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Sample records for lacz reporter gene

  1. Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy.

    PubMed

    Mahony, Donna; Karunaratne, Seetha; Rothnagel, Joseph A

    2002-04-01

    The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed. PMID:11994142

  2. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  3. Expression analysis of Dact1 in mice using a LacZ reporter.

    PubMed

    Suzuki, Daisuke; Leu, N Adrian; Brice, Angela K; Senoo, Makoto

    2014-05-01

    The Wnt signaling pathway is essential for cell fate decisions during embryonic development as well as for homeostasis after birth. Dapper antagonist of catenin-1 (Dact1) plays an important role during embryogenesis by regulating Wnt signaling pathways. Consequently, targeted disruption of the Dact1 gene in mice leads to perinatal lethality due to severe developmental defects involving the central nervous system, genitourinary system and distal digestive tract. However, the expression and potential function of Dact1 in other tissues during development and postnatal life have not been well studied. Here, we have generated reporter mice in which LacZ expression is driven by the Dact1 gene promoter and characterized Dact1-LacZ expression in embryos and adult tissues. Our data show that while Dact1-LacZ is expressed in multiple mesoderm- and neuroectoderm-derived tissues during development, high expression of Dact1-LacZ is restricted to a small subset of adult tissues, including the brain, eye, heart, and some reproductive organs. These results will serve as a basis for future investigation of Dact1 function in Wnt-mediated organogenesis and tissue homeostasis. PMID:24681206

  4. Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives.

    PubMed

    Shaw-White, J R; Denko, N; Albers, L; Doetschman, T C; Stringer, J R

    1993-01-01

    Transgenes in mice often exhibit different expression patterns in different transgenic lines. While the basis for this phenomenon is not understood, it is widely believed that the site at which the transgene becomes integrated into the mouse genome is a major factor in determining the pattern of expression. Most transgenic mice have been produced by microinjection of DNA into the male pronucleus, which results in integration of tandem arrays of the transgene at random chromosomal sites. In the experiments described in this report, electroporation of embryonic stem (ES) cells was used to place single copies of a lacZ transgene into either random sites or into the HPRT (hypoxanthine phosphoribosyl transferase) locus of the mouse genome. Expression of lacZ was assayed by histochemical staining for Escherichia coli beta-galactosidase activity in ES cells and in differentiated derivatives obtained by teratocarcinoma formation. Several of the randomly integrated cell lines expressed lacZ at high levels in a variety of cell types present in the tumours, but most notably in epithelial cells. Targeted cell lines with lacZ in opposite orientation to the direction of HPRT gene transcription also expressed well in epithelial cells, but the targeted cell lines did not express in a wider variety of cell types than some of the nontargeted cell lines. Targeted cell lines transcribing lacZ in the same orientation as HPRT transcription did not express high levels of lacZ in any differentiated cell type. Analysis of transcripts suggested that this orientation effect may have been the result of transcriptional interference perpetrated by the HPRT gene promoter. PMID:8513334

  5. Gene fusions with lacZ by duplication insertion in the radioresistant bacterium Deinococcus radiodurans

    SciTech Connect

    Lennon, E.; Minton, K.W. )

    1990-06-01

    Deinococcus radiodurans is the most-studied species of a eubacterial family characterized by extreme resistance to DNA damage. We have focused on developing molecular biological techniques to investigate the genetics of this organism. We report construction of lacZ gene fusions by a method involving both in vitro splicing and the natural transformation of D. radiodurans. Numerous fusion strains were identified by expression of beta-galactosidase. Among these fusion strains, several were inducible by exposure to the DNA-damaging agent mitomycin C, and four of the inducible fusion constructs were cloned in Escherichia coli. Hybridization studies indicate that one of the damage-inducible genes contains a sequence reiterated throughout the D. radiodurans chromosome. Survival measurements show that two of the fusion strains have increased sensitivity to mitomycin C, suggesting that the fusions within these strains inactivate repair functions.

  6. A gene expression resource generated by genome-wide lacZ profiling in the mouse

    PubMed Central

    Tuck, Elizabeth; Estabel, Jeanne; Oellrich, Anika; Maguire, Anna Karin; Adissu, Hibret A.; Souter, Luke; Siragher, Emma; Lillistone, Charlotte; Green, Angela L.; Wardle-Jones, Hannah; Carragher, Damian M.; Karp, Natasha A.; Smedley, Damian; Adams, Niels C.; Bussell, James N.; Adams, David J.; Ramírez-Solis, Ramiro; Steel, Karen P.; Galli, Antonella; White, Jacqueline K.

    2015-01-01

    ABSTRACT Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource. PMID:26398943

  7. p21-LacZ reporter mice reflect p53-dependent toxic insult

    SciTech Connect

    Vasey, Douglas B. Wolf, C. Roland; MacArtney, Thomas; Brown, Ken; Whitelaw, C. Bruce A.

    2008-03-15

    There is an urgent need to discover less toxic and more selective drugs to treat disease. The use of transgenic mice that report on toxic insult-induced transcription can provide a valuable tool in this regard. To exemplify this strategy, we have generated transgenic mice carrying a p21-LacZ transgene. Transgene activity reflected endogenous p21 gene activation in various tissues, displayed compound-specific spatial expression signatures in the brain and immune tissues and enabled p53-dependent and p53-independent responses to be identified. We discuss the application of these mice in delineating the molecular events in normal cellular growth and disease and for the evaluation of drug toxicity.

  8. A novel LacZ reporter mouse reveals complex regulation of the progesterone receptor promoter during mammary gland development.

    PubMed

    Ismail, Preeti M; Li, Jie; DeMayo, Francesco J; O'Malley, Bert W; Lydon, John P

    2002-11-01

    To further our understanding of progesterone (P) as an endocrine mammogen, a PR(lacz) knockin mouse was generated in which the endogenous progesterone receptor (PR) promoter directly regulated lacZ reporter expression. The PR(lacz) mouse revealed PR promoter activity was restricted to the epithelial compartment during the prenatal and postnatal stages of mammary gland development. At puberty, PR promoter activity was unexpectedly robust and restricted to the body cells within the terminal end buds and to the luminal epithelial cells in the subtending ducts. In the adult, the preferential localization of PR(lacz) positive cells to the distal regions of ductal side branches provided a cellular context to the recognized mandatory role of P in ductal side-branching, and segregation of these cells from cells that undergo proliferation supported an intraepithelial paracrine mode of action for P in branching morphogenesis. Toward the end of pregnancy, the PR(lacz) mouse disclosed a progressive attenuation in PR promoter activity, supporting the postulate that the preparturient removal of the proliferative signal of P is a prerequisite for the emergence of a functional lactating mammary gland. The data suggest that PR expression before pregnancy is to ensure the specification and spatial organization of ductal and alveolar progenitor cell lineages, whereas abrogation of PR expression before lactation is required to enable terminal differentiation of the mammary gland. PMID:12403837

  9. Isolation and Characterization of Three Streptococcus pneumoniae Transformation-Specific Loci by Use of a lacZ Reporter Insertion Vector

    PubMed Central

    Pestova, Ekaterina V.; Morrison, Donald A.

    1998-01-01

    Although more than a dozen new proteins are produced when Streptococcus pneumoniae cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random lacZ transcriptional fusion library was constructed in S. pneumoniae by using the insertional lacZ reporter vector pEVP3. Screening the library for clones with competence-specific β-galactosidase (β-Gal) production yielded three insertion mutants with induced β-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the lacZ reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts of Bacillus subtilis com operons. One locus contained open reading frames (ORFs) homologous to the comEA and comEC genes in B. subtilis but lacked a comEB homolog. A second locus contained four ORFs with homology to the B. subtilis comG gene ORFs 1 to 4, but comG gene ORFs 5 to 7 were replaced in S. pneumoniae with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an erm cassette for gene disruptions. PMID:9573156

  10. Folding LacZ in the Periplasm of Escherichia coli

    PubMed Central

    Dwyer, Robert S.; Malinverni, Juliana C.; Boyd, Dana; Beckwith, Jon

    2014-01-01

    Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic β-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli. PMID:25002543

  11. Folding LacZ in the periplasm of Escherichia coli.

    PubMed

    Dwyer, Robert S; Malinverni, Juliana C; Boyd, Dana; Beckwith, Jon; Silhavy, Thomas J

    2014-09-01

    Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic β-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli. PMID:25002543

  12. Expression analysis of mouse Rhobtb3 using a LacZ reporter and preliminary characterization of a knockout strain.

    PubMed

    Lutz, Julia; Grimm-Günter, Eva-Maria S; Joshi, Pooja; Rivero, Francisco

    2014-11-01

    RhoBTB3 is an atypical member of the Rho family of small GTPases. It localizes at the Golgi apparatus and endosomes and is involved in vesicle trafficking and in targeting proteins for degradation in the proteasome. Previous studies using Northern blot analysis showed that Rhobtb3 is ubiquitously expressed in adult mice, but expression is particularly high in brain, heart and uterus. The gene is also expressed between embryonic days 11.5 and 17.5. To investigate the specific cell types that express this gene across tissues, both in the embryo and in the adult organism, we have made use of a gene trap mouse strain that expresses the LacZ gene under the transcriptional control of the endogenous Rhobtb3 promoter. Histochemical detection of β-galactosidase expression revealed a profile characterized by nearly ubiquitous expression of Rhobtb3 in the embryo, but with particularly high levels in bone, cartilage, all types of muscle, testis and restricted areas of the nervous system. In the adult, expression persists at much lower levels in cardiac muscle, the tunica media of blood vessels and cartilage and at high levels in the seminiferous tubules. A general preliminary characterization of this gene trap mouse strain revealed reduced viability, a postnatal growth defect and reduced testis size. Our results should pave the way for future studies aimed at investigating the roles of RhoBTB3 in tissue development and in cardiac, vascular and testicular function. PMID:24923387

  13. Effective gene transfer of lacZ and P0 into Schwann cells of P0-deficient mice.

    PubMed

    Guénard, V; Schweitzer, B; Flechsig, E; Hemmi, S; Martini, R; Suter, U; Schachner, M

    1999-01-15

    Mutations in the gene encoding for the myelinating Schwann cell protein P0 have been linked to inherited peripheral neuropathies, including the Charcot-Marie-Tooth type 1B disease (CMT1B) and Dejerine-Sottas syndrome (DSS). Recently generated mice deficient in the P0 gene (P0-/- mice) resemble cases of CMT1B and DSS with impaired myelin dosage (Martini et al., 1995a). Potential approaches to treat such diseases include the introduction of the normal gene in the nerves of strongly affected patients. In the present study we used P0-/- mice to evaluate the efficiency of a replication-defective, E1-deleted adenovirus vector carrying the lacZ (Ad-RSV-lacZ) or P0 (Ad-RSV-P0) gene to infect abnormally myelinating Schwann cells. The Ad-RSV-lacZ vector suspension was injected into the left sciatic nerve ofPO-/- mice and the nerves examined for beta-galactosidase activity by X-gal histochemistry. Contralateral nerves injected with vehicle solution or non-injected served as controls. Beta-galactosidase activity was detected in nerves injected with the Ad-RSV-lacZ vector up to 2 weeks post-injection. Immunosuppressing the mice with FK506 to decrease the infiltration of activated T-cells in infected nerves lengthened beta-galactosidase activity to 8 weeks, the longest time point examined. Ultrastructural analysis indicated that X-gal crystals were present mostly in abnormally myelinating Schwann cells. These findings demonstrate that an adenovirus vector can successfully infect Schwann cells in P0-/- mice and expression can be maintained for several weeks. The Ad-RSV-P0 suspension was then injected in the sciatic nerve of immunosuppressed P0-/- mice. Two and four weeks post-injection both P0 mRNA and protein could be detected by in situ hybridization and Western blotting in some of the nerves. Furthermore, P0 protein expression was observed in myelin-like structures and onion bulb-like cells by immunohistochemistry. These results indicate that Schwann cells in P0-/- mice can be induced to produce P0 protein after gene transfer. Genetic repair of abnormal Schwann cells by using adenovirus vectors might be a possible technique to treat animal models of inherited peripheral neuropathies. PMID:9890631

  14. Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-05-01

    To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria. PMID:23353051

  15. RHIZOSPHERE COLONIZATION OF OILSEED RAPE BY PSEUDOMONAS ALCALIGENES A9(LACZ)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Root colonization of oilseed rape by Pseudomonas alcaligenes A9(LacZ) in rhizosphere microcosms was investigated with the aid of the lacZ marker gene. Rape seeds were pelletized with A9(LacZ), an effective bacterial strain for promotion of rape seedling growth . Results indicated that A9(LacZ) popu...

  16. Gene transfer of lacZ into avian neural tube and neural crest cells by retroviral infection of grafted embryonic tissues.

    PubMed

    Stocker, K M; Brown, A M; Ciment, G

    1993-01-01

    We describe here a new method for transferring genes into cells of the neural tube and neural crest of early avian embryos in vivo. Using the marker gene lacZ as an example, we infected dissected neural tubes from Hamburger-Hamilton stage 12-13 quail embryos with a replication-defective retrovirus carrying lacZ during a 2 hr period of exposure to the virus in culture. Infected neural tubes were then grafted into uninfected host chicken embryos in ovo and, after continued development for several days, the chimeric embryos were processed for beta-galactosidase histochemistry to identify the progeny of infected cells. We show that virus-infected neural tubes grafted isotopically into the trunk region of host embryos gave rise to cells of both the spinal cord and neural crest. Infected neural crest cells localized within dorsal root ganglia, sympathetic ganglia, peripheral nerves, and within the skin, where they were likely to give rise to melanocytes. These data are consistent with those using other cell marking techniques applied to the neural crest, indicating that retrovirus infection in culture, grafting, and beta-galactosidase expression has a neutral effect on neural crest cell migration and localization. These results indicate the heterospecific grafting of early avian tissues infected with retroviruses carrying foreign genes may be an effective strategy for testing the biological role of various gene products during development. PMID:8380875

  17. Identification of the lacZ insertion site and beta-galactosidase expression in transgenic chickens.

    PubMed

    Mozdziak, Paul E; Wu, Qian; Bradford, Jennifer M; Pardue, Samuel L; Borwornpinyo, Suparerk; Giamario, Carol; Petitte, James N

    2006-04-01

    The quail:chick chimera system is a classical research model in developmental biology. An improvement over the quail:chick chimera system would be a line of transgenic chickens expressing a reporter gene. Transgenic chickens carrying lacZ and expressing bacterial beta-galactosidase have been generated, but complete characterization of the insertion event and characterization of beta-galactosidase expression have not previously been available. The genomic sequences flanking the retroviral insertion site have now been identified by using inverse polymerase chain reaction (PCR), homozygous individuals have been identified by using PCR-based genotyping, and beta-galactosidase expression has been evaluated by using Western analysis and histochemistry. Based upon the current draft of the chicken genome, the viral insertion carrying the lacZ gene has been located on chromosome 11 within the predicted gene for neurotactin/fractalkine (CX3CL1); neurotactin mRNA expression appears to be missing from the brain of homozygous individuals. When Generation 2 (G2) lacZ-positive individuals were inter-mated, they generated 361 G3 progeny; 82 were homozyous for lacZ (22.7%), 97 were wild-type non-transgenic (26.9%), and 182 (50.4%) were hemizygous for lacZ. Western analysis revealed the highest expression in the muscle and liver. With the identification of homozygous birds, the line of chickens is now designated NCSU-Blue1. PMID:16408197

  18. Isolation and characterization of Bacillus subtilis genomic lacZ fusions induced during partial purine starvation.

    PubMed Central

    Saxild, H H; Jensen, C L; Hubrechts, P; Hammer, K

    1994-01-01

    Random genomic Bacillus subtilis lacZ fusions were screened in order to identify the possible existence of regulons responding to the stimuli generated by partial purine starvation. A leaky pur mutation (purL8) was isolated and used to generate the partial purine starvation conditions in the host strain used for screening. On the basis of their induction during partial purine starvation, seven genomic lacZ fusions were isolated. None of the fusions map in loci previously reported to contain purine-regulated genes. One fusion maps very close to the citB locus and may very well be a citB fusion. The fusions were divided into two types on the basis of their response to complete starvation for either ATP or GTP or both components at the same time. Except for one, type 2 fusions were induced by specific starvation for ATP and by simultaneous starvation for ATP and GTP, but not by specific GTP starvation in a gua strain or by GTP starvation induced by the addition of decoyinine. Type 1 fusions were equally well induced by all three kinds of purine starvation including GTP starvation induced by decoyinine. Further subdivisions of the fusions were obtained on the basis of their responses to the spo0A gene product. A total of five fusions showed that spo0A affected expression. One class was unable to induce lacZ expression in the absence of the spo0A gene product, whereas the other class had increased lacZ expression during partial purine starvation in a spo0A background. PMID:8288519

  19. Mef2c-F10N enhancer driven β-galactosidase (LacZ) and Cre recombinase mice facilitate analyses of gene function and lineage fate in neural crest cells.

