed by sortase A.

    Science.gov (United States)

    Levary, David A; Parthasarathy, Ranganath; Boder, Eric T; Ackerman, Margaret E

    2011-04-06

    Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  2. Protein-protein fusion catalyzed by sortase A.

    Directory of Open Access Journals (Sweden)

    David A Levary

    Full Text Available Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  3. Fusion expression of mutated cecropin CMIV in E. coli

    Institute of Scientific and Technical Information of China (English)

    谢维; 邱奇峰; 董雪吟; 华子春; 徐贤秀

    1997-01-01

    A cDNA coding mutated cecropin CMIV from Bombyx mori was synthesized according to its amino acid sequense using E .coli biased codons .The gene was cloned into the fusion expression vector pEZZ318 and was expressed in E .coli HB101.The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product .The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.

  4. Full-Quantum chemical calculation of the absorption maximum of bacteriorhodopsin: a comprehensive analysis of the amino acid residues contributing to the opsin shift

    Science.gov (United States)

    Hayashi, Tomohiko; Matsuura, Azuma; Sato, Hiroyuki; Sakurai, Minoru

    2012-01-01

    Herein, the absorption maximum of bacteriorhodopsin (bR) is calculated using our recently developed method in which the whole protein can be treated quantum mechanically at the level of INDO/S-CIS//ONIOM (B3LYP/6-31G(d,p): AMBER). The full quantum mechanical calculation is shown to reproduce the so-called opsin shift of bR with an error of less than 0.04 eV. We also apply the same calculation for 226 different bR mutants, each of which was constructed by replacing any one of the amino acid residues of the wild-type bR with Gly. This substitution makes it possible to elucidate the extent to which each amino acid contributes to the opsin shift and to estimate the inter-residue synergistic effect. It was found that one of the most important contributions to the opsin shift is the electron transfer from Tyr185 to the chromophore upon excitation. We also indicate that some aromatic (Trp86, Trp182) and polar (Ser141, Thr142) residues, located in the vicinity of the retinal polyene chain and the β-ionone ring, respectively, play an important role in compensating for the large blue-shift induced by both the counterion residues (Asp85, Asp212) and an internal water molecule (W402) located near the Schiff base linkage. In particular, the effect of Trp86 is comparable to that of Tyr185. In addition, Ser141 and Thr142 were found to contribute to an increase in the dipole moment of bR in the excited state. Finally, we provide a complete energy diagram for the opsin shift together with the contribution of the chromophore-protein steric interaction. PMID:27493528

  5. Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

    Directory of Open Access Journals (Sweden)

    Projan Steve

    2003-09-01

    Full Text Available Abstract Background Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H, is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost in the amplified library. Results Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. Conclusion Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the

  6. A New Target in Non-small Cell Lung Cancer: EML4-ALK Fusion Gene%非小细胞肺癌治疗的新靶点:EML4-ALK融合基因

    Institute of Scientific and Technical Information of China (English)

    王慧娟; 袁静

    2011-01-01

    It was only 3 years ago that the fusion gene between echinoderm microtubule-associated protein-like4 (EML4) and anaplastic lymphoma kinase (ALK) has been identified in a subset of non-small cell lung cancer (NSCLC).EML4-ALK is most often detected in never smokers with lung adenocarcinoma and has unique pathologic features.EML4ALK fusion gene is oncogenic, which could be suppressed by ALK-inhibitor through blocking the downstream signaling passway of EML4-ALK.This review will focus on the molecular structure, function, biology, detection method and the diagnostic and therapeutic meaning of EML4-ALK of lung cancer.%3年前棘皮动物微管相关类蛋白4 (echinoderm microtubule-associated protein-like4,EML4)与间变性淋巴瘤激酶 (anaplastic lymphoma kinase,ALK)融合基因被发现存在于部分非小细胞肺癌(non-small cell lung cancer,NSCLC)中.该融合基因常见于不吸烟的肺腺癌患者,有其独特的病理学特征,可以诱导肿瘤生成.ALK抑制剂能够作用于该基因的下游信号传导通路并拮抗其促肿瘤生成活性.本文旨在介绍EML4-ALK基因的结构、功能、生物学特征、检测方法 及其在肺癌诊断治疗中的意义.

  7. Response of the gypsy moth, Lymantria dispar to transgenic poplar, Populus simonii x P. nigra, expressing fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide.

    Science.gov (United States)

    Cao, Chuan-Wang; Liu, Gui-Feng; Wang, Zhi-Ying; Yan, Shan-Chun; Ma, Ling; Yang, Chuan-Ping

    2010-01-01

    The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω-ACTX-Ar1 sequence coding for an ω-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The growth and development of L. dispar were significantly affected by continually feeding on the transgenic poplar, with the larval instars displaying significantly shorter developmental times than those fed on nontransgenic poplar, but pupation was delayed. Mortality was higher in populations fed transgenic poplar leaves, than for larvae fed nontransgenic poplar leaves. The cumulative mortality during all stages of larvae fed transgenic leaves was 92% compared to 16.7% of larvae on nontransgenic leaves. The highest mortality observed was 71.7% in the last larval instar stage. A two-choice test showed that fifth-instar larvae preferred to feed on nontransgenic leaves at a ratio of 1:1.4. Feeding on transgenic leaves had highly significant negative effects on relative growth of larvae, and the efficiency of conversion of ingested and digested food. Activity of major midgut proteinases was measured using substrates TAME and BTEE showed significant increases in tryptase and chymotrypsinlike activity (9.2- and 9.0-fold, respectively) in fifth-instar larvae fed on transgenic leaves over control. These results suggest transgenic poplar is resistant to L. dispar, and the mature L. dispar may be weakened by the transgenic plants due to Bt protoxins activated by elevated major midgut proteinase activity. The new transgenic poplar expressing fusion protein genes of Bt and a new spider insecticidal peptide are good candidates for managing gypsy moth.