    PubMed

    Aoto, Kazushi; Sandell, Lisa L; Butler Tjaden, Naomi E; Yuen, Kobe C; Watt, Kristin E Noack; Black, Brian L; Durnin, Michael; Trainor, Paul A

    2015-06-01

    Neural crest cells (NCC) comprise a multipotent, migratory stem cell and progenitor population that gives rise to numerous cell and tissue types within a developing embryo, including craniofacial bone and cartilage, neurons and glia of the peripheral nervous system, and melanocytes within the skin. Here we describe two novel stable transgenic mouse lines suitable for lineage tracing and analysis of gene function in NCC. Firstly, using the F10N enhancer of the Mef2c gene (Mef2c-F10N) linked to LacZ, we generated transgenic mice (Mef2c-F10N-LacZ) that express LacZ in the majority, if not all migrating NCC that delaminate from the neural tube. Mef2c-F10N-LacZ then continues to be expressed primarily in neurogenic, gliogenic and melanocytic NCC and their derivatives, but not in ectomesenchymal derivatives. Secondly, we used the same Mef2c-F10N enhancer together with Cre recombinase to generate transgenic mice (Mef2c-F10N-Cre) that can be used to indelibly label, or alter gene function in, migrating NCC and their derivatives. At early stages of development, Mef2c-F10N-LacZ and Mef2c-F10N-Cre label NCC in a pattern similar to Wnt1-Cre mice, with the exception that Mef2c-F10N-LacZ and Mef2c-F10N-Cre specifically label NCC that have delaminated from the neural plate, while premigratory NCC are not labeled. Thus, our Mef2c-F10N-LacZ and Mef2c-F10N-Cre transgenic mice provide new resources for tracing migratory NCC and analyzing gene function in migrating and differentiating NCC independently of NCC formation. PMID:25794678

  20. Levels of mRNAs which code for small, acid-soluble spore proteins and their LacZ gene fusions in sporulating cells of Bacillus subtilis.

    PubMed Central

    Mason, J M; Fajardo-Cavazos, P; Setlow, P

    1988-01-01

    The levels of mRNAs from genes (sspA, B and E) which code for major small, acid-soluble, spore proteins of Bacillus subtilis have been determined, as well as the levels of mRNAs from ssp-lacZ gene fusions. Increasing the gene dosage of ssp-lacZ fusions resulted in parallel increases in both the ssp-lacZ mRNA level and the rate of b-galactosidase accumulation. Similarly, an 11-fold increase in sspE gene dosage gave a comparable increase in sspE mRNA, but at most a 1.5-fold increase in the amount of sspE gene product accumulated. In contrast, an 11-fold increase in the dosage of the sspA or B genes had no significant effect on the level of total sspA plus sspB mRNA, but did alter the ratios of these mRNAs as well as the amount of their gene products, to reflect the altered ratio of the two genes. These results suggest that intact ssp genes, but not ssp-lacZ gene fusions, are subject to feedback regulation of gene expression, with this regulation of the sspA and B genes effected by modulation of mRNA levels, while the feedback regulation of the sspE gene is at the post-transcriptional level. Images PMID:2456528

  1. p53 deficiency alters the yield and spectrum of radiation-induced lacZ mutants in the brain of transgenic mice

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Kanazawa, N.; Lutze-Mann, L.; Winegar, R. A.

    2001-01-01

    Exposure to heavy particle radiation in the galacto-cosmic environment poses a significant risk in space exploration and the evaluation of radiation-induced genetic damage in tissues, especially in the central nervous system, is an important consideration in long-term manned space missions. We used a plasmid-based transgenic mouse model system, with the pUR288 lacZ transgene integrated in the genome of every cell of C57Bl/6(lacZ) mice, to evaluate the genetic damage induced by iron particle radiation. In order to examine the importance of genetic background on the radiation sensitivity of individuals, we cross-bred p53 wild-type lacZ transgenic mice with p53 nullizygous mice, producing lacZ transgenic mice that were either hemizygous or nullizygous for the p53 tumor suppressor gene. Animals were exposed to an acute dose of 1 Gy of iron particles and the lacZ mutation frequency (MF) in the brain was measured at time intervals from 1 to 16 weeks post-irradiation. Our results suggest that iron particles induced an increase in lacZ MF (2.4-fold increase in p53+/+ mice, 1.3-fold increase in p53+/- mice and 2.1-fold increase in p53-/- mice) and that this induction is both temporally regulated and p53 genotype dependent. Characterization of mutants based on their restriction patterns showed that the majority of the mutants arising spontaneously are derived from point mutations or small deletions in all three genotypes. Radiation induced alterations in the spectrum of deletion mutants and reorganization of the genome, as evidenced by the selection of mutants containing mouse genomic DNA. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported owing to technical limitations in most other mutation assays.

  2. Molecular cloning of Mu d(bla lacZ) transcriptional and translational fusions.

    PubMed Central

    Wanner, B L

    1987-01-01

    The vector pBW2 was made to selectively clone chimeric plasmids with chromosomal Mu d(bla lacZ) transcriptional or translational fusions. It was tetracycline resistant and had the carboxyl-terminal end of bla distal to its PstI site. Because ligation of PstI-digested chromosomal DNA of a Mu d(bla lacZ) insertion with pBW2 restored bla, ampicillin-resistant chimeric plasmids were selectable. These plasmids had the Mu d bla amino terminus and simultaneously acquired other Mu d sequences including lacZ, the chromosomal fusion joint, and the DNA adjacent to the nearest chromosomal PstI site. The plasmid pBW2 was useful in the molecular cloning of several psi and pho::lacZ(Mu d) fusions, as well as chromosomal genes located near Mu d insertions. PMID:3032905

  3. Detection of antistaphylococcal and toxic compounds by biological assay systems developed with a reporter Staphylococcus aureus strain harboring a heat inducible promoter - lacZ transcriptional fusion.

    PubMed

    Chanda, Palas Kumar; Ganguly, Tridib; Das, Malabika; Lee, Chia Yen; Luong, Thanh T; Sau, Subrata

    2007-11-30

    Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cell-wall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region (P(g)) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the P(g)-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that P(g) in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced P(g) efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals. PMID:18047789

  4. Advances in directed molecular evolution of reporter genes.

    PubMed

    Xiong, Ai-Sheng; Peng, Ri-He; Zhuang, Jing; Davies, Jim; Zhang, Jian; Yao, Quan-Hong

    2012-06-01

    Many reporter genes, such as gfp, gusA, and lacZ, are widely used for research into plants, animals, and microorganisms. Reporter genes, which offer high levels of sensitivity and convenience of detection, have been utilized in transgenic technology, promoter analysis, drug screening, and other areas. Directed molecular evolution is a powerful molecular tool for the creation of designer proteins for industrial and research applications, including studies of protein structure and function. Directed molecular evolution is based mainly on in vitro recombination methods, such as error-prone PCR and DNA shuffling. The strategies of directed evolution of enzyme biocatalysts have been the subject of several recent reviews. Here, we briefly summarize successes in the field of directed molecular evolution of reporter genes and discuss some of the applications. PMID:21774740

  5. Insights Into Mutagenesis Using Escherichia coli Chromosomal lacZ Strains That Enable Detection of a Wide Spectrum of Mutational Events

    PubMed Central

    Seier, Tracey; Padgett, Dana R.; Zilberberg, Gal; Sutera, Vincent A.; Toha, Noor; Lovett, Susan T.

    2011-01-01

    Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3′ exonucleases targeted to the lagging strand. The loss of 3′ exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F′ element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes. PMID:21441210

  6. Systemic RNAi delivery to the muscles of ROSA26 mice reduces lacZ expression.

    PubMed

    Wei, Jessica; Chamberlain, Joel R

    2014-01-01

    RNAi has potential for therapeutically downregulating the expression of dominantly inherited genes in a variety of human genetic disorders. Here we used the ROSA26 mouse, which constitutively expresses the bacterial lacZ gene in tissues body wide, as a model to test the ability to downregulate gene expression in striated muscles. Recombinant adeno-associated viral vectors (rAAVs) were generated that express short hairpin RNAs (shRNAs) able to target the lacZ mRNA. Systemic delivery of these rAAV6 vectors led to a decrease of β-galactosidase expression of 30-50-fold in the striated muscles of ROSA26 mice. However, high doses of vectors expressing 21 nucleotide shRNA sequences were associated with significant toxicity in both liver and cardiac muscle. This toxicity was reduced in cardiac muscle using lower vector doses. Furthermore, improved knockdown in the absence of toxicity was obtained by using a shorter (19 nucleotide) shRNA guide sequence. These results support the possibility of using rAAV vectors to deliver RNAi sequences systemically to treat dominantly inherited disorders of striated muscle. PMID:25127128

  7. [Promoter recognition and beta-galactosidase reporter gene expression in Rhodococcus].

    PubMed

    Liu, Changchun; Yu, Huimin; Yuchao, M; Pan, Wenyu; Luo, Hui; Shen, Zhongyao

    2009-09-01

    The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the beta-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus. PMID:19938479

  8. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.

    PubMed

    Muramatsu, T; Mizutani, Y; Ohmori, Y; Okumura, J

    1997-01-13

    By using three nonviral transfection methods, i.e., microparticle bombardment, lipofection and electroporation, the transfection efficiency and the expression intensity of a lacZ reporter gene were compared in developing chicken embryos in ovo. Of the three transfection methods employed, electroporation conferred the strongest expression of the bacterial lacZ gene with similar transfection efficiency. The results suggest that as far as transient gene expression is concerned, electroporation would provide a useful and efficient nonviral means of foreign gene transfection to somatic cells of living chicken embryos in ovo. PMID:9016787

  9. Progress in gene targeting and gene therapy for retinitis pigmentosa

    SciTech Connect

    Farrar, G.J.; Humphries, M.M.; Erven, A.

    1994-09-01

    Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectors for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.

  10. An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola.

    PubMed Central

    Lindgren, P B; Frederick, R; Govindarajan, A G; Panopoulos, N J; Staskawicz, B J; Lindow, S E

    1989-01-01

    We have constructed derivatives of the transposon Tn3 that allow an ice nucleation gene (inaZ) to be used as 'reporter' of the transcriptional activity of genes into which it is inserted. In these derivatives (Tn3-Ice and Tn3-Spice), the lacZYA sequences of transposon Tn3-HoHo1 were replaced with inaZ lacking its native promoter. The ice nucleation activity of virB::inaZ fusions in the correct transcriptional orientation was inducible by acetosyringone, a plant metabolite which activates the vir operon of Agrobacterium tumefaciens Ti plasmids, while fusions in the opposite orientation were unresponsive to the inducer. Tn3-Spice was also used to investigate the expression of a cluster of genes (hrp) which control pathogenicity and hypersensitivity elicited by Pseudomonas syringae pv. phaseolicola. An inducible region was identified which is expressed at low levels in vitro but becomes activated when the bacteria come into contact with the susceptible host, bean. Activation of this region occurred within 2 h post-inoculation and was nearly complete by the time the bacteria began to multiply in the leaf tissue. The inaZ reporter appears to be at least 10(5)-fold more sensitive than lacZ in P.s.phaseolicola. Thus, the inaZ fusion system provides a sensitive, convenient and inexpensive tool for the study of bacterial gene expression, particularly during plant pathogenesis, and should be generally useful as a reporter gene system in Gram-negative bacteria. PMID:2548841

  11. INDUCTION OF A MYCOPLASMA GALLISEPTICUM PMGA GENE IN THE CHICKEN TRACHEAL RING ORGAN CULTURE MODEL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To search for Mycoplasma gallisepticum genes involved in colonization of the poultry respiratory tract, a transposon containing a promoterless lacZ gene was employed as a transcriptional reporter. The transposon was used to randomly mutagenize the chromosome of the M. gallisepticum S6 strain and a ...

  12. Effect of methyl methanesulfonate on hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9

    PubMed Central

    Kumar, Vineet; Ara, Gulshan; Afzal, Mohammad; Siddique, Yasir Hasan

    2011-01-01

    Methyl methanesulfonate (MMS) is an anti-carcinogenic drug and its toxicity has been reported in various experimental models. The hsp70s are a family of ubiquitously expressed heat shock proteins. In the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals. Nowadays emphasis is given to the use of alternatives to mammals in testing, research and education. The European Centre for the Validation of Alternative Methods (EVCAM) has recommended the use of Drosophila as an alternative model for scientific studies. Almost all living organisms possess proteins with a similar structure to that of hsp70s. In the present study, the toxicity of MMS was evaluated by quantifying hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9, at different doses and hours of exposure. We studied the effect of 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS at 2, 4, 24 and 48 hours of exposure on hsp70 expression by using the soluble O-nitrophenyl-β-D-galactopyranoside (ONPG) assay and on establishing the tissue damage by the Trypan blue exclusion assay in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9. A dose-dependent increase in the expression of hsp70 was observed at 0.25, 0.50, and 0.75 µl/ml of MMS compared to the control. At the highest dose, i.e. 1.0 µl/ml of MMS, the activity of hsp70 was decreased due to tissue damage. PMID:22058658

  13. Characterization of Platelet-Derived Growth Factor-A Expression in Mouse Tissues Using a lacZ Knock-In Approach

    PubMed Central

    Andrae, Johanna; Gouveia, Leonor; He, Liqun; Betsholtz, Christer

    2014-01-01

    Expression of the platelet-derived growth factor A-chain gene (Pdgfa) occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH) has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfaex4COIN, which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ) reporter under endogenous Pdgfa transcription control. The generated Pdgfaex4COIN-INV-lacZ allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfaex4COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4COIN-INV-lacZ recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology. PMID:25166724

  14. TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts

    PubMed Central

    Kucab, Jill E.; Zwart, Edwin P.; van Steeg, Harry; Luijten, Mirjam; Schmeiser, Heinz H.; Phillips, David H.; Arlt, Volker M.

    2016-01-01

    3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:C > T:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:C > T:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers’ lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity. PMID:26723900

  15. Mapping oxytocin receptor gene expression in the mouse brain and mammary gland using an oxytocin receptor-LacZ reporter mouse.

    PubMed

    Gould, B R; Zingg, H H

    2003-01-01

    The hypothalamic nonapeptide oxytocin (OT) has an established role as a circulating hormone but can also act as a neurotransmitter and as a neuromodulator by interacting with its central OT receptor (OTR). To understand the role of the OTR in the mouse brain we investigated the expression of the OTR gene at the cellular level. We targeted the lacZ reporter gene to the OTR gene locus downstream of the endogenous OTR regulatory elements. Using lactating mouse mammary gland as a control for OTR promoter directed specificity of lacZ gene expression, X-gal histochemistry on tissue sections confirmed that gene expression was restricted to the myoepithelial cells. We also identified for the first time in mice the expression of the OTR gene in neighbouring adipocytes. Further, investigation in the mouse brain identified numerous nuclei containing neurons expressing the OTR gene. Whilst some of these regions had been described for rat or sheep, the OTR-LacZ reporter mouse enabled the identification of novel sites of central OTR gene expression. These regions include the accessory olfactory bulb, the medial septal nucleus, the posterolateral cortical amygdala nucleus, the posterior aspect of the basomedial amygdala nucleus, the medial part of the supramammillary nucleus, the dorsotuberomammillary nucleus, the medial and lateral entorhinal cortices, as well as specific dorsal tegmental, vestibular, spinal trigeminal, and solitary tract subnuclei. By mapping the distribution of OTR gene expression, depicted through histochemical detection of beta-galactosidase, we were able to identify single OTR gene expressing neurons and small neuron clusters that would have remained undetected by conventional approaches. These novel sites of OTR gene expression suggest additional functions of the oxytocinergic system in the mouse. These results lay the foundation for future investigation into the neural role of the OTR and provide a useful model for further study of oxytocin functions in the mouse. PMID:14596857

  16. Frameshift mutagenicity of aromatic amines related to aminofluorene in a lacZ reversion assay in E. coli

    SciTech Connect

    Hoffmann, G.R.; Janel-Bintz, R.; Fuchs, R.P.P.

    1997-10-01

    We studied in the mutagenicity of three aromatic amines in a lacZ reversion assay in E. coli: 2-nitrofluorene (NF), N-2-acetylaminofluorene (AAF), and N-hydroxy-N-2-acetylaminofluorene (NHA). Mutations that confer the Lac{sup +} phenotype were measured using an F{prime} factor from strain CC109 of Cupples et al. The F{prime} contains a lacZ mutation that reverts by a -2 frameshift at a site of repetitive dinucleotides (CG{sub 5} to CG{sub 4}). The F{prime} was transferred into strains carrying an LPS{sup d} mutation that increases permeability to aromatic amines and a plasmid (pYG219) that contains the Salmonella nat gene, which confers N- and O-acetyltransferase (NAT/OAT) activity. Mutagenesis was measured by papillation assays and quantitative reversion assays. The results show that the LPS{sup d} mutation, conferring enhanced permeability, facilitates measuring the mutagenicity of aromatic amines but is not absolutely required, in that a lower level of mutagenicity is detected in LPS{sup +} strains. The NAT/OAT activity conferred by pYG219 strongly potentiates the mutagenicity of NF and NHA. The mutagenicity of NF is undoubtedly ascribable to aminofluorene (AF) adducts: The mutagenicity of NHA may be due either to AAF adducts or to AF adducts produced by deacetylation. Surprisingly, AAF was weakly mutagenic in a NAT/OAT LPS{sup d} strain even without metabolic activation by a mammalian cytochrome P450.

  17. Comparison between S+L- assay and LacZ marker rescue assay for detecting replication-competent gammaretroviruses.

    PubMed

    Hashimoto-Gotoh, A; Yoshikawa, R; Miyazawa, T

    2015-09-01

    To avoid contamination of adventitious gammaretroviruses in biological products such as vaccines, it is necessary to check the master seed cells for manufacturing. There are several assays to detect infectious gammaretroviruses. Among these, sarcoma-positive, leukemia-negative (S+L-) assay is a classical infectivity assay, which is often recommended in governmental guidelines. The S+L- cells used in S+L- assay generate unique focus upon the infection of replication-competent gammaretroviruses. Although S+L- assay is well recognized for the detection, their applicability is questionable in some cases. On the other hand, LacZ marker rescue (LMR) assay detects infectious gammaretroviruses by transducing LacZ marker gene to the target cells, which shows lacZ-positive foci if the infectious virus is present. In this study, we compared LMR and S+L- assays for detection of a variety of endogenous and exogenous gammaretroviruses. As results, LMR assay could detect all gammaretroviruses examined. On the other hand, S+L- assay using feline S+L- cells, termed QN10S, could not detect porcine endogenous retrovirus (PERV) subgroups A/B. Further, S+L- mink cells could not detect feline leukemia virus subgroups B in addition to PERV-A/B. These data indicate that LMR assay is better suited to detect wider range of gammaretroviruses. PMID:26164289

  18. Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM.

    PubMed

    Douglas, Grace L; Klaenhammer, Todd R

    2011-10-01

    Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression. PMID:21873486

  19. A cryptic ribosome binding site, false signals in reporter systems and avoidance of protein translation chaos.

    PubMed

    Sachadyn, Paweł; Stanisławska-Sachadyn, Anna; Kabat, Ewa Maria; Zielińska, Aleksandra; Kur, Józef

    2009-09-10

    The expression of reporter gene may be induced by activation of cryptic signalling sequences, as we found while constructing the mutS-lacZ fusion gene. We cloned the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. The clones expected to produce beta-galactosidase as the C-terminal fusion were selected for the complementation of beta-galactosidase activity in a lacZ deficient E. coli strain. Surprisingly, one of the clones, though displaying beta-galactosidase activity, did not produce the fusion protein. As shown by DNA sequencing a 92 bp fragment in the 3' part of mutS gene was substituted by a 19 bp sequence. As the consequence of the resultant frameshift, a truncated MutS peptide was translated instead of beta-galactosidase fusion. The cloned lacZ gene lacked its ribosome binding site, so lacZ expression could be explained by activation of a cryptic ribosome binding site in the 3' end of mutS gene. This observation shows that fusion domains in reporter systems are possible to produce accidentally misleading signals. This observation also suggests that some triplets like AGG and AGA, present in the canonical ribosome binding sequence, are rarely used codons to prevent chaotic protein translation. PMID:19607865

  20. Magnetic Resonance Reporter Gene Imaging

    PubMed Central

    Lee, Sheen-Woo; Lee, Sang-Hoon; Biswal, Sandip

    2012-01-01

    Molecular imaging has undergone an explosive advancement in recent years, due to the tremendous research efforts made to understand and visualize biological processes. Molecular imaging by definition assesses cellular and molecular processes in living subjects, with the targets of following metabolic, genomic, and proteomic events. Furthermore, reporter gene imaging plays a central role in this field. Many different approaches have been used to visualize genetic events in living subjects, such as, optical, radionuclide, and magnetic resonance imaging. Compared with the other techniques, magnetic resonance (MR)-based reporter gene imaging has not occupied center stage, despite its superior three-dimensional depictions of anatomical details. In this article, the authors review the principles and applications of various types of MR reporter gene imaging technologies and discuss their advantages and disadvantages. PMID:22539936

  1. Integrated approach to the in vivo genotoxic effects of a titanium dioxide nanomaterial using LacZ plasmid-based transgenic mice.

    PubMed

    Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Costa, Pedro M; Alverca, Elsa; Zwart, Edwin; de Jong, Wim H; Fessard, Valérie; Lavinha, João; Silva, Maria J

    2014-07-01

    Titanium dioxide (TiO2 ) nanomaterials (NMs) are widely used in a diversity of products including cosmetics, pharmaceuticals, food, and inks, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. Previous studies on the genotoxicity of TiO2 have reported discrepant or inconclusive findings in both in vitro and in vivo systems. This study explores the in vivo genotoxic potential of a well-characterized uncoated TiO2 NM with an average diameter of 22 nm (NM-102, from JRC repository) using several genotoxicity endpoints in the LacZ plasmid-based transgenic mouse model. Mice were exposed by intravenous injection to two daily doses of NM-102: 10 and 15 mg/kg of body weight/day. Micronuclei were analyzed in peripheral blood reticulocytes 42 hr after the last treatment. DNA strand breaks (comet assay) and gene mutations were determined in the spleens and livers of the same animals 28 days after the last treatment. Histopathological and cytological analyses were also performed in liver samples. Genotoxic effects were not detected in mice exposed to the nanosized TiO2 under the experimental conditions used, despite a moderate inflammatory response that was observed in the liver. Considering the biopersistence of TiO2 in mouse liver and the moderate inflammatory response, the possibility of a secondary genotoxic effect at higher doses and in conditions that result in a stronger inflammatory response, for example, within a longer time window, should be investigated further. PMID:24590610

  2. Generation and characterization of a novel neural crest marker allele, Inka1-LacZ, reveals a role for Inka1 in mouse neural tube closure

    PubMed Central

    Reid, Bethany S.; Sargent, Thomas D.; Williams, Trevor

    2010-01-01

    Previous studies identified Inka1 as a gene regulated by AP-2α in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock-in allele. Inka1-LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1−/− mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP-2α is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not. PMID:20175189

  3. Adenoviral-mediated gene transfer to rabbit synovium in vivo.

    PubMed Central

    Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L

    1993-01-01

    Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791

  4. Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila.

    PubMed

    Auer, Jasmin S; Nagel, Anja C; Schulz, Adriana; Wahl, Vanessa; Preiss, Anette

    2015-12-01

    In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H) (MAPK-) (ko) and a phospho-mimetic Su(H) (MAPK-ac) isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg (BE) -lacZ). In general, Su(H) (MAPK-) (ko) induced a stronger response than wild-type Su(H), whereas the response to Su(H) (MAPK-ac) was very weak. Notch target genes cut, wingless and vg (BE) -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER (act) ) or the MAPK (rl (SEM) ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones. PMID:26702412

  5. Reporter gene imaging: potential impact on therapy.

    PubMed

    Serganova, Inna; Blasberg, Ronald

    2005-10-01

    Positron emission tomography (PET)-based molecular-genetic imaging in living organisms has enjoyed exceptional growth over the past 5 years; this is particularly striking since it has been identified as a new discipline only within the past decade. Positron emission tomography is one of three imaging technologies (nuclear, magnetic resonance and optical) that has begun to incorporate methods that are established in molecular and cell biology research. The convergence of these disciplines and the wider application of multi-modality imaging are at the heart of this success story. Most current molecular-genetic imaging strategies are "indirect," coupling a "reporter gene" with a complimentary "reporter probe." Reporter gene constructs can be driven by constitutive promoter elements and used to monitor gene therapy vectors and the efficacy of trans gene targeting and transduction, as well as to monitor adoptive cell-based therapies. Inducible promoters can be used as "sensors" to regulate the magnitude of reporter gene expression and can be used to provide information about endogenous cell processes. Reporter systems can also be constructed to monitor mRNA stabilization and specific protein-protein interactions. Promoters can be cell specific and restrict transgene expression to certain tissue and organs. The translation of reporter gene imaging to specific clinical applications is discussed. Several examples that have potential for patient imaging studies in the near future include monitoring adenoviral-based gene therapy, oncolytic herpes virus therapy, adoptive cell-based therapies and Salmonella-based tumor-targeted cancer therapy and imaging. The primary translational applications of noninvasive in vivo reporter gene imaging are likely to be (a) quantitative monitoring of the gene therapy vector and the efficacy of transduction in clinical protocols, by imaging the location, extent and duration of transgene expression; (b) monitoring cell trafficking, targeting, replication and activation in adoptive therapies, involving ex vivo transduction of harvested immune-competent cells and stem/progenitor cells; (c) assessments of endogenous molecular events using different reporter gene imaging technologies following the development of safe, efficient and target-specific vectors for "diagnostic transductions." PMID:16243653

  6. LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance

    PubMed Central

    Juers, Douglas H; Matthews, Brian W; Huber, Reuben E

    2012-01-01

    This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the “missing” N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of β-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon. PMID:23011886

  7. Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains.

    PubMed

    Davis, T O; Henderson, I; Brehm, J K; Minton, N P

    2000-01-01

    Non-proteolytic, Group II strains of Clostridium botulinum are of particular concern to the food industry because of their ability to survive and grow in REPFEDs (refrigerated processed foods of extended durability). Their analysis would benefit from the availability of a gene transfer system. In the present study we have been able, for the first time, to demonstrate transformation in a representative Group II strain, ATCC 25765. Initial attempts to transform ATCC 25765 with existing clostridial cloning vectors (pMTL540E and pMTL500E) were, however, prevented by a restriction barrier. Through a combination of classical and molecular approaches we were able to show that strain ATCC 25765 possesses a restriction endonuclease (Cbol) and a methylase activity (M. Cbol) which have the same specificity as Mspl and M.Mspl, respectively. Cbol cleaves the palindrome 5'-CCGG-3' to generate a 3'-GC sticky end, whilst M.Cbol specifically methylates the external C residue. An E. coli host was generated which expressed a Bacillus subtilis methylase enzyme (M.BsuF1) with equivalent specificity to M.Cbol. Plasmids (pMTL540E and pMTL500E) prepared in this strain were subsequently shown to be capable of transforming ATCC 25765. The highest frequencies (0.8 X 10(4) transformants per microg of DNA) were obtained when cells were cultivated in media supplemented with 1% (w/v) glycine, and when the electroporation was undertaken at 10 kV/cm, 25 microF and at 400 ohms. Having developed an effective transformation procedure, we went on to construct reporter cassettes based on the Thermanaerobacterium sulfurigenes lacZ and the Vibrio fischeri luxAB genes. Using the former, and promoter regions isolated from the botulinum toxin genes, we have obtained preliminary evidence that reporter genes may be used to evaluate the physiological factors that affect toxin production in the food environment. PMID:10937489

  8. New insights into the regulation of the pac gene from Escherichia coli W ATCC 11105.

    PubMed

    Roa, A; García, J L

    1999-08-01

    The regulation of the pac gene encoding the penicillin G acylase of Escherichia coli W ATCC 11015 has been investigated by a molecular approach using lacZ as a reporter gene. This analysis revealed that a region of 170 bp located upstream of the pac structural gene contains the regulatory sequences that control its expression. The cAMP receptor protein is involved not only in the catabolite repression of penicillin G acylase production caused by glucose but also in the induction of pac gene expression by phenylacetic acid. Primer extension analyses have demonstrated that the transcription of the pac gene can be initiated from at least three different promoters. Although all these promoters are functional, their relative activity depends on the transcribed gene, the P1 and P3 promoters being more active in the presence of the pac gene, whereas the P2 promoter was stronger when the upstream region of the pac gene was fused to the lacZ reporter. A deletion of the region surrounding the -10 box of the P3 promoter produced a constitutive expression of the fused gene indicating that this sequence is required for phenylacetic acid induction and suggesting that the expression of the pac gene is regulated by a repression mechanism. This work reveals that the regulation of the pac gene is more complex than previously envisioned and provides new clues to investigate further this interesting regulatory system. PMID:10436917

  9. Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

    PubMed Central

    Dryselius, Rikard; Nikravesh, Abbas; Kulyté, Agne; Goh, Shan; Good, Liam

    2006-01-01

    Background A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria. PMID:17118182

  10. The Chicken Gene Nomenclature Committee report.

    PubMed

    Burt, David W; Carrë, Wilfrid; Fell, Mark; Law, Andy S; Antin, Parker B; Maglott, Donna R; Weber, Janet A; Schmidt, Carl J; Burgess, Shane C; McCarthy, Fiona M

    2009-01-01

    Comparative genomics is an essential component of the post-genomic era. The chicken genome is the first avian genome to be sequenced and it will serve as a model for other avian species. Moreover, due to its unique evolutionary niche, the chicken genome can be used to understand evolution of functional elements and gene regulation in mammalian species. However comparative biology both within avian species and within amniotes is hampered due to the difficulty of recognising functional orthologs. This problem is compounded as different databases and sequence repositories proliferate and the names they assign to functional elements proliferate along with them. Currently, genes can be published under more than one name and one name sometimes refers to unrelated genes. Standardized gene nomenclature is necessary to facilitate communication between scientists and genomic resources. Moreover, it is important that this nomenclature be based on existing nomenclature efforts where possible to truly facilitate studies between different species. We report here the formation of the Chicken Gene Nomenclature Committee (CGNC), an international and centralized effort to provide standardized nomenclature for chicken genes. The CGNC works in conjunction with public resources such as NCBI and Ensembl and in consultation with existing nomenclature committees for human and mouse. The CGNC will develop standardized nomenclature in consultation with the research community and relies on the support of the research community to ensure that the nomenclature facilitates comparative and genomic studies. PMID:19607656

  11. Recombinant Marek's disease virus (MDV)-derived lymphoblastoid cell lines: regulation of a marker gene within the context of the MDV genome.

    PubMed

    Parcells, M S; Dienglewicz, R L; Anderson, A S; Morgan, R W

    1999-02-01

    Marek's disease is a herpesvirus (Marek's disease virus [MDV])-induced pathology of chickens characterized by paralysis and the rapid appearance of T-cell lymphomas. Lymphoblastoid cell lines (LBCLs) derived from MDV-induced tumors have served as models of MDV latency and transformation. We have recently reported the construction of mutant MDVs having a deletion (M. S. Parcells et al., J. Virol. 69:7888-7898, 1995) and an insertion (A. S. Anderson et al., J. Virol. 72:2548-2553, 1998) within the unique short region of the virus genome. These mutant MDVs retained oncogenicity, and LBCLs have been established from the mutant-induced tumors. We report the characterization of these cell lines with respect to (i) virus structure within and reactivated from the cell lines, (ii) surface antigen expression, (iii) kinetics of MDV and marker gene induction, (iv) localization and colocalization of induced MDV antigens and beta-galactosidase (beta-Gal), and (v) methylation status of the region of lacZ insertion in recombinant- and non-recombinant-derived cell lines. Our results indicate that (i) recombinant-derived cell lines contain no parental virus, (ii) the established cell lines are predominantly CD4(+) CD8(-), (iii) the percentage of Lac-expressing cells is low (1 to 3%) but increases dramatically upon 5'-iododeoxyuridine (IUdR) treatment, (iv) lacZ expression is induced with the same kinetics as several MDV lytic-phase genes (pp38, US1, gB, gI, and US10), and (v) the regulation of lacZ expression is not mediated by methylation. Furthermore, the MDV-encoded oncoprotein, Meq, could be detected in cells expressing beta-Gal and various lytic antigens but did not appear to be induced by IUdR treatment. Our results indicate that regulation of the lacZ marker gene can serve as sensitive measure of virus lytic-phase induction and the reactivation from latency. PMID:9882341

  12. A source of artifact in the lacZ reversion assay in Escherichia coli.

    PubMed

    Hoffmann, George R; Gray, Carol L; Lange, Paulina B; Marando, Christie I

    2015-06-01

    The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its utility for discerning effects of weak mutagens may be compromised by the artifact. PMID:26046973

  13. Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes.