  8. Economics of fusion research

    Energy Technology Data Exchange (ETDEWEB)

    None, None

    1977-10-15

    This report provides the results of a study of methods of economic analysis applied to the evaluation of fusion research. The study recognizes that a hierarchy of economic analyses of research programs exists: standard benefit-cost analysis, expected value of R and D information, and expected utility analysis. It is shown that standard benefit-cost analysis, as commonly applied to research programs, is inadequate for the evaluation of a high technology research effort such as fusion research. A methodology for performing an expected value analysis is developed and demonstrated and an overview of an approach to perform an expected utility analysis of fusion research is presented. In addition, a potential benefit of fusion research, not previously identified, is discussed and rough estimates of its magnitude are presented. This benefit deals with the effect of a fusion research program on optimal fossil fuel consumption patterns. The results of this study indicate that it is both appropriate and possible to perform an expected value analysis of fusion research in order to assess the economics of a fusion research program. The results indicate further that the major area of benefits of fusion research is likely due to the impact of a fusion research program on optimal fossil fuel consumption patterns and it is recommended that this benefit be included in future assessments of fusion research economics.

  9. Materials research for fusion

    Science.gov (United States)

    Knaster, J.; Moeslang, A.; Muroga, T.

    2016-05-01

    Fusion materials research started in the early 1970s following the observation of the degradation of irradiated materials used in the first commercial fission reactors. The technological challenges of fusion energy are intimately linked with the availability of suitable materials capable of reliably withstanding the extremely severe operational conditions of fusion reactors. Although fission and fusion materials exhibit common features, fusion materials research is broader. The harder mono-energetic spectrum associated with the deuterium-tritium fusion neutrons (14.1 MeV compared to hydrogen and helium as transmutation products that might lead to a (at present undetermined) degradation of structural materials after a few years of operation. Overcoming the historical lack of a fusion-relevant neutron source for materials testing is an essential pending step in fusion roadmaps. Structural materials development, together with research on functional materials capable of sustaining unprecedented power densities during plasma operation in a fusion reactor, have been the subject of decades of worldwide research efforts underpinning the present maturity of the fusion materials research programme.

  10. Detection of BCR/ABL Fusion Gene by Fluorescence In Situ Hybridization and Its Clinical Application%荧光原位杂交技术检测BCR/ABL融合基因的临床应用

    Institute of Scientific and Technical Information of China (English)

    周瑞莲; 莫耀禧; 蓝梅; 林金盈

    2011-01-01

    This study was aimed to investigate the clinical value of detecting BCR/ABL fusion gene by fluorescence in situ hybridization (FISH). The conventional cytogenetic test and detection of BCR/ABL fusion gene by FISH for bone marrow of patients with newly diagnosed chronic myeloproliferative disease or myelodysplastic and myeloproliferative disorders, acute lymphocytic leukemia and chronic myelogeneous leukemia (CML) after allogeneic hematopoietic stem cell transplantation were carried out. The results showed that ( 1 ) out of 46 newly diagnosed as chronic myeloproliferative dsease or myelodysplastic and myeloproliferative disorders, 22 cases were diagnosed as CML, the FISH detection showed all positive (100% ), while cytogenetic test showed 86.4% (19/22) positive, in the other 24 patients who were diagnosed as other chronic myeloproliferative disease or myelodysplastic and myeloproliferative disorders, BCR/ABL fusion gene all were be detected as regative 100% by FISH, while the cytogenetic test of bone marrow in 3 cases supported the diagnosis of CML, and the diagnosis of myelodysplastic disorder in 1 case; (2) in 3 out of 7 acute lymphocytic leukemia cases the BCR/ABL fusion gene could not be detected by FISH; (3) the BCR/ABL fusion gene could be detected by FISH in 2 cases of CML received allogeneic hematopoietic stem cell transplantation, with abnormal threshold 6.5% and 1. 2% respectively. It is concluded that the detection of BCR/ABL fusion gene by FISH is sensitive and reliable, which is very important for the diagnosis and differential dignosis of chronic myeloproliferative disorders, myelodysplastic and myeloproliferative disease, as well as definite diagnosis of Ph* acute lymphoblastic leukemia. This method also has an important significance for monitor of minimal residual disease in CML patients received allogeneic hematopoietic stem cell transplantation.%本研究探讨应用荧光原位杂交技术( FISH)检测BCR/ABL融合基因在临床中的应用价

  11. Cell fusion in tumor progression: the isolation of cell fusion products by physical methods

    Directory of Open Access Journals (Sweden)

    Vincitorio Massimo

    2011-09-01

    Full Text Available Abstract Background Cell fusion induced by polyethylene glycol (PEG is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks. Results Here we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia. Conclusions In the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

  12. Promoter hypermethylation of the retinoic acid receptor beta2 gene is frequent in acute myeloid leukaemia and associated with the presence of CBFβ-MYH11 fusion transcripts

    DEFF Research Database (Denmark)

    Rethmeier, Anita; Aggerholm, Anni; Olesen, Lene Hyldahl;

    2006-01-01

    was unmethylated in 10/10 bone marrow and 7/7 blood samples from healthy individuals, the gene was hypermethylated in 43% of the AML patients. The RARbeta2 degree of promoter methylation differed between and within individuals, and the mRNA transcription levels of the gene varied inter-individually by a factor...

  13. Non-Invasive Gene Therapy of Experimental Parkinson’s Disease

    Science.gov (United States)

    2006-09-01

    predicted by the “ Rule of 5” (Lipinski et al., 1997). The adverse effect of molecular weight on membrane perme- ation is not observed if the molecular...for the broad spectrum of opsin pro- moter driven gene expression in the eye is that the other ocu- lar structures are embryologically related to the...ciliary body is embryologically related to the photoreceptor cells of the retina. Transfection of iris and ciliary body with the Crx homeobox gene

  14. Novel ZEB2-BCL11B Fusion Gene Identified by RNA-Sequencing in Acute Myeloid Leukemia with t(2;14(q22;q32.

    Directory of Open Access Journals (Sweden)

    Synne Torkildsen

    Full Text Available RNA-sequencing of a case of acute myeloid leukemia with the bone marrow karyotype 46,XY,t(2;14(q22;q32[5]/47,XY,idem,+?4,del(6(q13q21[cp6]/46,XY[4] showed that the t(2;14 generated a ZEB2-BCL11B chimera in which exon 2 of ZEB2 (nucleotide 595 in the sequence with accession number NM_014795.3 was fused to exon 2 of BCL11B (nucleotide 554 in the sequence with accession number NM_022898.2. RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript. All functional domains of BCL11B are retained in the chimeric protein. Abnormal expression of BCL11B coding regions subjected to control by the ZEB2 promoter seems to be the leukemogenic mechanism behind the translocation.

  15. Muon Catalyzed Fusion

    Science.gov (United States)

    Armour, Edward A.G.

    2007-01-01

    Muon catalyzed fusion is a process in which a negatively charged muon combines with two nuclei of isotopes of hydrogen, e.g, a proton and a deuteron or a deuteron and a triton, to form a muonic molecular ion in which the binding is so tight that nuclear fusion occurs. The muon is normally released after fusion has taken place and so can catalyze further fusions. As the muon has a mean lifetime of 2.2 microseconds, this is the maximum period over which a muon can participate in this process. This article gives an outline of the history of muon catalyzed fusion from 1947, when it was first realised that such a process might occur, to the present day. It includes a description of the contribution that Drachrnan has made to the theory of muon catalyzed fusion and the influence this has had on the author's research.

  16. 检测循环前列腺癌细胞中TMPRSS2:ERG融合基因%Detection of TMPRSS2:ERG fusion gene in circulating prostate cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xueying Mao; Dan Berney; David M. Prowse; Yong-Jie Lu; Greg Shaw; Sharon Y. James; Patraicia Purkis; Sakunthala C. Kudahetti; Theodora Tsigani; Saname Kia; Bryan D. Young; R. Tim D. Oliver

    2008-01-01

    目的:研究前列腺癌病人的循环癌细胞(CTC)中TMPRSS2:ERG融合基因的存在及其与肿瘤转移之间的潜在关系.方法:利用RT-PCR,在27例前列腺切除术中得到的前列腺癌活检标本中检测TMPRSS2:ERG和TMPRSS2:ETV1转录子出现的频率,在15名晚期雄激素非依赖病人的循环癌细胞中检测TMPRSS2:ERGG转录子出现的频率.利用荧光原位杂交技术(FISH)分析10个CTC样本(取自15个CTC样本)中导致TMPRSS2:ERG融合的ERG基因组截短情况.结果:在44%的样本中发现了TMPRSS2:ERGG转录子,但是没有检测到TMPRSS2:ETV1转录子的表达.FISH分析结果显示在10例CTC样本的6例中发现染色体重组影响了ERG基因,包括一例在原癌位置上发生了TMPRSS2:ERG融合.可是,在15例CTC样本中没有检测到TMPRSS2:ERG转录子,包括用FISH检测的10例.结论:虽然需要进一步研究来确认TMPRSS2:ERG融合与前列腺癌转移之间的关系,但是通过FISH分析ERG基因基因组截短是一种有效的监测CTC的出现和前列腺癌潜在转移的方法.%Aim: To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. Methods: We analyzed the frequency of TMPRSS2:ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2:ERG fusion in 10 of the 15 CTC samples. Results: TMPRSS2:ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2:ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the

  17. [Sequence analysis of the matrix protein and fusion protein genes of a field peste des petits ruminants virus strain from Tibet, China].

    Science.gov (United States)

    Bao, Jing-Yue; Zhao, Wen-Ji; Wang, Zhi-Liang; Li, Lin; Wu, Guo-Zhen; Wu, Xiao-Dong; Liu, Chun-Ju; Wang, Jun-Wei; Liu, Yu-Tian; Li, Jin-Ming; Wang, Ying-Li

    2010-07-01

    The nucleotide sequences of M and F genes from a field strain of peste des petits ruminants virus (PPRV) ("China/Tib/Gej/07-30") was firstly determined. The M gene was 1 483 nucleotides in length with a single open reading frame (ORF), encoding a protein of 335 amino acids. The F gene was 2411 nucleotides in length, encoding a protein of 546 amino acids. The resulting nucleotide sequence and the deduced amino acid sequences were compared with the homologous regions of other PPRV isolates. The nucleotide sequences of M and F genes of the "China/Tib/Gej/07-30" was 92.4%-97.7% and 85.5%-96.1% identical to other PPRV isolates, respectively, while a homology of 97.0%-98.2% and 94.3%-98.2% could be observed at the amino acids level respectively. Several sequence motifs in the M and F genes had been identified on the basis of conservation in the PPRVs and the morbilliviruses. The 3' untranslated region of M gene was 443 nucleotides in length with 82.4%-93.5% identical to other PPRV isolates. The 5' untranslated region of F gene was 634 nucleotides in length with 76.2%-91.7% identical to other PPRV isolates.