    PubMed

    Totten, D C; Vuong, M; Litvinova, O V; Jinwal, U K; Gulia-Nuss, M; Harrell, R A; Beneš, H

    2013-02-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes. PMID:23241066

  14. Repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy.

    PubMed Central

    Ghazizadeh, S; Carroll, J M; Taichman, L B

    1997-01-01

    Retrovirus-mediated gene transfer is commonly used in gene therapy protocols and has the potential to provide long-term expression of the transgene. Although expression of a retrovirus-delivered transgene is satisfactory in cultured cells, it has been difficult to achieve consistent and high-level expression in vivo. In this investigation, we explored the possibility of modulating transgene expression by host-derived cytokines. Normal human keratinocytes and dermal fibroblasts were transduced with recombinant retroviruses expressing a reporter gene (lacZ). Treatment of transduced cells with a proinflammatory cytokine, gamma interferon (IFN-gamma), significantly reduced lacZ expression to less than 25% of that of nontreated cells. The inhibition was concentration dependent (peak at 5 ng/ml) and time dependent (maximal at 16 h for transcript and 24 h for protein); expression remained repressed in the continued presence of IFN-gamma but returned to normal levels 24 h after IFN-gamma withdrawal. The decrease in beta-galactosidase activity appeared to result from decrease in steady-state lacZ mRNA levels. Inhibitors of transcription and translation blocked IFN-gamma-induced repression, suggesting involvement of newly synthesized protein intermediates. Similar results were obtained by treatment of transduced cells with IFN-alpha but not with other proinflammatory cytokines, including tumor necrosis factor alpha, interleukin-2 (IL-1), IL-4, and granulocyte colony-stimulating factor. Although the level of lacZ mRNA was reduced by >70% following IFN treatment, the rate of lacZ transcription was not significantly different from that for nontreated cells. These results suggest that IFN-mediated regulation of transgene expression is at a posttranscriptional level. Interestingly, IFN-gamma also suppressed transgene expression driven by a cellular promoter (involucrin) inserted in an internal position in the retroviral vector. The presence of the overlapping 3' untranslated regions in transcripts initiated from the internal promoter and the long terminal repeat is suggestive of a posttranscriptional regulation, likely at the level of RNA stabilization. These results provide direct evidence for modulatory effects of IFNs on retrovirus-mediated transgene expression and suggest that gene therapy results may be altered by host inflammatory responses. PMID:9371574

  15. Frequencies of mutagenic translesion DNA synthesis over cisplatin-guanine intra-strand crosslinks in lacZ plasmids propagated in human cells.

    PubMed

    Fujikawa, Yoshihiro; Kawanishi, Masanobu; Kuraoka, Isao; Yagi, Takashi

    2014-08-01

    Cisplatin (cis-diamminedichloroplatinum(II)), a widely used anticancer drug, forms inter- and intra-strand DNA crosslinks. The major intra-strand crosslinks are Pt adducts at 1,2-d(GpG) and 1,3-d(GpNpG) (Pt-GG and Pt-GNG, respectively). Although most of the intra-strand crosslinks are removed by the nucleotide excision repair (NER), the remaining crosslinks can cause mutations through the translesion DNA synthesis (TLS) during chromosome replication. To understand the precise mechanism of cisplatin mutagenesis in human cells, the plasmid carrying a single Pt-GG or 1,3-d(GpTpG) crosslink (Pt-GTG) site-specifically in lacZ gene was constructed and propagated in NER-defective xeroderma pigmentosum cells. The plasmids retrieved from the cells were introduced into indicator bacterial cells to access frequencies of TLS and mutations. The experiments revealed that Pt-GTG blocked DNA replication more strongly and caused more mutations (29.1%) than Pt-GG (1.7%). Most mutations were G to A or T base changes at 5' G residue in the Pt-GTG crosslinks. These results indicate that the Pt-GTG crosslinks become effective obstacles for cancer cell division, and have an important role for cisplatin cancer therapy. PMID:25344160

  16. Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

    PubMed Central

    Yaghoubi, Shahriar S.; Campbell, Dean O.; Radu, Caius G.; Czernin, Johannes

    2012-01-01

    Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials. PMID:22509201

  17. Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter

    PubMed Central

    Williams, Scott S.; Cobo-Stark, Patricia; Hajarnis, Sachin; Aboudehen, Karam; Shao, Xinli; Richardson, James A.; Patel, Vishal

    2014-01-01

    Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1β, which is required for activity in transfected cells. Mutation of the HNF-1β-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1β. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1β is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues. PMID:24899057

  18. Mutagenesis induced by targeted alpha therapy using 213Bi-cDTPA-9.2.27 in lacZ transgenic mice.

    PubMed

    Allen, Barry J; So, Trina; Abbas Rizvi, Syed M; Song, Emma Y; Fernandez, Harvey R; Lutz-Mann, Louise

    2009-05-01

    Targeted alpha therapy utilizes alpha-emitting radionuclides conjugated to monoclonal antibodies to allow specific irradiation of cancer cells whilst sparing normal, healthy tissues. The mutagenic potential of (213)Bi conjugated to a human melanoma antigen-specific antibody (9.2.27) was examined using an in vivo transgenic mouse model containing multiple copies of a lacZ target gene in every cell, allowing the quantification and comparison of mutagenesis in different organs. Mice received an ip injection of 16.65 MBq of (213)Bi-cDTPA-9.2.27, and were sacrificed at 24 h, 1 w and 4 w post-injection. Pharmacokinetic studies gave the absorbed and effective doses for each organ. The mutant frequency and mutant spectra were analysed for the brain, spleen and kidneys. The brain and spleen did not show significant increases in induced mutation frequencies compared to spontaneous background levels or changes in mutant spectra, these results being independent of p53 status. However, elevated mutation frequencies and persistent size change mutations were observed in the kidneys, but are not significant at the p = 0.05 level. The effect of p53 status was also evident, as p53 heterozygotes displayed higher mutation frequencies than their wild-type counterparts, suggesting a reduction in the p53 gene may lead to an increased susceptibility to mutagenesis. These effects were time dependent and levels returned to those of the controls at 4 w post-irradiation, albeit with a predominant residue of size mutations. These effects were observed at activities very much higher than those expected for the therapy of human patients. As such, the induction of secondary cancer with the (213)Bi-cDTPA-9.2.27 alpha immunoconjugate is not expected to be a significant problem in the clinic. PMID:19337032

  19. Simultaneous gene inactivation and promoter reporting in cyanobacteria.

    PubMed

    Chen, Kangming; Xu, Xinyi; Gu, Liping; Hildreth, Michael; Zhou, Ruanbao

    2015-02-01

    Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms. PMID:25434810

  20. Luciferase as a reporter of gene activity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since their development and introduction in the early days of plant genetic engineering, reporter genes have established a proven track record as effective tools for exploring the molecular underpinnings of gene regulation. When driven by appropriate genetic control systems (e.g. transcriptional pr...

  1. Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction.

    PubMed

    Gordon, J W

    2001-04-01

    The risk of insertion of adenovirus gene therapy DNA into female germ cells during the course of somatic gene therapy was stringently tested in the mouse by injecting up to 10(10) infectious particles directly into the ovary and by incubating naked oocytes in a solution of 2 x 10(8) particles/ml for 1 h prior to in vitro fertilization (IVF). The vector used was a recombinant adenovirus carrying the bacterial lacZ gene driven by the cytomegalovirus promoter (Adbeta-gal). Ovaries were stained for LacZ activity, or immunochemically for LacZ, 5-7 days after injection. Although very large amounts of LacZ activity and protein were detected, all positive staining was in the thecal portion of the ovary, with no staining seen in oocytes. In another series of experiments, mice with injected ovaries were mated, and preimplantation embryos or fetuses were analyzed either for LacZ expression or by PCR for lacZ DNA. None of 202 preimplantation embryos stained positively for LacZ and none of 58 fetuses were positive for DNA by PCR analysis. Finally, more than 1400 eggs were fertilized after exposure to the vector prior to IVF and stained as morulae for LacZ activity. Fewer than 2% of the embryos stained positively for LacZ, and experiments indicated that the staining was due to incomplete washing of the eggs prior to IVF. These data provide strong evidence that adenoviruses cannot infect oocytes and that the risk of female germ-line transduction with such vectors is very low. PMID:11319918

  2. A saturation screen for cis-acting regulatory DNA in the Hox genes of Ciona intestinalis

    SciTech Connect

    Keys, David N.; Lee, Byung-in; Di Gregorio, Anna; Harafuji, Naoe; Detter, Chris; Wang, Mei; Kahsai, Orsalem; Ahn, Sylvia; Arellano, Andre; Zhang, Quin; Trong, Stephan; Doyle, Sharon A.; Satoh, Noriyuki; Satou, Yutaka; Saiga, Hidetoshi; Christian, Allen; Rokhsar, Dan; Hawkins, Trevor L.; Levine, Mike; Richardson, Paul

    2005-01-05

    A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

  3. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    NASA Technical Reports Server (NTRS)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  4. Design and interpretation of microRNA-reporter gene activity.

    PubMed

    Carroll, Adam P; Tooney, Paul A; Cairns, Murray J

    2013-06-15

    MicroRNAs (miRNAs) are small noncoding RNA molecules that act as sequence specificity guides to direct post-transcriptional gene silencing. In doing so, miRNAs regulate many critical developmental processes, including cellular proliferation, differentiation, migration, and apoptosis, as well as more specialized biological functions such as dendritic spine development and synaptogenesis. Interactions between miRNAs and their miRNA recognition elements occur via partial complementarity, rendering tremendous redundancy in targeting such that miRNAs are predicted to regulate 60% of the genome, with each miRNA estimated to regulate more than 200 genes. Because these predictions are prone to false positives and false negatives, there is an ever present need to provide material support to these assertions to firmly establish the biological function of specific miRNAs in both normal and pathophysiological contexts. Using schizophrenia-associated miR-181b as an example, we present detailed guidelines and novel insights for the rapid establishment of a streamlined miRNA-reporter gene assay and explore various design concepts for miRNA-reporter gene applications, including bidirectional miRNA modulation. In exemplifying this approach, we report seven novel miR-181b target sites for five schizophrenia candidate genes (DISC1, BDNF, ENKUR, GRIA1, and GRIK1) and dissect a number of vital concepts regarding future developments for miRNA-reporter gene assays and the interpretation of their results. PMID:23481915

  5. Another reading of NAS gene report

    SciTech Connect

    Colwell, R.K.

    1988-06-01

    This commentary, which was prepared at the request of the Public Affairs Committee of the Ecological Society of America, suggest that the summary sections of the report focus on reassuring the public and oversimplify complex issues. The author comments on positive applications of genetic engineering such as the hardening of species threatened with extinction and biodegradation of industrial wastes. However, he warns that the biologists' ability to manipulate an organism's genome has exceeded the knowledge of the ecology of those species. Schemes to exploit agriculturally marginal habitats may have severe ecological consequences such as changes in community structure. Engineering the organism for a specific function would ensure the ability of the organism to carry out its intended function without negative impact on the environment. Genetic engineering promises to be an important component of a larger, integrated approach towards stopping the deterioration of the global ecosystem.

  6. Depletion of poly(ADP-ribose) polymerase-1 reduces host cell reactivation of a UV-damaged adenovirus-encoded reporter gene in human dermal fibroblasts.

    PubMed

    Ghodgaonkar, Medini M; Zacal, Natalie; Kassam, Shaqil; Rainbow, Andrew J; Shah, Girish M

    2008-04-01

    In response to ultraviolet radiation (UV), mammalian cells rapidly activate a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP), and we recently showed that one of the causes for PARP-activation is UV-induced direct DNA photolesions which are repaired by nucleotide excision repair process (NER). To determine whether PARP can play a role in NER, we stably depleted PARP in NER-proficient human skin fibroblasts GM637 by DNA vector-based RNAi. In these cells, we examined host cell reactivation (HCR) of UVB or UVC-irradiated recombinant adenovirus AdCA35lacZ, encoding a beta-galactosidase (beta-gal) reporter gene. The depletion of PARP decreased the HCR of UVB- or UVC-damaged reporter gene to a similar extent, indicating the role of PARP in NER. Moreover, PARP-depletion reduced the HCR capacity of the NER-competent GM637 cells to a level closer to that in the XP-C and CS-B cell lines, which are deficient in the lesion recognition steps of the global genome repair (GGR) and transcription-coupled repair (TCR) sub-pathways of NER, respectively. In order to identify the potential role of PARP in these two sub-pathways of NER from that of its known role in base excision repair (BER) of UVB-induced oxidant damage, we depleted PARP from XP-C and CS-B cells and examined HCR of the reporter gene damaged by UVB, UVC or photoactivated methylene blue, the latter causing predominantly 8-oxo-2'-deoxyguanosine damage that is repaired by BER. Interestingly, a decreased HCR due to PARP-depletion was observed in both the NER-deficient cell lines in response to virus damaged by these three agents, albeit with different kinetics from 12 to 44h after infection. The role of PARP in NER was highlighted by a decreased clonogenic survival of UV-irradiated NER-competent GM637 cells depleted of PARP. Our results, while confirming the role of PARP in base excision repair, suggest a novel role of PARP in both the GGR and TCR sub-pathways of NER. PMID:18289944

  7. Genetics in methylotrophic bacteria: Appendix. Final report

    SciTech Connect

    Lidstrom, M.E.

    1998-09-01

    This research has focused primarily on promoters in Methylobacterium extorquens AM1 and in methanotrophic bacteria. In Methylobacterium extorquens work continued on the moxF promoter. The author constructed chromosomal lacZ fusions of this promoter to avoid the regulation problems of plasmid-borne fragments and has shown that this is regulated normally in the chromosome. She has constructed lacZ fusions to some of the mox genes involved in the synthesis of the cofactor, PQQ, in order to carry out similar analysis of transcription of PQQ genes. The author has continued to isolate mox genes in methanotrophs for the purpose of studying their promoters and transcriptional regulation.

  8. Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

    PubMed

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2013-02-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis. PMID:22992803

  9. Photoacoustic imaging of gene expression using tyrosinase as a reporter gene

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Forbrich, Alexander; Harrison, Tyler; Hitt, Mary; Zemp, Roger J.

    2011-03-01

    Optical reporter genes, such as green fluorescence protein, are powerful research tools that allow visualization of gene expression. We have successfully used tyrosinase as a reporter gene for photoacoustic imaging. Tyrosinase is the key regulatory enzyme in the production of melanin which has a broad optical absorption spectrum. MCF-7 cells were stably transfected with tyrosinase under the control of an inducible promoter. For photoacoustic experiments, MCF-7 cells were resuspended at 108 cells/mL and injected in 700 μm (inner diameter) plastic tubing. Photoacoustic signal of MCF-7 cells expressing tyrosinase were >20-fold greater than those of untransfected MCF-7 cells. Photoacoustic signal of tyrosinaseexpressing MCF-7 cells were approximately 2-fold lesser and greater than those of blood at 576 and 650 nm, respectively, suggesting that photoacoustic signal from blood and tyrosinase-expressing cells can be separated by dualwavelength analysis. Photoacoustic signal from tyrosinase-expressing MCF-7 cells covered by chicken tissue could even be detected at a laser penetration depth of 4 cm, suggesting that tyrosinase can be used to image gene expression in relatively deep tissues. The current data suggests that tyrosinase is a strong reporter gene for photoacoustic imaging.

  10. Evaluating Reported Candidate Gene Associations with Polycystic Ovary Syndrome

    PubMed Central

    Pau, Cindy; Saxena, Richa; Welt, Corrine Kolka

    2013-01-01

    Objective To replicate variants in candidate genes associated with PCOS in a population of European PCOS and control subjects. Design Case-control association analysis and meta-analysis. Setting Major academic hospital Patients Women of European ancestry with PCOS (n=525) and controls (n=472), aged 18 to 45 years. Intervention Variants previously associated with PCOS in candidate gene studies were genotyped (n=39). Metabolic, reproductive and anthropomorphic parameters were examined as a function of the candidate variants. All genetic association analyses were adjusted for age, BMI and ancestry and were reported after correction for multiple testing. Main Outcome Measure Association of candidate gene variants with PCOS. Results Three variants, rs3797179 (SRD5A1), rs12473543 (POMC), and rs1501299 (ADIPOQ), were nominally associated with PCOS. However, they did not remain significant after correction for multiple testing and none of the variants replicated in a sufficiently powered meta-analysis. Variants in the FBN3 gene (rs17202517 and rs73503752) were associated with smaller waist circumferences and variant rs727428 in the SHBG gene was associated with lower SHBG levels. Conclusion Previously identified variants in candidate genes do not appear to be associated with PCOS risk. PMID:23375202

  11. Successful Transfection of Genes Using AAV-2/9 Vector in Swine Coronary and Peripheral Arteries

    PubMed Central

    Pankajakshan, Divya; Makinde, Toluwalope O.; Gaurav, Rohit; Del Core, Michael; Hatzoudis, George; Pipinos, Iraklis; Agrawal, Devendra K.

    2011-01-01

    Background Gene therapy has attracted attention for its potential to treat several cardiovascular diseases. The use of adeno-associated viral (AAV) vectors to facilitate therapeutic gene transfer to suppress intimal hyperplasia is a promising concept. The objective of this study was to analyze the in vivo transduction of a novel recombinant AAV-2/9 vector with SM22α promoter, containing β-galactosidase gene (Lac Z) or green fluorescent protein (GFP) as reporter genes, to the medial layer smooth muscle cells (SMCs) of swine coronary and peripheral arteries. Methods The AAV2/9 vector containing SM22α (1×1013 pfu) were administered into carotid/femoral/coronary arteries of domestic swine using irrigating balloon catheter-based gene delivery. Following gene transfer, cryosections of arteries were processed for X-Gal and GFP analysis. Fluorescence microscopy and Western blotting were done to analyze the GFP expression in the SMCs. Results LacZ mRNA expression was visualized in the medial layer 7 days after vector administration. The GFP expression was detected at 7th day and lasted for at least 2 months showing the longer-lasting expression of the AAV2/9-vector. Control arteries did not show any expression of GFP or LacZ. There was no significant effect of AAV2/9 viral transduction on serum amylase, fibrinogen and serum CRP levels. Conclusion These finding support the use of AAV2/9 as a vector to effectively transduce a gene in SMCs of coronary and peripheral arteries without causing inflammation. PMID:21529824

  12. Ferritin reporter used for gene expression imaging by magnetic resonance

    SciTech Connect

    Ono, Kenji; Fuma, Kazuya; Tabata, Kaori; Sawada, Makoto

    2009-10-23

    Magnetic resonance imaging (MRI) is a minimally invasive way to provide high spatial resolution tomograms. However, MRI has been considered to be useless for gene expression imaging compared to optical imaging. In this study, we used a ferritin reporter, binding with biogenic iron, to make it a powerful tool for gene expression imaging in MRI studies. GL261 mouse glioma cells were over-expressed with dual-reporter ferritin-DsRed under {beta}-actin promoter, then gene expression was observed by optical imaging and MRI in a brain tumor model. GL261 cells expressing ferritin-DsRed fusion protein showed enhanced visualizing effect by reducing T2-weighted signal intensity for in vitro and in vivo MRI studies, as well as DsRed fluorescence for optical imaging. Furthermore, a higher contrast was achieved on T2-weighted images when permeating the plasma membrane of ferritin-DsRed-expressing GL261. Thus, a ferritin expression vector can be used as an MRI reporter to monitor in vivo gene expression.

  13. Design considerations for incorporating sodium iodide symporter reporter gene imaging into prostate cancer gene therapy trials.

    PubMed

    Siddiqui, Farzan; Barton, Kenneth N; Stricker, Hans J; Steyn, Phillip F; Larue, Susan M; Karvelis, Kastytis C; Sparks, Richard B; Kim, Jae Ho; Brown, Stephen L; Freytag, Svend O

    2007-04-01

    This study was done to aid in the design of a phase I gene therapy trial in patients with prostate cancer. We determined the dosimetric characteristics of our reporter gene system when coupled with intravenous administration of radioactive sodium pertechnetate (Na(99m) TcO(4)) and determined the feasibility of using human sodium iodide symporter (hNIS) as a reporter gene to study the dynamics of adenoviral transgene expression in a large animal tumor. A replication-competent Ad5-yCD/mutTK(SR39) rep-hNIS adenovirus was injected into the prostate gland of dogs for dosimetry purposes, and into a canine soft tissue sarcoma (STS) for imaging purposes. After resection of the prostate, the amount of (99m)TcO(4)() sequestered in the prostate was determined, the radiation dose absorbed by the prostate and nontarget critical organs was calculated, and hNIS reporter gene expression was imaged in the STS by single-photon emission computed tomography (SPECT). On the basis of the findings from 25 dogs, the amount of (99m)TcO (4)() sequestered in the prostate ranged from 13 to 276 muCi. Using the highest value observed, absorbed radiation dose to critical organs was calculated and found to be below U.S. Food and Drug Administration limits for diagnostic imaging. Also, (99m)TcO (4)() uptake was readily detected by SPECT and found to persist in vivo for at least 4 days. On the basis of our dosimetry calculations, up to five imaging procedures can be safely performed in humans after intraprostatic injection of the Ad5-yCD/mutTK(SR39)rep-hNIS adenovirus and the hNIS reporter gene system can be used to study the dynamics of adenoviral gene therapy vectors in large animal tumors. PMID:17408358

  14. Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli.

    PubMed Central

    Bharatan, Shanti M; Reddy, Manjula; Gowrishankar, J

    2004-01-01

    A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state. PMID:15020459

  15. Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli.

    PubMed

    Bharatan, Shanti M; Reddy, Manjula; Gowrishankar, J

    2004-02-01

    A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state. PMID:15020459

  16. Robust reconstruction of gene expression profiles from reporter gene data using linear inversion

    PubMed Central

    Zulkower, Valentin; Page, Michel; Ropers, Delphine; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    Motivation: Time-series observations from reporter gene experiments are commonly used for inferring and analyzing dynamical models of regulatory networks. The robust estimation of promoter activities and protein concentrations from primary data is a difficult problem due to measurement noise and the indirect relation between the measurements and quantities of biological interest. Results: We propose a general approach based on regularized linear inversion to solve a range of estimation problems in the analysis of reporter gene data, notably the inference of growth rate, promoter activity, and protein concentration profiles. We evaluate the validity of the approach using in silico simulation studies, and observe that the methods are more robust and less biased than indirect approaches usually encountered in the experimental literature based on smoothing and subsequent processing of the primary data. We apply the methods to the analysis of fluorescent reporter gene data acquired in kinetic experiments with Escherichia coli. The methods are capable of reliably reconstructing time-course profiles of growth rate, promoter activity and protein concentration from weak and noisy signals at low population volumes. Moreover, they capture critical features of those profiles, notably rapid changes in gene expression during growth transitions. Availability and implementation: The methods described in this article are made available as a Python package (LGPL license) and also accessible through a web interface. For more information, see https://team.inria.fr/ibis/wellinverter. Contact: Hidde.de-Jong@inria.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26072511

  17. Development of a LacZ-based transcriptional reporter system for use with Moraxella catarrhalis.

    PubMed

    Evans, Amanda S; Pybus, Christine; Hansen, Eric J

    2013-03-01

    The lack of a transcriptional reporter system for use in Moraxella catarrhalis has hindered studies of gene regulation in this pathogen. PCR and recombinant DNA methods were used to insert a multicloning site (MCS) and promoterless full-length Escherichia coli lacZ gene, flanked by transcriptional terminators both immediately upstream and downstream, into the M. catarrhalis recombinant plasmid pWW115. Insertion into the MCS in the newly constructed plasmid pASE222 of M. catarrhalis promoter regions controlled by either a repressor (i.e., NsrR) or activator (i.e., PhoB) yielded transcriptional fusion constructs that were appropriately responsive to signal inputs dependent on the host strain genotype, as measured quantitatively by means of a Miller β-galactosidase assay. The transcriptional reporter plasmid pASE222 should prove to be a useful tool for rapid screening of factors affecting gene expression in M. catarrhalis. PMID:23219721

  18. Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea.

    PubMed

    O'Brien, Jason M; Walker, Mike; Sivathayalan, Ahalya; Douglas, George R; Yauk, Carole L; Marchetti, Francesco

    2015-05-01

    The transgenic rodent mutation assay was used to compare the dose-response relationship of lacZ mutant frequency (MF) in spermatogonial stem cells exposed acutely or subchronically to N-ethyl-N-nitrosourea (ENU). Muta(™) Mouse males were exposed orally to 0, 25, 50, or 100 mg/kg ENU for acute exposures and 0, 1, 2, or 5 mg/(kg day) for 28-day subchronic exposures. LacZ MF was measured in sperm collected 70 days post-exposure to target spermatogonial stem cells. Dose-response data were fit to linear, quadratic, exponential, or power models. Acute exposure resulted in a dose-dependent increase in MF that was significant (P < 0.05) at all doses tested and was best described by a quadratic dose-response model that was linear in the low dose range. In contrast, similar total doses fragmented over a 28-day subchronic exposure only resulted in a significant increase in lacZ MF at the highest dose tested. Therefore, the subchronic no observable genotoxic effect level (NOGEL) was 2 mg/(kg day) (or 56 mg/kg total dose). The subchronic dose-response was best described by the exponential and power models, which were sublinear in the low dose range. Benchmark dose lower confidence limits (BMDLs) for acute and subchronic exposure were 3.0 and 1.0 mg/(kg day) (or 27.4 mg/kg total dose), respectively. These findings are supportive of a saturable DNA repair mechanism as the mutagenic mode of action for ENU in spermatogonia and imply that sufficiently low exposures would not cause appreciable genotoxic effects over background. This may have important implications for the quantitative risk assessment of germ cell mutagens. PMID:25598316

  19. Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis.

    PubMed Central

    Jaacks, K J; Healy, J; Losick, R; Grossman, A D

    1989-01-01

    We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. PMID:2502532

  20. A Brain-Specific Homeobox Gene, Bsx, Is Essential for Proper Postnatal Growth and Nursing▿

    PubMed Central

    McArthur, Tara; Ohtoshi, Akihira

    2007-01-01

    To investigate in vivo roles of a murine hypothalamic homeobox gene, Bsx, we generated and analyzed two mutant alleles, BsxΔHD and BsxlacZ. BsxΔHD lacks the homeodomain, and BsxlacZ is an insertion of a lacZ reporter gene. Bsx-lacZ expression was detected in the hypothalamus and pineal gland and reiterates Bsx expression. Bsx homozygous mutant mice were born at the expected Mendelian ratio, but their growth was impaired. Offspring from Bsx homozygous mutant females exhibited a low survival rate due to a nursing defect. Mammary glands of the mutant females developed normally during pregnancy; however, they involuted quickly after parturition. These results demonstrate that Bsx is required for postnatal growth and maintenance of lactating mammary glands. Thus, mouse Bsx is likely involved in systemic control of suppression of apoptosis of postpartum mammary epithelial cells. PMID:17485440

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

    SciTech Connect

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable 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.

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

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    Novel double and triple fusion reporter gene constructs harboring distinct imagable 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.

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

    DOEpatents

    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. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel cloning vector to aid in the construction of ß-galactosidase reporter systems for gene expression studies in lactose metabolizing strains of Shiga toxin producing Escherichia coli is described. The plasmid allows construction of translational fusions of cloned gene promoters with a short seg...

  5. Hazardous effects of effluent from the chrome plating industry: 70 kDa heat shock protein expression as a marker of cellular damage in transgenic Drosophila melanogaster (hsp70-lacZ).

    PubMed Central

    Mukhopadhyay, Indranil; Saxena, Daya Krishna; Chowdhuri, Debapratim Kar

    2003-01-01

    Hazardous effects of an effluent from the chrome plating industry were examined by exposing transgenic Drosophila melanogaster (hsp70-lacZ) to various concentrations (0.05, 0.1, 1.0, 10.0, and 100.0 micro L/mL) of the effluent through diet. The emergence pattern of adult flies was affected, along with impaired reproductive performance at the higher dietary concentrations of the effluent. Interestingly, the effect of the effluent was more pronounced in male than in female flies. The effect of the effluent on development of adult flies was concurrent with the expression pattern of the heat shock protein 70 gene (hsp70), both in larval tissues and in the reproductive organs of adult flies. We observed a dose- and time-dependent expression of hsp70 in third instar larvae exposed for different time intervals. Absence of hsp70 expression in larvae exposed to 0.1 micro L/mL of the effluent indicated that this is the highest nontoxic concentration for Drosophila. The stress gene assay in the reproductive organs of adult flies revealed hsp70 expression in the testis of male flies only. However, trypan blue dye exclusion tests in these tissues indicate tissue damage in the male accessory gland of adult flies, which was further confirmed by ultrastructural observations. In the present study we demonstrate the utility of transgenic Drosophila as an alternative animal model for evaluating hazardous effects of the effluent from the chrome plating industry and further reveal the cytoprotective role of hsp70 and its expression as an early marker in environmental risk assessment. PMID:14644668

  6. Photoacoustic molecular imaging of ferritin as a reporter gene

    NASA Astrophysics Data System (ADS)

    Ha, S.; Carson, A.; Kim, K.

    2012-02-01

    Spectral analysis of photoacoustic (PA) molecular imaging (PMI) of ferritin expressed in human melanoma cells (SK-24) was performed in vitro. Ferritin is a ubiquitously expressed protein which stores iron that can be detected by PA imaging, allowing ferritin to act as a reporter gene. To over-express ferritin, SK-24 cells were co-transfected with plasmid expressing Heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using LipofectamineTM 2000. Non-transfected SK-24 cells served as a negative control. Fluorescent imaging of EGFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation in SK-24 cells, a focused high frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (<20mJ/cm2), was used to scan the PA signal from 680 nm to 950 nm (in 10 nm increments) from the surface of the 6-well culturing plate. PA signal intensity from H-FT transfected SK-24 cells was not different from that of non-transfected SK-24 cells at wavelengths less than 770 nm, but was over 4 dB higher than non-transfected SK-24 cells at 850 ~ 950 nm. Fluorescent microscopy indicates significant accumulation of ferritin in H-FT transfected SK-24 cells, with little ferritin expression in non-transfected SK-24 cells. The PA spectral analysis clearly differentiates transfected SK-24 cells from nontransfected SK-24 cells with significantly increased iron signal at 850 ~ 950 nm, and these increased signals were associated with transfection of H-FT plasmid. As such, the feasibility of ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a new concept for PA imaging, and may provide various opportunities for molecular imaging and basic science research.

  7. Regulation of the EDG84A gene by FTZ-F1 during metamorphosis in Drosophila melanogaster.

    PubMed Central

    Murata, T; Kageyama, Y; Hirose, S; Ueda, H

    1996-01-01

    The transcription factor FTZ-F1 is a member of the nuclear hormone receptor superfamily and is transiently expressed during the mid- and late prepupal periods in Drosophila melanogaster. A putative pupal cuticle gene, EDG84A, is expressed slightly following FTZ-F1 expression during the prepupal period and carries a strong FTZ-F1 binding site between bases 100 and 92 upstream of its transcription start site. In this study, EDG84A mRNA was found to be prematurely expressed upon heat induction of FTZ-F1 in prepupae carrying the heat shock promoter-FTZ-F1 cDNA fusion gene construct. Transgenic fly lines having the 0.8-kb region of the EDG84A promoter fused to lacZ expressed the reporter gene in a tissue- and stage-specific manner. Base substitutions in the FTZ-F1 binding site within the 0.8-kb promoter abolished expression of lacZ. These results strongly suggest that the EDG84A gene is a direct target of FTZ-F1. Deletion studies of the cis-regulatory region of the EDG84A gene revealed that space-specific expression in imaginal disc-derived epidermis is controlled by the region between bp -408 and -104 from the transcription start site. The region between bp -408 and -194 is necessary to repress expression in a posterior part of the body, while the region between bp -193 and -104 carries a positive element for activation in an anterior part of the body. These results suggest that FTZ-F1 governs expression of the EDG84A gene in conjunction with putative tissue-specific regulators. PMID:8887679

  8. Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila

    PubMed Central

    Auer, Jasmin S.; Nagel, Anja C.; Schulz, Adriana; Wahl, Vanessa; Preiss, Anette

    2015-01-01

    In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H)MAPK-ko and a phospho-mimetic Su(H)MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vgBE-lacZ). In general, Su(H)MAPK-ko induced a stronger response than wild-type Su(H), whereas the response to Su(H)MAPK-ac was very weak. Notch target genes cut, wingless and vgBE-lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DERact) or the MAPK (rlSEM) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones. PMID:26702412

  9. Identification and characterization of hydrogen peroxide-sensitive mutants of Escherichia coli: genes that require OxyR for expression.