  18. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Liu, Yan-Hong [Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin, Heilongjiang 150081 (China); Li, Yan; Wang, Jia-Ye [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Hattori, Toshio [Department of Emerging Infectious Diseases, Division of Internal Medicine, Graduate School of Medicine, Tohoku University, Sendai 9808574 (Japan); Ling, Hong, E-mail: lingh@ems.hrbmu.edu.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Department of Parasitology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China); Zhang, Feng-Min, E-mail: fengminzhang@yahoo.com.cn [Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081 (China); Key Lab of Heilongjiang Province for Infection and Immunity, Key Lab of Heilongjiang Province Education Bureau for Etiology, Harbin, Heilongjiang 150081 (China)

    2010-01-22

    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

  19. Magnetic fusion technology

    CERN Document Server

    Dolan, Thomas J

    2014-01-01

    Magnetic Fusion Technology describes the technologies that are required for successful development of nuclear fusion power plants using strong magnetic fields. These technologies include: ? magnet systems, ? plasma heating systems, ? control systems, ? energy conversion systems, ? advanced materials development, ? vacuum systems, ? cryogenic systems, ? plasma diagnostics, ? safety systems, and ? power plant design studies. Magnetic Fusion Technology will be useful to students and to specialists working in energy research.

  20. Fusion research principles

    CERN Document Server

    Dolan, Thomas James

    2013-01-01

    Fusion Research, Volume I: Principles provides a general description of the methods and problems of fusion research. The book contains three main parts: Principles, Experiments, and Technology. The Principles part describes the conditions necessary for a fusion reaction, as well as the fundamentals of plasma confinement, heating, and diagnostics. The Experiments part details about forty plasma confinement schemes and experiments. The last part explores various engineering problems associated with reactor design, vacuum and magnet systems, materials, plasma purity, fueling, blankets, neutronics

  1. Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

    National Research Council Canada - National Science Library

    Schnitzler, Christine E; Pang, Kevin; Powers, Meghan L; Reitzel, Adam M; Ryan, Joseph F; Simmons, David; Tada, Takashi; Park, Morgan; Gupta, Jyoti; Brooks, Shelise Y; Blakesley, Robert W; Yokoyama, Shozo; Haddock, Steven Hd; Martindale, Mark Q; Baxevanis, Andreas D

    2012-01-01

    ...). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism...

  2. Magnetic fusion reactor economics

    Energy Technology Data Exchange (ETDEWEB)

    Krakowski, R.A.

    1995-12-01

    An almost primordial trend in the conversion and use of energy is an increased complexity and cost of conversion systems designed to utilize cheaper and more-abundant fuels; this trend is exemplified by the progression fossil fission {yields} fusion. The present projections of the latter indicate that capital costs of the fusion ``burner`` far exceed any commensurate savings associated with the cheapest and most-abundant of fuels. These projections suggest competitive fusion power only if internal costs associate with the use of fossil or fission fuels emerge to make them either uneconomic, unacceptable, or both with respect to expensive fusion systems. This ``implementation-by-default`` plan for fusion is re-examined by identifying in general terms fusion power-plant embodiments that might compete favorably under conditions where internal costs (both economic and environmental) of fossil and/or fission are not as great as is needed to justify the contemporary vision for fusion power. Competitive fusion power in this context will require a significant broadening of an overly focused program to explore the physics and simbiotic technologies leading to more compact, simplified, and efficient plasma-confinement configurations that reside at the heart of an attractive fusion power plant.

  3. Frontiers in fusion research

    CERN Document Server

    Kikuchi, Mitsuru

    2011-01-01

    Frontiers in Fusion Research provides a systematic overview of the latest physical principles of fusion and plasma confinement. It is primarily devoted to the principle of magnetic plasma confinement, that has been systematized through 50 years of fusion research. Frontiers in Fusion Research begins with an introduction to the study of plasma, discussing the astronomical birth of hydrogen energy and the beginnings of human attempts to harness the Sun's energy for use on Earth. It moves on to chapters that cover a variety of topics such as: * charged particle motion, * plasma kinetic theory, *

  4. Magnetic-confinement fusion

    Science.gov (United States)

    Ongena, J.; Koch, R.; Wolf, R.; Zohm, H.

    2016-05-01

    Our modern society requires environmentally friendly solutions for energy production. Energy can be released not only from the fission of heavy nuclei but also from the fusion of light nuclei. Nuclear fusion is an important option for a clean and safe solution for our long-term energy needs. The extremely high temperatures required for the fusion reaction are routinely realized in several magnetic-fusion machines. Since the early 1990s, up to 16 MW of fusion power has been released in pulses of a few seconds, corresponding to a power multiplication close to break-even. Our understanding of the very complex behaviour of a magnetized plasma at temperatures between 150 and 200 million °C surrounded by cold walls has also advanced substantially. This steady progress has resulted in the construction of ITER, a fusion device with a planned fusion power output of 500 MW in pulses of 400 s. ITER should provide answers to remaining important questions on the integration of physics and technology, through a full-size demonstration of a tenfold power multiplication, and on nuclear safety aspects. Here we review the basic physics underlying magnetic fusion: past achievements, present efforts and the prospects for future production of electrical energy. We also discuss questions related to the safety, waste management and decommissioning of a future fusion power plant.