    PubMed Central

    Mukhopadhyay, S; Schellhorn, H E

    1997-01-01

    Escherichia coli produces an inducible set of proteins that protect the cell from exogenous peroxide stress. A subset of these genes is induced by hydrogen peroxide and is controlled at the transcriptional level by the OxyR protein. To identify additional genes involved in protection from hydrogen peroxide, a library of random transcriptional fusions of lambda(plac)Mu53 was screened for hydrogen peroxide sensitivity and 27 such mutants were identified. These fusions were transduced into nonlysogenic strains to ensure that the phenotypes observed were the result of a single mutation. The mutants were grouped into three classes based on the expression of the lacZ fusion during growth in oxyR+ and deltaoxyR backgrounds. The expression of the lacZ fusion in 8 mutants was independent of OxyR, 10 mutants required OxyR for expression, and 6 mutants showed reduced levels of expression in the presence of OxyR. OxyR dependence varied from 2- to 50-fold in these mutants. The OxyR-dependent phenotype was complemented by a plasmid-borne copy of oxyR gene in all mutants. Three mutants exhibited dual regulation by OxyR and RpoS. We sequenced the fusion junctions of several of these mutants and identified the genetic loci responsible for the hydrogen peroxide-sensitive (hps) phenotype. In this study, we report the identification of several genes that require OxyR for expression, including hemF (encoding coproporphyrinogen III oxidase), rcsC (encoding a sensor-regulator protein of capsular polysaccharide synthesis genes), and an open reading frame, f497, that is similar to arylsulfatase-encoding genes. PMID:8990283

  10. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1995-01-01

    Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.

  11. A fast-evolving human NPAS3 enhancer gained reporter expression in the developing forebrain of transgenic mice

    PubMed Central

    Kamm, Gretel B.; López-Leal, Rodrigo; Lorenzo, Juan R.; Franchini, Lucía F.

    2013-01-01

    The developmental brain gene NPAS3 stands out as a hot spot in human evolution because it contains the largest number of human-specific, fast-evolving, conserved, non-coding elements. In this paper we studied 2xHAR142, one of these elements that is located in the fifth intron of NPAS3. Using transgenic mice, we show that the mouse and chimp 2xHAR142 orthologues behave as transcriptional enhancers driving expression of the reporter gene lacZ to a similar NPAS3 expression subdomain in the mouse central nervous system. Interestingly, the human 2xHAR142 orthologue drives lacZ expression to an extended expression pattern in the nervous system. Thus, molecular evolution of 2xHAR142 provides the first documented example of human-specific heterotopy in the forebrain promoted by a transcriptional enhancer and suggests that it may have contributed to assemble the unique properties of the human brain. PMID:24218632

  12. Photoacoustic microscopy of tyrosinase reporter gene in vivo

    NASA Astrophysics Data System (ADS)

    Krumholz, Arie; Vanvickle-Chavez, Sarah J.; Yao, Junjie; Fleming, Timothy P.; Gillanders, William E.; Wang, Lihong V.

    2011-08-01

    Photoacoustic tomography is a hybrid modality based on optical absorption excitation and ultrasonic detection. It is sensitive to melanin, one of the primary absorbers in skin. For cells that do not naturally contain melanin, melanin production can be induced by introducing the gene for tyrosinase, the primary enzyme responsible for expression of melanin in melanogenic cells. Optical resolution photoacoustic microscopy was used in the ex vivo study reported here, where the signal from transfected cells increased by more than 10 times over wild-type cells. A subsequent in vivo experiment was conducted to demonstrate the capability of photoacoustic microscopy to spectrally differentiate between tyrosinase-catalyzed melanin and various other absorbers in tissue.

  13. (Genetic engineering with a gene encoding a soybean storage protein). Progress report

    SciTech Connect

    Beachy, R.N.

    1985-01-01

    Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the ..cap alpha..'-subunit of ..beta..-conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ACR)

  14. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals.

    PubMed Central

    Casadaban, M J; Chou, J; Cohen, S N

    1980-01-01

    We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments. Images PMID:6162838

  15. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy1

    PubMed Central

    Ruan, Hangjun; Su, Hua; Hu, Lily; Lamborn, Kathleen R; Kan, YW; Deen, Dennis F

    2001-01-01

    Abstract The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen) produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE) from the erythropoietin gene (Epo), which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1). Under anoxic conditions, nine copies of HRE (9XHRE) yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV) in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy. PMID:11494119

  16. Identification of genes in anonymous DNA sequences. Final report: Report period, 15 April 1993--15 April 1994

    SciTech Connect

    Fields, C.A.

    1994-09-01

    This Report concludes the DOE Human Genome Program project, ``Identification of Genes in Anonymous DNA Sequence.`` The central goals of this project have been (1) understanding the problem of identifying genes in anonymous sequences, and (2) development of tools, primarily the automated identification system gm, for identifying genes. The activities supported under the previous award are summarized here to provide a single complete report on the activities supported as part of the project from its inception to its completion.

  17. A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

    PubMed

    Pereira, Luiz Miguel; Baroni, Luciana; Yatsuda, Ana Patrícia

    2014-03-01

    Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii. PMID:24440296

  18. In vivo translation of a region within the rrnB 16S rRNA gene of Escherichia coli.

    PubMed Central

    Berg, K L; Squires, C L; Squires, C

    1987-01-01

    In this study we show that a segment of the Escherichia coli rrnB 16S gene can be translated in vivo. Other laboratories have previously reported that there are internal transcription and translation signals and open reading frames within the E. coli rrnB rRNA operon. Their studies revealed a translation start signal followed by a 252-base-pair open reading frame (ORF16) within the 16S gene and detected a promoter (p16) in the same general region by using in vitro RNA polymerase binding and transcription initiation assays. By using plasmid gene fusions of ORF16 to lacZ we showed that an ORF16'-'beta-galactosidase fusion protein was made in vivo. Transcripts encoding the fusion protein were expressed either from the rrnB p1p2 control region or from a hybrid trp-lac promoter (tacP), but the amount of expression was considerably less than for a lacZ control plasmid. We used fusions to the cat gene to show that p16 is one-half as active as lacP. Deletions were used to show that p16 is located within ORF16 and thus cannot promote a transcript encoding the ORF16 peptide. A comparison of sequences from different organisms shows that ORF16 and p16 lie in a highly conserved region of the procaryotic 16S RNA structure. The first 20 amino acids of ORF16 are conserved in most eubacterial and plant organellar sequences, and promoter activity has been detected in this region of the Caulobacter crescentus sequence by other workers. Images PMID:2435709

  19. Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2

    PubMed Central

    Forbes, Elaine M; Nieduszynska, Siân R; Brunton, Fiona K; Gibson, Joanne; Glover, L Anne; Stansfield, Ian

    2007-01-01

    Background In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot. Results The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA. Conclusion This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2. PMID:17961216

  20. Development of Timd2 as a reporter gene for MRI

    PubMed Central

    Patrick, P. Stephen; Rodrigues, Tiago B.; Kettunen, Mikko I.; Lyons, Scott K.; Neves, André A.

    2015-01-01

    Purpose To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T‐cell immunoglobulin and mucin domain containing protein 2), using T1‐ and T2‐weighted imaging. Methods Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron‐loaded or manganese‐loaded ferritin, were imaged using T1‐ and T2‐weighted MRI. Mice were also implanted subcutaneously with Timd2‐expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2‐weighted imaging. Results Timd2‐expressing cells, but not control cells, showed a large increase in both R2 and R1 in vitro following incubation with iron‐loaded and manganese‐loaded ferritin, respectively. Expression of Timd2 had no effect on cell viability or proliferation; however, manganese‐loaded ferritin, but not iron‐loaded ferritin, was toxic to Timd2‐expressing cells. Timd2‐expressing xenografts in vivo showed much smaller changes in R2 following injection of iron‐loaded ferritin than the same cells incubated in vitro with iron‐loaded ferritin. Conclusion Timd2 has demonstrated potential as an MRI reporter gene, producing large increases in R2 and R1 with ferritin and manganese‐loaded ferritin respectively in vitro, although more modest changes in R2 in vivo. Manganese‐loaded apoferritin was not used in vivo due to the toxicity observed in vitro. Magn Reson Med, 2015. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Magn Reson Med 75:1697–1707, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance. PMID:25981669

  1. Optical imaging of reporter gene expression using a positron-emission-tomography probe

    NASA Astrophysics Data System (ADS)

    Liu, Hongguang; Ren, Gang; Liu, Shuanglong; Zhang, Xiaofen; Chen, Luxi; Han, Peizhen; Cheng, Zhen

    2010-11-01

    Reporter gene/reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene/reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene/nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine ([18F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene/reporter probe techniques in medical research.

  2. Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator

    PubMed Central

    2012-01-01

    Background Glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNAAsp. Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. Results The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. Conclusions The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri. PMID:23035718

  3. The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants.

    PubMed

    Piruzian, E S; Monzavi-Karbassi, B; Darbinian, N S; Goldenkova, I V; Kobets, N S; Mochulsky, A V

    1998-03-01

    In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering. PMID:9563842

  4. The application of luciferase as a reporter of environmental regulation of gene expression in mycobacteria.

    PubMed

    Gordon, S; Parish, T; Roberts, I S; Andrew, P W

    1994-11-01

    This paper describes the construction of pSG10, the first mycobacterial promoter probe shuttle vector to use the structural gene of a bacterial luciferase as a reporter gene. To examine the utility of using bacterial luciferase to measure gene expression in mycobacteria, the authors have used this vector to monitor the induction of the acetamidase gene promoter of Mycobacterium smegmatis. Luciferase proved to be a rapid, sensitive and easily assayable reporter of changes in gene activity in response to environment in mycobacteria. PMID:7765445

  5. Selective Disruption of Genes Transiently Induced in Differentiating Mouse Embryonic Stem Cells by Using Gene Trap Mutagenesis and Site-Specific Recombination

    PubMed Central

    Thorey, Irmgard S.; Muth, Katrin; Russ, Andreas P.; Otte, Jürgen; Reffelmann, Armin; von Melchner, Harald

    1998-01-01

    A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development. Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of β-galactosidase (β-Gal). As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected. Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo. Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of β-Gal expression. PMID:9566926

  6. Glycerophosphorylcholine regulates Haemophilus influenzae glpQ gene expression.

    PubMed

    Alrousan, Enas; Fan, Xin

    2015-05-01

    An important virulence strategy adopted by Haemophilus influenzae to establish a niche on the mucosal surface of the host is the phosphorylcholine (ChoP) decoration of its lipopolysaccharides, which promotes adherence to the host cells. Haemophilus influenzae is able to use glycerophosphorylcholine (GPC) from host for ChoP synthesis. Utilization of GPC requires glpQ, which encodes a glycerophosphodiester phosphodiesterase enzyme. In this study, we investigate the transcriptional regulation of glpQ gene using real-time PCR and transcriptional fusion of H. influenzae glpQ promoter to the Escherichia coli lacZ reporter gene. The glpQ promoter activities were examined under environmental conditions including changes in temperature, oxygen, high salt and minimal growth medium. Our data showed that under room temperature and anaerobic conditions, the glpQ gene expression levels were significantly higher than under other growth conditions. In addition, the glpQ gene expression levels were upregulated in the presence of GPC. These results suggest that H. influenzae may upregulate glpQ expression in response to different environments it encounters during infection, from the airway surfaces (room temperature) to deep tissues (anaerobic). Upregulation of glpQ by GPC may allow efficient use of abundant GPC from mammalian cells by H. influenzae as a source of nutrient and for ChoP decoration of lipopolysaccharide that facilitates bacterial adhesion to host cells and growth during infection. PMID:25837816

  7. Using a Single Fluorescent Reporter Gene to Infer Half-Life of Extrinsic Noise and Other Parameters of Gene Expression

    PubMed Central

    Komorowski, Michał; Finkenstädt, Bärbel; Rand, David

    2010-01-01

    Abstract Fluorescent and luminescent proteins are often used as reporters of transcriptional activity. Given the prevalence of noise in biochemical systems, the time-series data arising from these is of significant interest in efforts to calibrate stochastic models of gene expression and obtain information about sources of nongenetic variability. We present a statistical inference framework that can be used to estimate kinetic parameters of gene expression, as well as the strength and half-life of extrinsic noise from single fluorescent-reporter-gene time-series data. The method takes into account stochastic variability in a fluorescent signal resulting from intrinsic noise of gene expression, kinetics of fluorescent protein maturation, and extrinsic noise, which is assumed to arise at transcriptional level. We use the linear noise approximation and derive an explicit formula for the likelihood of observed fluorescent data. The method is embedded in a Bayesian paradigm, so that certain parameters can be informed from other experiments allowing portability of results across different studies. Inference is performed using Markov chain Monte Carlo. Fluorescent reporters are primary tools to observe dynamics of gene expression and the correct interpretation of fluorescent data is crucial to investigating these fundamental processes of cellular life. As both magnitude and frequency of the noise may have a dramatic effect on the cell fitness, the quantification of stochastic fluctuation is essential to the understanding of how genes are regulated. Our method provides a framework that addresses this important question. PMID:20550887

  8. Mannan-conjugated adenovirus enhanced gene therapy effects on murine hepatocellular carcinoma cells in vitro and in vivo.

    PubMed

    Liu, Zhongbing; Ke, Famin; Duan, Chenggang; Lan, Huan; Li, Juan; Gao, Cen; Li, Jinwei; Zhong, Zhirong

    2013-08-21

    The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. For some patients for whom surgical treatments are not appropriate, one can only rely on chemotherapy. In the conventional chemotherapy, side effects usually occurred in most cases due to high toxicity levels. Moreover, the development of drug resistance toward chemotherapeutic agents often prevents the successful long-term use of chemotherapy for HCC. Gene therapy represents the exciting biotechnological advance that may revolutionize the conventional fashion of cancer treatment. Overexpression of phosphatase and tensin homologue (PTEN) in cancer cells carrying deletion/mutant type of it can induce the apoptosis of cancer cells and inhibit cell proliferation. In this work, in order to make full use of the high transfectivity of adenovirus, we managed to conjugate the polysaccharide mannan (polymannose) to the surface of the adenovirus chemically under appropriate oxidizing conditions to prepare the mannan-modified adenovirus (Man-Ad5-PTEN). The cytotoxicity and anticancer activity of Man-Ad5-PTEN were assessed in vitro. Reporter gene expression of LacZ transferred by Man-Ad5-LacZ was verified on mannose receptor-deficient NIH/3T3 cells versus mannose receptor-efficient macrophages. Hepatocellular carcinoma cell lines transduced by mannan-modified adenovirus were assayed for cell cycle, apoptosis, invasion, and migration. Further, we detected the antitumor effect on intraperitoneal H22 tumor-bearing mice treated by Man-Ad5-PTEN alone or combined with chemotherapeutic agent of doxorubicin. The results demonstrated that cell growth suppression was not observed in Chang normal hepatocyte cells and the cell killing by Man-Ad5-PTEN is tumor selective. Further, the results showed that the strategy of mannan conjugation could enhance adenovirus-mediated PTEN gene therapy effects on murine hepatocellular carcinoma cells in vitro and in vivo. PMID:23937094

  9. Two medfly promoters that have originated by recent gene duplication drive distinct sex, tissue and temporal expression patterns.

    PubMed Central

    Christophides, G K; Livadaras, I; Savakis, C; Komitopoulou, K

    2000-01-01

    Genes encoding predominantly male-specific serum polypeptides (MSSPs) in the medfly Ceratitis capitata are members of a multigene family that are structurally similar to the genes encoding odorant binding proteins of insects. To study the transcriptional regulation of the genes MSSP-alpha2 and MSSP-beta2, overlapping fragments of their promoters, containing the 5' UTRs and 5' flanking regions, were fused to the lacZ reporter gene and introduced into the medfly genome via Minos-mediated germline transformation. Transgenic flies were functionally assayed for beta-galactosidase activity. Despite their extensive sequence similarity, the two gene promoters show distinct expression patterns of the reporter gene, consistent with previously reported evidence for analogous transcriptional activity of the corresponding endogenous genes. The MSSP-alpha2 promoter drives gene expression specifically in the fat body of the adult males, whereas the MSSP-beta2 promoter directs gene expression in the midgut of both sexes. In contrast, similar transformation experiments in Drosophila melanogaster showed that both promoters drive the expression of the reporter gene in the midgut of adult flies of both sexes. Thus, the very same MSSP-alpha2 promoter fragment directs expression in the adult male fat body in Ceratitis, but in the midgut of both sexes in Drosophila. Our data suggest that through the evolution of the MSSP gene family a limited number of mutations that occurred within certain cis-acting elements, in combination with new medfly-specific trans-acting factors, endowed these recently duplicated genes with distinct sex-, tissue-, and temporal-specific expression patterns. PMID:10978283

  10. Toxic potential of municipal solid waste leachates in transgenic Drosophila melanogaster (hsp70-lacZ): hsp70 as a marker of cellular damage.