  5. Fusion of Nonionic Vesicles

    DEFF Research Database (Denmark)

    Bulut, Sanja; Oskolkova, M. Z.; Schweins, R.

    2010-01-01

    We present an experimental study of vesicle fusion using light and neutron scattering to monitor fusion events. Vesicles are reproducibly formed with an extrusion procedure using an single amphiphile triethylene glycol mono-n-decyl ether in water. They show long-term stability for temperatures...... around 20 C, but at temperatures above 26 C we observe an increase in the scattered intensity due to fusion. The system is unusually well suited for the study of basic mechanisms of vesicle fusion. The vesicles are flexible with a bending rigidity of only a few k(H)T. The monolayer spontaneous curvature...

  6. Short-wavelength sensitive opsin (SWS1 as a new marker for vertebrate phylogenetics

    Directory of Open Access Journals (Sweden)

    Müller Johannes

    2006-11-01

    Full Text Available Abstract Background Vertebrate SWS1 visual pigments mediate visual transduction in response to light at short wavelengths. Due to their importance in vision, SWS1 genes have been isolated from a surprisingly wide range of vertebrates, including lampreys, teleosts, amphibians, reptiles, birds, and mammals. The SWS1 genes exhibit many of the characteristics of genes typically targeted for phylogenetic analyses. This study investigates both the utility of SWS1 as a marker for inferring vertebrate phylogenetic relationships, and the characteristics of the gene that contribute to its phylogenetic utility. Results Phylogenetic analyses of vertebrate SWS1 genes produced topologies that were remarkably congruent with generally accepted hypotheses of vertebrate evolution at both higher and lower taxonomic levels. The few exceptions were generally associated with areas of poor taxonomic sampling, or relationships that have been difficult to resolve using other molecular markers. The SWS1 data set was characterized by a substantial amount of among-site rate variation, and a relatively unskewed substitution rate matrix, even when the data were partitioned into different codon sites and individual taxonomic groups. Although there were nucleotide biases in some groups at third positions, these biases were not convergent across different taxonomic groups. Conclusion Our results suggest that SWS1 may be a good marker for vertebrate phylogenetics due to the variable yet consistent patterns of sequence evolution exhibited across fairly wide taxonomic groups. This may result from constraints imposed by the functional role of SWS1 pigments in visual transduction.

  7. Gene therapy rescues cone structure and function in the 3-month-old rd12 mouse: a model for midcourse RPE65 leber congenital amaurosis.

    Science.gov (United States)

    Li, Xia; Li, Wensheng; Dai, Xufeng; Kong, Fansheng; Zheng, Qinxiang; Zhou, Xiangtian; Lü, Fan; Chang, Bo; Rohrer, Bärbel; Hauswirth, William W; Qu, Jia; Pang, Ji-jing

    2011-01-01

    RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin-specific antibodies. Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin-positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin-positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials.

  8. Brains, genes, and primates.

    Science.gov (United States)

    Izpisua Belmonte, Juan Carlos; Callaway, Edward M; Caddick, Sarah J; Churchland, Patricia; Feng, Guoping; Homanics, Gregg E; Lee, Kuo-Fen; Leopold, David A; Miller, Cory T; Mitchell, Jude F; Mitalipov, Shoukhrat; Moutri, Alysson R; Movshon, J Anthony; Okano, Hideyuki; Reynolds, John H; Ringach, Dario; Sejnowski, Terrence J; Silva, Afonso C; Strick, Peter L; Wu, Jun; Zhang, Feng

    2015-05-06

    One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators, and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive, and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward.

  9. Estimating neural background input with controlled and fast perturbations: A bandwidth comparison between inhibitory opsins and neural circuits

    Directory of Open Access Journals (Sweden)

    David Eriksson

    2016-08-01

    Full Text Available To test the importance of a certain cell type or brain area it is common to make a lack of function experiment in which the neuronal population of interest is inhibited. Here we review physiological and methodological constraints for making controlled perturbations using the corticothalamic circuit as an example. The brain with its many types of cells and rich interconnectivity offers many paths through which a perturbation can spread within a short time. To understand the side effects of the perturbation one should record from those paths. We find that ephaptic effects, gap-junctions, and fast chemical synapses are so fast that they can react to the perturbation during the few milliseconds it takes for an opsin to change the membrane potential. The slow chemical synapses, astrocytes, extracellular ions and vascular signals, will continue to give their physiological input for around 20 milliseconds before they also react to the perturbation. Although we show that some pathways can react within milliseconds the strength/speed reported in this review should be seen as an upper bound since we have omitted how polysynaptic signals are attenuated. Thus the number of additional recordings that has to be made to control for the perturbation side effects is expected to be fewer than proposed here. To summarize, the reviewed literature not only suggests that it is possible to make controlled lack of function experiments, but, it also suggests that such a lack of function experiment can be used to measure the context of local neural computations.

  10. Gene ranking and signaling pathway of schizophrenia susceptible genes based on data fusion arithmetic%基于数据融合算法的精神分裂症易感基因排序及其信号通路研究

    Institute of Scientific and Technical Information of China (English)

    杨铠冰; 冀燃; 王美琴; 张敏; 张大保

    2015-01-01

    目的:利用已有的研究结果,采用数据融合分析方法,对与已知的精神分裂症易感基因有密切关系的候选基因进行统计分析,得出基因排序及其信号通路的结果,以此证明数据融合算法能够有效筛选精神分裂症易感基因。方法根据已知的与精神分裂症相关的基因,利用 Endeavour工具,通过数据融合算法将候选基因排序,并用 DAVID 数据库对基因排序结果进行功能注释和富集分析。结果利用数据融合算法获得的排序较高的基因与已有研究进行比较,证实排名第二的 NRG3和排名第三的AKT1与精神分裂症有密切联系。其他排名较高的基因可以作为潜在的疾病易感基因,为精神分裂症的研究提供参考。结论数据融合算法能够准确评价候选基因与精神分裂症的联系,使用该方法能有效地对精神分裂症易感基因进行筛选和判定。%Objective Based on previous studies,this paper applies data fusion arithmetic to extract the potential susceptible genes and signaling pathway related to schizophrenia, which proves that data fusion algorithm is an effective method to filter schizophrenia susceptible genes. Methods By making use of Endeavour tool which is based on data fusion method,the candidate genes were ranked according to their similarity with the tralning genes. Then DAVID database was used to implement the functional annotation and enrichment analysis for the extracted genes. Results Compared with previous studies,the second NRG3 and third AKT1 which are obtalned from higher ranking candidate genes,are closely related to schizophrenia. Other higher ranking genes could be used as potential disease-susceptibility genes to provide reference for the research of schizophrenia. Conclusions Data fusion arithmetic can help to detect the potential schizophrenia candidate genes objectively and correctly,and this method is a powerful tool for identifying schizophrenia

  11. Cell fusion and nuclear fusion in plants.

    Science.gov (United States)

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall.

  12. Incidence and clinical relevance of TEL/AML1 fusion genes in children with acute lymphoblastic leukemia enrolled in the German and Italian multicenter therapy trials. Associazione Italiana Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Study Group.

    Science.gov (United States)

    Borkhardt, A; Cazzaniga, G; Viehmann, S; Valsecchi, M G; Ludwig, W D; Burci, L; Mangioni, S; Schrappe, M; Riehm, H; Lampert, F; Basso, G; Masera, G; Harbott, J; Biondi, A

    1997-07-15

    The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9;22) and t(4;11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12;21)(p13;q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12;21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12;21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre-B immunophenotype. Among the B-lineage subgroup, the t(12;21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (> or = 1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children

  13. 融合基因VH-mms13的构建及其蛋白表达鉴定%Construction of fusion gene VH-mms13 and identification of its protein

    Institute of Scientific and Technical Information of China (English)

    杨柳青; 孔登; 王雪耘; 王晓红; 孟丽; 王小柯

    2015-01-01

    Objective To construct the prokaryotic expression vector pET30a( +)-VH-mms13 and identification of its protein after induced with IPTG.Method Heavy chain variable region VH gene of typeⅣcollagenase monoclonal antibody and magnetosome membrane protein gene mms13 were amplified separately,the fusion gene VH-linker-mms13 were synthesized by SOE-PCR technique and inserted into pET30a ( +) plasmid, which was confirmed by restriction enzyme digest and sequencing.Then the recombinant plasmid pET30a ( +)-VH-mms13 was transform into E.coli DE3 and induced with 0.4 mmol/L IPTG.The fused protein was identified by SDS-PAGE and Western blot.Results The length of fusion gene VH-mms13 was 738 bp,and the sequence was correct.After induced with IPTG,the fused protein was found in the inclusion body and Western blot results suggested that the fused protein can bind with His-tag antibody specifically.Conclusion Expression vector pET30a ( +)-VH-mms13 is successfully constructed and the fusion protein has good immunogenicity,which lay the foundation for the development of biomagnetism-targeted drug.%目的:构建原核表达载体pET30a(+)-VH-mms13,诱导表达后鉴定融合蛋白表达。方法分别扩增单克隆抗体基因的重链可变区VH基因和细菌磁小体膜蛋白基因mms13基因,采用重叠延伸PCR技术(splicing by overlap extension ,SOE-PCR)构建融合基因VH-linker-mms13,并将融合基因插入pET30a(+)载体,酶切、测序验证;将重组质粒导入大肠杆菌DE3中,0.4 mmol/L异丙基疏代半乳糖苷( Isopropyl β-D-thiogalactoside ,IPTG)诱导表达,产物经SDS-PAGE电泳和Western blot 双重鉴定。结果 PCR鉴定构建的融合基因VH-mms13大小为738 bp,与理论值相符,测序结果表明序列无误;转入DE3经IPTG诱导,在包含体中检测到融合蛋白表达;Western blot结果显示该表达蛋白可与His-tag抗体特异性结合,蛋白大小符合融合

  14. Nuclear fusion inside condense matters

    Institute of Scientific and Technical Information of China (English)

    HE Jing-tang

    2007-01-01

    This article describes in detail the nuclear fusion inside condense matters--the Fleischmann-Pons effect, the reproducibility of cold fusions, self-consistentcy of cold fusions and the possible applications.

  15. Novel TNS3-MAP3K3 and ZFPM2-ELF5 fusion genes identified by RNA sequencing in multicystic mesothelioma with t(7;17)(p12;q23) and t(8;11)(q23;p13).