    PubMed

    Bhargav, Devyani; Pratap Singh, Mahendra; Murthy, Ramesh Chandra; Mathur, N; Misra, Divya; Saxena, Daya Krishna; Kar Chowdhuri, Debapratim

    2008-02-01

    Municipal solid wastes (MSWs) are one of the major sources of environmental pollution. Leachates from these wastes might contaminate the water sources and affect quality of environment. The study was carried out to determine the possible toxic effects of leachates from MSW in transgenic Drosophila melanogaster (hsp70-lacZ). Third instar larvae exposed to 1.0-3.0% of these leachates at different time intervals were examined for hsp70 expression, oxidative stress enzyme activities, proteotoxicity, tissue damage along with effect on emergence and reproduction. Maximum hsp70 expression was observed in the larvae exposed to highly acidic leachates. Overwhelming of hsp70 expression in the exposed larvae caused a concomitant decline in total protein content and a significant elevation in oxidative stress enzymes and lipid peroxidation (LPO) product. The leachates caused a significant delay in emergence of flies and affected the reproductive performance of the flies at the tested concentrations. The present study highlights the toxic potential of MSW leachates and the advantage of Drosophila as a model to evaluate the impact of leachates at organismal and cellular levels, also advocating Hsp70 as the first tier indicator of toxicity. PMID:17300838

  11. Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

    PubMed

    Ho, Pei-i; Yue, Kimberley; Pandey, Pramod; Breault, Lyne; Harbinski, Fred; McBride, Aaron J; Webb, Brian; Narahari, Janaki; Karassina, Natasha; Wood, Keith V; Hill, Adam; Auld, Douglas S

    2013-05-17

    Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes. PMID:23485150

  12. Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors

    PubMed Central

    Al Ali, Sally; Baldanta, Sara; Fernández-Escobar, Mercedes; Guerra, Susana

    2016-01-01

    Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. PMID:27213433

  13. Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

    PubMed Central

    Garcia, Miguel; Pimentel, Madalena; Moniz-Pereira, José

    2002-01-01

    A mycobacteriophage Ms6 strong promoter region (Plys) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem σ70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region Plys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation. PMID:12003945

  14. First Report of the Multiresistance Gene cfr in Streptococcus suis

    PubMed Central

    Wang, Yang; Li, Dexi; Song, Li; Liu, Yang; He, Tao; Liu, Hebing; Wu, Congming

    2013-01-01

    The multiresistance gene cfr was identified for the first time in streptococci, namely, in porcine Streptococcus suis isolate S10. The cfr gene was detected on the ∼100-kb plasmid pStrcfr, where it was bracketed by two copies of the novel insertion sequence ISEnfa5, located in the same orientation. The detection of a cfr- and ISEnfa5-containing amplicon by inverse PCR suggests that ISEnfa5 may play a role in the dissemination of cfr. PMID:23733472

  15. Signal transduction pathways that regulate CAB gene expression. Progress report

    SciTech Connect

    Chory, J.

    1993-12-31

    We have completed the initial genetic and phenotypic characterization of several classes of new mutants that affect CAB gene expression. The doc mutants (for dark overexpression of cab) are characterized by elevated levels of CAB gene expression in the dark; however, unlike the previously isolated de-etiolated mutants (also isolated in my lab), the doc mutants still appear etiolated. The doc alleles define 3 loci, each of which maps to a separate chromosome. The details of the mutant isolation scheme and the genetic and phenotypic description of these new mutants are described. The second class of mutants, the gun mutants (for genomes uncoupled) show accumulation of CAB mRNA in the absence of chloroplast gene expression and development. Thus, the normally tightly coordinated expression between the chloroplast and nuclear genes that encode chloroplast-destined proteins has been uncoupled. We have shown that the Arabidopsis HY3 locus encodes the type B phytochrome apoprotein gene and have characterized the phenotypes of null hy3 alleles to ascertain a role for this phytochrome in Arabidopsis development. We have also isolated and characterized a number of alleles of the phytochrome A gene.

  16. Multiple reporter gene assays for the assessment and estimation of chemical toxicity.

    PubMed

    Takahashi, Junko; Iwahashi, Hitoshi

    2004-01-01

    To detect chemical toxicity, we are making new bioassay systems that use promoters selected from yeast DNA microarray experiments. We performed multiple reporter gene assays using the promoters of these genes; the promoter regions were inserted upstream of green fluorescence protein (GFP). In this report, six genes (HSP26, MET17, YLL057C, FIT2, CUP1 and OYE3) were selected and assays were carried out for 55 chemicals. The promoters of these genes showed different responses to chemicals within 4 h. This result indicates that this technique enables us to predict the toxicity of chemicals in the environment and to understand toxicities of newly synthesized chemicals. PMID:15746902

  17. The plant mitochondrial mat-r gene/nad1 gene complex. Progress report

    SciTech Connect

    Wolstenholme, D.R.

    1994-06-01

    The authors have completed sequencing the segments (totalling 19 kb, both complementary strands) of the maize mtDNA molecule that encode the entire NADH dehydrogenase subunit (nadl) gene. They have identified nucleotides in mature transcripts of the nadl gene that are edited and have generated clones of cDNAs of entire mature (fully spliced) nadl transcripts. They have examined the relative rates of splicing in transcripts of the four nadl gene group II introns and begun examining nadl intron cDNAs to determine the extent and distribution of RNA edits in introns, in order to evaluate the possibility that intron excision and exon splicing might be editing independent.

  18. Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications.

    PubMed

    Zampini, Massimiliano; Mur, Luis A J; Rees Stevens, Pauline; Pachebat, Justin A; Newbold, C James; Hayes, Finbarr; Kingston-Smith, Alison

    2016-01-01

    Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology. PMID:27220405

  19. Inflammatory bowel disease gene discovery. CRADA final report

    SciTech Connect

    1997-09-09

    The ultimate goal of this project is to identify the human gene(s) responsible for the disorder known as IBD. The work was planned in two phases. The desired products resulting from Phase 1 were BAC clone(s) containing the genetic marker(s) identified by gene/Networks, Inc. as potentially linked to IBD, plasmid subclones of those BAC(s), and new genetic markers developed from these plasmid subclones. The newly developed markers would be genotyped by gene/Networks, Inc. to ascertain evidence for linkage or non-linkage of IBD to this region. If non-linkage was indicated, the project would move to investigation of other candidate chromosomal regions. Where linkage was indicated, the project would move to Phase 2, in which a physical map of the candidate region(s) would be developed. The products of this phase would be contig(s) of BAC clones in the region exhibiting linkage to IBD, as well as plasmic subclones of the BACs and further genetic marker development. There would also be continued genotyping with new polymorphic markers during this phase. It was anticipated that clones identified and developed during these two phases would provide the physical resources for eventual disease gene discovery.

  20. Bioluminescent reporters for catabolic gene expression and pollutant bioavailability

    SciTech Connect

    Heitzer, A.; DiGrazia, P.M.; Sayler, G.S. . Center for Environmental Biotechnology); Burlage, R.S. )

    1991-01-01

    The application of visualized catabolic nah-gene expression using a luxCDABE gene fusion provides a valuable method to measure quantitatively and specifically naphthalene and salicylate bioavailability. It has been demonstrated that the physiological state of the test culture together with the intrinsic regulation mechanisms of the naphthalene degradation pathway as well as the physiological aspects of the lux gene fusion have to be taken into account. The method presented provides a high potential for in situ bioprocess monitoring. In addition, the results obtained with immobilized cells provide a basis for the development of biosensors for environmental applications in specific pollutant monitoring in waste streams and soil slurry systems but, as a general method, also for more conventional biotechnological process control. 8 refs., 2 figs., 1 tab.

  1. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies

    PubMed Central

    Lu, Yujie; Darne, Chinmay D.; Tan, I-Chih; Zhu, Banghe; Hall, Mary A.; Lazard, ZaWaunyka W.; Davis, Alan R.; Simpson, LaShan; Sevick-Muraca, Eva M.; Olmsted-Davis, Elizabeth A.

    2013-01-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models. PMID:24104323

  2. Sleeping Beauty Transposon Vectors in Liver-directed Gene Delivery of LDLR and VLDLR for Gene Therapy of Familial Hypercholesterolemia.

    PubMed

    Turunen, Tytteli A K; Kurkipuro, Jere; Heikura, Tommi; Vuorio, Taina; Hytönen, Elisa; Izsvák, Zsuzsanna; Ylä-Herttuala, Seppo

    2016-03-01

    Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH. PMID:26670130

  3. Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis.

    PubMed

    Liu, Y; Abaibou, H; Fletcher, H M

    2000-11-01

    Several reports have supported the association of Porphyromonas gingivalis with periodontal disease. Genetic studies are vital for understanding the relative importance of virulence factors in this organism. Thus, gene reporters may prove useful for the study of gene expression in this organism. We have investigated the use of the green fluorescent protein (GFP), bacterial luciferase, and bifunctional xylosidase/arabinosidase enzyme (XA) as reporters of gene expression in P. gingivalis. Fusion cassettes containing the promoterless tetracycline resistant gene [tetA(A)Q2] and the promoterless gfp, luxAB, or xa gene were placed under the control of the rgpA promoter in P. gingivalis W83 using recombinational allelic exchange. The rgpA gene encodes for an arginine-specific protease in P. gingivalis. No GFP activity was detected in P. gingivalis isogenic mutants carrying the rgpA::gfp-tetA(Q)2 fusion construct. Luciferase activity in P. gingivalis mutants carrying the rgpA::luxAB-tetA(Q)2 fusion was only detected in the presence of exogenous FMNH(2). xa gene expression in P. gingivalis with the rgpA::xa-tetA(Q)2 fusion construct was detected in crude extracts using rho-nitrophenol derivatives as substrate and on agar plates with methylumbelliferyl derivatives under long-wave ultraviolet light. This indicates that both luxAB and xa genes can be used as reporters of gene expression in P. gingivalis. However, only the xa gene can be used as a noninvasive reporter gene. PMID:11078651

  4. MAMMALIAN CELL GENE MUTATION ASSAYS WORKING GROUP REPORT

    EPA Science Inventory

    Mammalian cell gene mutation assays have been used for many years and the diversity of the available systems attests to the varied methods found to grow mammalian dells and detect mutations. s part of the International Workshop on Standardization of Genotoxicity Test Procedures, ...

  5. Structural Explanation for Allolactose (lac Operon Inducer) Synthesis by lacZ β-Galactosidase and the Evolutionary Relationship between Allolactose Synthesis and the lac Repressor

    PubMed Central

    Wheatley, Robert W.; Lo, Summie; Jancewicz, Larisa J.; Dugdale, Megan L.; Huber, Reuben E.

    2013-01-01

    β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-β-galactosidase) that traps allolactose. Various lines of evidence indicate that the glucose of the trapped allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol) and l-ribose in the site and kinetic binding studies with substituted β-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795–803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of β-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of β-galactosidase played an important role in lac operon evolution. PMID:23486479

  6. Isolation and characterization of Vsx1, a novel mouse CVC paired-like homeobox gene expressed during embryogenesis and in the retina.

    PubMed

    Ohtoshi, A; Justice, M J; Behringer, R R

    2001-08-10

    Gastrula stage mouse embryo RNA was screened by degenerate RT-PCR to yield a novel paired-like homeobox gene. The open reading frame encoded by the cDNA was most similar to human VSX1. Mouse Vsx1 encodes a protein of 363 amino acid residues that contains a CVC domain that was originally identified as a conserved motif among mouse CHX10, goldfish VSX-1 and C. elegans CEH-10. Linkage analysis showed that mouse Vsx1 mapped to the distal region of chromosome 2. RT-PCR analysis detected mouse Vsx1 transcripts from gastrulation and post-gastrulation stage mouse embryos, suggesting a role for Vsx1 during mouse embryogenesis. Analysis of the eyes of mouse chimeras generated with embryonic stem cells in which a lacZ reporter was targeted to the Vsx1 locus suggested that Vsx1 is expressed in the inner nuclear layer of the retina. PMID:11485319

  7. Analysis of Gene Targeting & Nonhomologous End-joining. Final Report

    SciTech Connect

    Haber, J. E.

    2002-11-30

    Overall, we identified a number of new proteins that participate in nonhomologous end-joining and also in telomere addition to the ends of broken chromosomes. We showed that NHEJ is severely reduced in cells expressing both yeast mating-type genes and then went on to identify the NEJ1 gene that was under this control. We showed the epistasis relations among a set of mutations that impair telomere addition and we showed that there are in fact two pathways to repair broken chromosomes in the absence of telomerase. We characterized the DNA damage checkpoint pathway in response to a single broken chromosome and characterized especially the adaptation of cells arrested by an unrepaired DSB. We demonstrated that the DNA damage response is nuclear-limited. We showed adaptation defects for Tid1and Srs2 proteins and showed that Srs2 was also recovery-defective, even when DNA was repaired.

  8. 5'-coding sequence of the nasA gene of Azotobacter vinelandii is required for efficient expression.

    PubMed

    Wang, Baomin; Wang, Yumei; Kennedy, Christina

    2014-10-01

    The operon nasACBH in Azotobacter vinelandii encodes nitrate and nitrite reductases that sequentially reduce nitrate to nitrite and to ammonium for nitrogen assimilation into organic molecules. Our previous analyses showed that nasACBH expression is subject to antitermination regulation that occurs upstream of the nasA gene in response to the availability of nitrate and nitrite. In this study, we continued expression analyses of the nasA gene and observed that the nasA 5'-coding sequence plays an important role in gene expression, as demonstrated by the fact that deletions caused over sixfold reduction in the expression of the lacZ reporter gene. Further analysis suggests that the nasA 5'-coding sequence promotes gene expression in a way that is not associated with weakened transcript folding around the translational initiation region or codon usage bias. The findings from this study imply that there exists potential to improve gene expression in A. vinelandii by optimizing 5'-coding sequences. PMID:25110215

  9. A specific library of randomly integrated reporter genes for the isolation of inducible functions by cell sorting

    SciTech Connect

    Lapeyre, J.N.; Marini, F.; Gratzner, H.G. AMC ImmunoDiagnostics, Houston, TX )

    1993-01-01

    A library of cells containing randomly integrated reporter genes has been constructed. The purpose of this library is to enable the isolation of genes of interest which are inducible by radiation, biological response modifiers, cytokines, or other agents. These genes are located near reporter genes which can be induced by the upstream promoter of the gene of interest. The reporter gene, Lac Z, was randomly inserted into the genome by retroviral transduction and subsequent selection of the neo[sup r] gene with gentamycin. Studies of radiation inducible genes were undertaken, whereby cells with the radiation sensitive function were isolated by sorting the cells fluorescent after staining with the beta gal substrate, fluorescein digalactoside (FDG). This gene-tagging approach is an improvement over the cDNA library subtraction protocol in that a single library of cells with random marker gene integration can be repeatedly and sequentially probed by sorting under different, selective conditions, dependent upon the genes to be characterized.

  10. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

    PubMed

    West, David B; Engelhard, Eric K; Adkisson, Michael; Nava, A J; Kirov, Julia V; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J; Lloyd, Kent C

    2016-02-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  11. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

    PubMed Central

    Adkisson, Michael; Nava, A. J.; Kirov, Julia V.; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J.; Lloyd, Kent C.

    2016-01-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  12. Regulation of spo0H, a gene coding for the Bacillus subtilis sigma H factor.

    PubMed Central

    Weir, J; Predich, M; Dubnau, E; Nair, G; Smith, I

    1991-01-01

    The Bacillus spo0H gene codes for sigma H, which, as part of the RNA polymerase holoenzyme E sigma H, is responsible for the transcription of several genes which are expressed at the beginning of the sporulation process. In this communication, we examined the regulation of the spo0H gene of Bacillus subtilis by using lacZ reporter gene assays, quantitative RNA determinations, and Western immunoassay. The expression of the spo0H gene increases as the culture enters the mid-logarithmic stage of growth. This increased expression requires the genes spo0A, spo0B, spo0E, and spo0F, and the requirement for at least spo0A and spo0B can be bypassed when the abrB gene is mutated. The expression of the spo0H gene is constitutive in the presence of the abrB mutation, being expressed at higher levels during vegetative growth. In addition, the sof-1 mutation, in the spo0A structural gene, can bypass the need for spo0F in spo0H expression. The transcriptional start site of spo0H was determined by using RNA made in vivo as well as in vitro. These studies indicate that spo0H is transcribed by the major vegetative RNA polymerase, E sigma A. spo0H RNA and sigma H levels during growth are not identical to each other or to the pattern of expression of spoVG, a gene transcribed by E sigma H. This suggests that spo0H is regulated posttranscriptionally and also that factors in addition to sigma H levels are involved in the expression of genes of the E sigma H regulon. Images PMID:1898930

  13. Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction.