    Science.gov (United States)

    Panagopoulos, Ioannis; Gorunova, Ludmila; Davidson, Ben; Heim, Sverre

    2015-02-28

    Multicystic mesothelioma is a rare disease of unknown etiology and pathogenesis. Nothing has been known about the cytogenetic and molecular genetic features of these tumors. Here we present the first cytogenetically analyzed multicystic mesothelioma with the karyotype 46,XX,t(7;17)(p13;q23),t(8;11)(q23;p13). RNA-sequencing showed that the t(7;17)(p13;q23) generated a chimeric TNS3-MAP3K3 gene, which codes for a chimeric protein kinase, as well as the reciprocal MAP3K3-TNS3 in which the region of TNS3 coding for the SH2_Tensin_like region and the tensin phosphotyrosine-binding domain is under the control of the MAP3K3 promoter. The other translocation, t(8;11)(q23;p13), generated a chimeric ZFPM2-ELF5 gene which codes for a chimeric transcription factor in which the first 40 amino acids of ELF5 are replaced by the first 100 amino acids of ZFPM2. RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcripts. The finding of acquired clonal chromosome abnormalities in cells cultured from the lesion and the presence of the TNS3-MAP3K3 chimeric protein kinase and the ZFPM2-ELF5 chimeric transcription factor confirm the neoplastic nature of multicystic mesothelioma. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Fusion of biological membranes

    Indian Academy of Sciences (India)

    K Katsov; M Müller; M Schick

    2005-06-01

    The process of membrane fusion has been examined by Monte Carlo simulation, and is found to be very different than the conventional picture. The differences in mechanism lead to several predictions, in particular that fusion is accompanied by transient leakage. This prediction has recently been verified. Self-consistent field theory is applied to examine the free energy barriers in the different scenarios.

  17. Sensor Data Fusion

    DEFF Research Database (Denmark)

    Plascencia, Alfredo; Stepán, Petr

    2006-01-01

    The main contribution of this paper is to present a sensor fusion approach to scene environment mapping as part of a Sensor Data Fusion (SDF) architecture. This approach involves combined sonar array with stereo vision readings.  Sonar readings are interpreted using probability density functions...

  18. Complementary Advanced Fusion Exploration

    Science.gov (United States)

    2005-08-01

    homographic computer vision image fusion, out-of-sequence measurement and track data handling, Nash bargaining approaches to sensor management... homographic fusion notions are identified together with the Nash approach, the pursuit-evasion approach to threat situation outcome determination, and the

  19. Controlled Nuclear Fusion.

    Science.gov (United States)

    Glasstone, Samuel

    This publication is one of a series of information booklets for the general public published by The United States Atomic Energy Commission. Among the topics discussed are: Importance of Fusion Energy; Conditions for Nuclear Fusion; Thermonuclear Reactions in Plasmas; Plasma Confinement by Magnetic Fields; Experiments With Plasmas; High-Temperature…

  20. Controlled thermonuclear fusion

    CERN Document Server

    Bobin, Jean Louis

    2014-01-01

    The book is a presentation of the basic principles and main achievements in the field of nuclear fusion. It encompasses both magnetic and inertial confinements plus a few exotic mechanisms for nuclear fusion. The state-of-the-art regarding thermonuclear reactions, hot plasmas, tokamaks, laser-driven compression and future reactors is given.

  1. Cell fusions in mammals

    DEFF Research Database (Denmark)

    Larsson, Lars-Inge; Bjerregaard, Bolette; Talts, Jan Fredrik

    2008-01-01

    Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appe...

  2. Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF%农杆菌介导油菜油体基因与碱性成纤维细胞生长因子融合基因转化油菜研究

    Institute of Scientific and Technical Information of China (English)

    徐岩; 肖艳双; 杜金霞; 汪洪; 郑伟; 李营; 庞实锋

    2009-01-01

    [Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Method] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed (Brassica campestris L.) by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows: explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

  3. Compact fusion reactors

    CERN Document Server

    CERN. Geneva

    2015-01-01

    Fusion research is currently to a large extent focused on tokamak (ITER) and inertial confinement (NIF) research. In addition to these large international or national efforts there are private companies performing fusion research using much smaller devices than ITER or NIF. The attempt to achieve fusion energy production through relatively small and compact devices compared to tokamaks decreases the costs and building time of the reactors and this has allowed some private companies to enter the field, like EMC2, General Fusion, Helion Energy, Lawrenceville Plasma Physics and Lockheed Martin. Some of these companies are trying to demonstrate net energy production within the next few years. If they are successful their next step is to attempt to commercialize their technology. In this presentation an overview of compact fusion reactor concepts is given.

  4. Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion.

    Directory of Open Access Journals (Sweden)

    Hyun Sik Jang

    Full Text Available Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.

  5. Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma.

    Science.gov (United States)

    Xie, Zhongqiu; Babiceanu, Mihaela; Kumar, Shailesh; Jia, Yuemeng; Qin, Fujun; Barr, Frederic G; Li, Hui

    2016-11-15

    Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

  6. Fusion of a viral antigen to invariant chain leads to augmented T-cell immunity and improved protection in gene-gun DNA-vaccinated mice

    DEFF Research Database (Denmark)

    Grujic, Mirjana; Holst, Peter J; Christensen, Jan P

    2009-01-01

    against lethal peripheral challenge. The current study questioned whether the same strategy, i.e. linkage of GP to an Ii chain, could be applied to a naked DNA vaccine. Following gene-gun immunization with the linked construct (DNA-IiGP), GP-specific CD4(+) T cells could not be detected by flow cytometry...

  7. Novel RNA hybridization method for the in situ detection of ETV1, ETV4, and ETV5 gene fusions in prostate cancer.

    Science.gov (United States)

    Kunju, Lakshmi P; Carskadon, Shannon; Siddiqui, Javed; Tomlins, Scott A; Chinnaiyan, Arul M; Palanisamy, Nallasivam

    2014-09-01

    The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.

  8. Cloning and fusion protein expression of the S100A4 gene in sika deer%梅花鹿S100A4基因的克隆及融合蛋白的表达

    Institute of Scientific and Technical Information of China (English)

    王大涛; 赵海平; 褚文辉; 杨福合; 邢秀梅; 王桂武; 李春义

    2011-01-01

    为了研究梅花鹿S100A4(S100 calcium binding protein A4)基因在鹿茸生长过程中的作用.用RT-PCR法从生茸骨膜细胞总RNA中克隆了梅花鹿S100A4基因,在NCBI中对基因序列进行比对;将完整的基因序列与逆转录病毒表达载体pLEGFP-Ci重组,获得了重组质粒pLEGFP-S100;用脂质体法将pLEGFP-S100与pVSV-G(被膜载体)共转染包装细胞GP2-293,获得重组病毒上清液,感染角柄骨膜细胞后逆转录病毒携带的基因进入宿主细胞.结果显示:S100A4基因是一个相对保守的基因,与多个物种的匹配度达到90%;重组逆转录病毒载体pLEGFP-S100可以形成重组逆转录病毒粒子,将S100A4基因导入靶细胞,并表达S100A4与GFP(Green fluorescent protein)的融合蛋白.%In order to better understand the function of S100 calcium binding protein A4 in antler development of sika deer ( Cervus nippon), we cloned S100A4 genes from total RNA of cultured antlerogenic periosteum cells using reverse transcription polymerase chain reaction ( RT-PCR), S100A4 gene sequences were compared with closely related animal species in NCBI. Full lengths of S100A4 genes were inserted into a vector plasmid pLEGFP-C1 ( retroviral express vector). The recombinant plasmid pLEGFP-S100 and pVSV-G (envelope vector) were co-transfected into GP2 -293 cells (packaging cell line ) using lipofectimin 2000, and the resultant viral supernatants were collected. The cultured pedicle periosteum cells were then infected with virus in the supernatants. Results showed that S100A4 gene was a relatively conserved gene, and had about 90% homology with several species. Recombinant retroviral vector pLEGFP-S100 could effectively deliver a gene into target cell line, and express a fusion GFP ( green fluorescent protein ) protein.

  9. ALK protein expression and gene fusion in bronchoscopic specimens of lung adenocarcinoma%检测肺腺癌活检标本间变性淋巴瘤激酶蛋白表达和基因融合

    Institute of Scientific and Technical Information of China (English)

    梁小龙; 王孟昭; 张静; 罗玉凤; 张淑英; 武莎斐; 刘媛媛; 曾瑄

    2014-01-01

    Objective To explore ALK protein expression and gene fusion in formalin-fixed and paraffin-embedded (FFPE) specimens obtained from lung cancer by bronchoscopy,and to investigate the relationship between ALK status and clinicopathological characteristics of the patients.Methods Seventyfour FFPE samples obtained from lung adenocarcinoma by bronchoscopy were tested for ALK protein expression and gene fusion respectively by immunohistochemistry (IHC) using Ventana D5F3 antibody and fluorescence in situ hybridization (FISH) using ALK break apart probe.Results sixty-five of the 74 samples were successfully tested by FISH (87.8%,65/74).There were 5 FISH-positive cases (7.7%,5/65),all with advanced stage carcinoma.Among these five FISH-positive cases,3 were IHC-positive (4.1%,3/74) and 2 IHC-negative cases.All the other 69 samples were IHC-negative,including nine FISH-uninformative samples (7 samples were less than 50 tumor cells and 2 samples with weak FISH signal).Both ALK IHC and FISH results were not correlated with age,sex,history of smoking,histological classification,differentiation and lymph node metastasis.Conclusions Bronchoscopic specimens of lung cancer can be used to detect ALK expression and gene fusion.Inmunohistochemistry in combination with FISH test may be more favorable for ALK test.%目的 探讨采用肺腺癌支气管镜活检标本检测间变性淋巴瘤激酶(ALK)蛋白表达和基因融合的可行性,及其与临床病理特征的关系.方法 74例福尔马林固定石蜡包埋的肺腺癌支气管镜活检标本,采用Bench Mark全自动免疫组化染色机和D5F3抗体试剂盒,以免疫组化法检测ALK蛋白的表达,采用Abbott ALK分离探针,以荧光原位杂交(FISH)法检测ALK基因融合.结果 74例标本中,65例成功地进行了FISH检测,成功检测率为87.8% (65/74);FISH阳性5例,阳性率为7.7%(5/65),均为中晚期低分化腺癌;其中免疫组化阳性3例,阳性率为4.1% (3/74);另2

  10. Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution

    OpenAIRE

    Arimura, Shin-ichi; Yamamoto, Junko; Aida, Gen Paul; Nakazono, Mikio; Tsutsumi, Nobuhiro

    2004-01-01

    The balance between mitochondrial fusion and fission influences the reticular shape of mitochondria in yeasts. Little is known about whether mitochondria fusion occurs in plants. Plant mitochondria are usually more numerous and more grain-shaped than animal mitochondria. blast searches of the nuclear and mitochondrial genome sequences of Arabidopsis thaliana did not find any obvious homologue of mitochondrial fusion genes found in animals and yeasts. To determine whether mitochondrial fusion ...

  11. DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time.

    Science.gov (United States)

    Chudley, Lindsey; McCann, Katy; Mander, Ann; Tjelle, Torunn; Campos-Perez, Juan; Godeseth, Rosemary; Creak, Antonia; Dobbyn, James; Johnson, Bernadette; Bass, Paul; Heath, Catherine; Kerr, Paul; Mathiesen, Iacob; Dearnaley, David; Stevenson, Freda; Ottensmeier, Christian

    2012-11-01

    We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA(27-35). We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2(+) patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2(-) control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4(+) and PSMA(27)-specific CD8(+) T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.0024