    PubMed

    Aoyama, Yutaka; Kobayashi, Koichi; Morishita, Yoshihiro; Maeda, Kengo; Murohara, Toyoaki

    2015-07-01

    The wnt signaling pathway plays important roles in development and in many diseases. Recently several reports suggest that non-canonical Wnt proteins contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on virus-induced myocarditis have not been explored. Here, we investigated the effect of Wnt11 protein in a model of myocarditis induced by coxsackievirus B3 (CVB3) using recombinant adeno-associated virus 9 (rAAV9). The effect of Wnt11 gene therapy on a CVB3-induced myocarditis model was examined using male BALB/c mice. Mice received a single intravenous injection of either rAAV9-Wnt11 or rAAV9-LacZ 2 weeks before intraperitoneal administration of CVB3. Intravenous injection of the rAAV9 vector resulted in efficient, durable, and relatively cardiac-specific transgene expression. Survival was significantly greater among rAAV9-Wnt11 treated mice than among mice treated with rAAV9-LacZ (87.5% vs. 54.1%, P < 0.05). Wnt11 expression also reduced the infiltration of inflammatory cells, necrosis of the myocardium, and suppressed the mRNA expression of inflammatory cytokines. This is the first report to show that Wnt11 expression improves the survival of mice with CVB3-induced myocarditis. AAV9-mediated Wnt11 gene therapy produces beneficial effects on cardiac function and increases the survival of mice with CVB3-induced myocarditis through the suppression of both infiltration of inflammatory cells and gene expression of inflammatory cytokines. PMID:25886696

  14. Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea.

    PubMed

    Gregor, D; Pfeifer, F

    2001-07-01

    The bgaH reading frame encoding a beta-galactosidase of 'Haloferax alicantei' was used as a reporter gene to investigate three different promoter regions derived from gvpA genes of Haloferax mediterranei (mc-gvpA) and Halobacterium salinarum (c-gvpA and p-gvpA) in Haloferax volcanii transformants. The fusion of bgaH at the start codon of each gvpA reading frame (A1-bgaH fusion genes) caused translational problems in some cases. Transformants containing constructs with fusions further downstream in the gvpA reading frame (A-bgaH) produced beta-galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The beta-galactosidase activities quantified by standard ONPG assays correlated well with the mRNA data determined with transformants containing the respective gvpA genes: the cA-bgaH fusion gene was completely inactive, the mcA-bgaH transformants showed low amounts of products, whereas the pA-bgaH fusion gene was constitutively expressed in the respective transformants. The transcription of each A-bgaH gene was activated by the homologous transcriptional activator protein GvpE. The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA-bgaH and mcA-bgaH, whereas the promoter of cA-bgaH was only activated by cGvpE. Among the three GvpE proteins tested, cGvpE appeared to be the strongest transcriptional activator. PMID:11429452

  15. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1991--May 31, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-05-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  16. MS-1 magA: Revisiting Its Efficacy as a Reporter Gene for MRI.

    PubMed

    Pereira, Sofia M; Williams, Steve R; Murray, Patricia; Taylor, Arthur

    2016-01-01

    Bacterial genes involved in the biomineralization of magnetic nanoparticles in magnetotactic bacteria have recently been proposed as reporters for magnetic resonance imaging (MRI). In such systems, the expression of the bacterial genes in mammalian cells purportedly leads to greater concentrations of intracellular iron or the biomineralization of iron oxides, thus leading to an enhancement in relaxation rate that is detectable via MRI. Here, we show that the constitutive expression of the magA gene from Magnetospirillum magnetotacticum is tolerated by human embryonic kidney (HEK) cells but induces a strong toxic effect in murine mesenchymal/stromal cells and kidney-derived stem cells, severely restricting its effective use as a reporter gene for stem cells. Although it has been suggested that magA is involved in iron transport, when expressed in HEK cells, it does not affect the transcription of endogenous genes related to iron homeostasis. Furthermore, the magA-induced enhancement in iron uptake in HEK cells is insignificant, suggesting this gene is a poor reporter even for cell types that can tolerate its expression. We suggest that the use of magA for stem cells should be approached with caution, and its efficacy as a reporter gene requires a careful assessment on a cell-by-cell basis. PMID:27118760

  17. Role of starvation genes in the survival of deep subsurface bacterial communities. Final report

    SciTech Connect

    Matin, A.; Schmidt, T.; Caldwell, D.

    1998-11-01

    The investigation dealt with several aspects of subsurface bacterial survival and their nature. Mutants of Pseudomonas putida, a common environmental bacterium with counterparts in the subsurface, were isolated by transposon mutagenesis. These mutants were highly sensitive to starvation stress. Reporter gene fusions also showed that these genes were starvation genes since they were induced several fold when the cultures were started. Since the regulatory religions (promoters) of starvation genes are of interest in bioremediation and in experiments designed to understand the roles of starvation genes in the maintenance of microbial community structure, the promoter of one of these genes (pstarv1, contained in strain MK107) was characterized in detail. As a preliminary to these studies, the growth characteristics of Pseudomonas putida MK1 and MK107 were compared for cells growing in batch cultures or as an attached monolayer in microstat cultures.

  18. Improved lux reporters for use in Staphylococcus aureus.

    PubMed

    Mesak, Lili Rosana; Yim, Grace; Davies, Julian

    2009-05-01

    The use of luxABCDE (lux) offers certain advantages over other reporters, such as: lacZ and xylE. It is real time and its signal generation is produced without the requirement for any additional substrates. In some bacteria such as Staphylococcus spp, light production by luciferase is restricted because of a limited availability of endogenous substrates such as fatty acid aldehyde. We describe the construction of promoterless-lux cloning vectors, pGYlux and pAmilux. S. aureus carrying B. subtilis xyl/tetO promoter fused to the lux genes of pGYlux gave up to a 2.5-fold enhancement of luminescence over S. aureus carrying the xyl/tetO promoter fused to lux genes of the previously published parent vector pAL2. Furthermore, pAmilux showed a 6-fold enhancement of lux expression when compared to pGYlux in S. aureus. This was achieved by cloning the constitutive ami promoter upstream of the luxCDE genes to increase endogenous fatty acid aldehyde production while maintaining its reporter functionality by fusing promoters to the luxAB genes. PMID:19399993

  19. Gene Therapy for Bladder Overactivity and Nociception with Herpes Simplex Virus Vectors Expressing Preproenkephalin

    PubMed Central

    Yokoyama, Hitoshi; Sasaki, Katsumi; Franks, Michael E.; Goins, William F.; Goss, James R.; de Groat, William C.; Glorioso, Joseph C.; Chancellor, Michael B.

    2009-01-01

    Abstract Interstitial cystitis/painful bladder syndrome (IC/PBS) is a major challenge to treat. We studied the effect of targeted and localized expression of enkephalin in afferent nerves that innervate the bladder by gene transfer using replication-defective herpes simplex virus (HSV) vectors in a rat model of bladder hyperactivity and pain. Replication-deficient HSV vectors encoding preproenkephalin, which is a precursor for Met- and Leu-enkephalin, or control vector encoding the lacZ reporter gene, were injected into the bladder wall of female rats. After viral vector injection, quantitative polymerase chain reaction showed high preproenkephalin transgene levels in bladder and dorsal root ganglia innervating the bladder in enkephalin vector-treated animals. Functionally, enkephalin vector-treated animals showed reductions in bladder hyperactivity and nociceptive behavior induced by intravesical application of capsaicin; however, vector-mediated expression of enkephalin did not alter normal voiding. This antinociceptive effect of enkephalin gene therapy was antagonized by naloxone hydrochloride administration. Together, our results with HSV vectors encoding preproenkephalin demonstrated physiological improvement in visceral pain induced by bladder irritation. Thus, gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as IC/PBS. PMID:20377371

  20. An Approach for Treating the Hepatobiliary Disease of Cystic Fibrosis by Somatic Gene Transfer

    NASA Astrophysics Data System (ADS)

    Yang, Yiping; Raper, Steven E.; Cohn, Jonathan A.; Engelhardt, John F.; Wilson, James M.

    1993-05-01

    Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.

  1. The profile of melatonin receptors gene expression and genes associated with their activity in colorectal cancer: a preliminary report.

    PubMed

    Ziółko, E; Kokot, T; Skubis, A; Sikora, B; Szota-Czyż, J; Kruszniewska-Rajs, C; Wierzgoń, J; Mazurek, U; Grochowska-Niedworok, E; Muc-Wierzgoń, M

    2015-01-01

    The antiproliferative and immunomodulatory effects of melatonin (MLT) have been demonstrated in a variety of neoplasms including colorectal cancer (CRC). In humans and other mammals, MLT acts on target tissues through membrane and retinoid nuclear receptors. The aim of this study was to evaluate transcription activity of melatonin receptors and genes associated with regulation of their activity in colorectal adenocarcinoma tissues in relation to clinical stage of cancer. A total of 24 pairs of surgically removed tumoral and healthy (marginal) tissue samples from colorectal cancer patients at clinical stages I-II and III-IV were collected. As an additional control, twenty normal samples were tak¬en from people whose large intestine tissues were reported as non-tumoral after colonoscopy. Expression of mRNA genes was studied by microarray HG-U133A analysis. The analysis of gene expression profile was performed using commercially available oligonucleotide microarrays of HG-U133A. High increase of MT1 mRNA expression levels in all cancerous samples vs non-cancerous tissues was observed. The MT2 mRNA expression levels increased slightly in marginal and malignant samples. Among the genes participating in the cascade of signal transfer in cells activated by MLT via melatonin receptors, we found encoding genes (GNA11, OXTR, TPH1) only for differentiating stage III - IV of CRC. Monitoring the expression levels of genes that are related to melatonin receptors may offer a strategy to anticipate tumour development and estimate the molecular changes that occur during carcinogenesis. The mechanism behind this association needs further elucidation. PMID:26753642

  2. Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging

    PubMed Central

    Qin, Chunxia; Cheng, Kai; Chen, Kai; Hu, Xiang; Liu, Yang; Lan, Xiaoli; Zhang, Yongxue; Liu, Hongguang; Xu, Yingding; Bu, Lihong; Su, Xinhui; Zhu, Xiaohua; Meng, Shuxian; Cheng, Zhen

    2013-01-01

    Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR) – the key enzyme in melanin production – was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo. PMID:23508226

  3. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1994-01-01

    Consistent with the long term goal of our research to understand the nature of the key enzymes in eukaryotic DNA replication we have characterized the properties of the wild type DNA polymerases of the {alpha}-family and their mutants. We have also provided evidence for the role of aphidicolin in the elongation process of the in vivo DNA replication in eukaryotic cells. We also developed a technology for planned prep from a large numbers of clones for direct screening by size or restriction digestion in order to facilitate our goals to clone the DNA polymerase gene.

  4. Dual luciferase gene reporter assays to study miRNA function.

    PubMed

    Clment, Thomas; Salone, Vronique; Rederstorff, Mathieu

    2015-01-01

    This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not a miRNA can bind to the UTR and regulate its expression. PMID:25791601

  5. Bacteriophage P1 Bof protein is an indirect positive effector of transcription of the phage bac-1 ban gene in some circumstances and a direct negative effector in other circumstances.

    PubMed Central

    Schaefer, T S; Hays, J B

    1991-01-01

    Previous genetic studies have suggested that the Bof protein of bacteriophage P1 can act as both a negative and a positive regulator of phage gene expression: in bof-1 prophages, the ref gene and a putative phage ssb gene are derepressed, but expression of an operator-semiconstitutive variant of the phage ban gene (bac-1) is markedly reduced. An explanation of this apparent duality is suggested by recent reports that Bof is a corepressor of genes that are regulated by the phage C1 repressor, including the autoregulated c1 gene itself. Here we show, by means of operon fusions to lacZ, that the balance points between Bof-mediated decreases in c1 expression and Bof-mediated increases in C1 efficacy are different among various C1-regulated genes. Thus, expression of Bof by P1 prophages affects some genes (e.g., bac-1 ban) positively, and others (e.g., ref) negatively. Even at bac-1 ban, where the positive indirect effect of Bof is physiologically dominant, Bof can be seen to act as a corepressor if C1 is supplied from a nonautoregulated (ptac-c1) source, eliminating the effect of Bof on C1 synthesis. PMID:1917872

  6. p53 Status and gene transfer experiments using CMV enhancer/promoter.

    PubMed

    Allamane, S; Ratel, D; Jourdes, P; Berger, F; Benabid, A L; Wion, D

    2001-01-12

    Comparison of transfection efficiencies between different commercial reagents or methods is a matter of major concern in the field of gene therapy. Transfection efficiencies are usually evaluated by the quantification of a reporter gene expression (i.e., luciferase or lacZ) whose expression is usually driven by the CMV promoter. However, this experimental approach does not consider the possible effects of the transfection on the activity of the promoter used to drive reporter genes expression. Using p53 null fibroblasts we show that transfection efficiency estimated by the use of pCMV-luc or pCMV-betagal plasmids may be dramatically affected by the cell p53 status. These data highlight the fact that differences in p53 levels may be one of the parameters involved in the variation of transfection efficiencies observed with different cell lines. Furthermore, they point to the fact that comparison of transfection efficiencies should distinguish differences in the efficiency of transfection from differences in the level of transcription of the transgene. Finally they suggest that the known p53 down-regulation of the CMV promoter should be considered in order to avoid the nonintentional construction of transfer vectors in which the expression of a transgene down-modulates its own promoter. PMID:11162475

  7. Utility of an appropriate reporter assay: Heliotrine interferes with GAL4/upstream activation sequence-driven reporter gene systems.

    PubMed

    Luckert, Claudia; Hessel, Stefanie; Lampen, Alfonso; Braeuning, Albert

    2015-10-15

    Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals. PMID:26212314

  8. Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model

    PubMed Central

    Yang, Feng-Yi; Chang, Wen-Yuan; Lin, Wei-Ting; Hwang, Jeng-Jong; Chien, Yi-Chun; Wang, Hsin-Ell; Tsai, Min-Lan

    2015-01-01

    The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2′-fluoro-2′-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases. PMID:26429860

  9. Serum response factor, an enriched cardiac mesoderm obligatory factor, is a downstream gene target for Tbx genes.

    PubMed

    Barron, Matthew R; Belaguli, Narasimhaswamy S; Zhang, Shu Xiang; Trinh, Mimi; Iyer, Dinaker; Merlo, Xanthi; Lough, John W; Parmacek, Michael S; Bruneau, Benoit G; Schwartz, Robert J

    2005-03-25

    We tested the idea that T-box factors direct serum response factor (SRF) gene activity early in development. Analysis of SRF-LacZ "knock-in" mice showed highly restricted expression in early embryonic cardiac and skeletal muscle mesoderm and neuroectoderm. Examination of the SRF gene for regulatory regions by linking the promoter and 5'-flanking sequences, up to 5.5 kb, failed to target LacZ transgene activity to the heart and the tail pre-somitic mesenchyme. However, linkage of a minimal SRF promoter with the SRF 3'-untranslated region (UTR), inundated with multimeric T-box binding sites (TBEs), restored robust reporter gene activity to embryonic heart and tail. Finer dissection of the 3'-UTR to a small cluster of TBEs also stimulated transgene activity in the cardiac forming region and the tail, however, when the TBEs contained within these DNA sequences were mutated, preventing Tbx binding, transgene activity was lost. Tbx2, Tbx5, and the cardiac-enriched MYST family histone acetyltransferase TIP60, were observed to be mutual interactive cofactors through the TIP60 zinc finger and the T-box of the Tbx factors. In SRF-null ES cells, TIP60, Tbx2, and Tbx5 were sufficient to stimulate co-transfected SRF reporter activity, however this activity required the presence of the SRF 3'-UTR. SRF gene transactivation was blocked by two distinct TIP60 mutants, in which either the histone acetyltransferase domain was inactivated or the Zn finger-protein binding domain was excised. Our study supports the idea that SRF embryonic cardiac gene expression is dependent upon the SRF 3'-UTR enhancer, Tbx2, Tbx5, and TIP60 histone acetyltransferase activity. PMID:15591049

  10. Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression

    PubMed Central

    Yang, Dongshan; Song, Jun; Zhang, Jifeng; Xu, Jie; Zhu, Tianqing; Wang, Zhong; Lai, Liangxue; Chen, Y. Eugene

    2016-01-01

    The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5′ RACE and 3′ RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits. PMID:27117226

  11. Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression.

    PubMed

    Yang, Dongshan; Song, Jun; Zhang, Jifeng; Xu, Jie; Zhu, Tianqing; Wang, Zhong; Lai, Liangxue; Chen, Y Eugene

    2016-01-01

    The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5' RACE and 3' RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits. PMID:27117226

  12. Isolation of Crt Mutants Constitutive for Transcription of the DNA Damage Inducible Gene Rnr3 in Saccharomyces Cerevisiae

    PubMed Central

    Zhou, Z.; Elledge, S. J.

    1992-01-01

    Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis. It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication. To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid. Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents. Of these, 200 are recessive and fall into 9 complementation groups. In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway. Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth. Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6. crt6-68 and crt7-240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature. The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response. crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs. These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network. PMID:1516817

  13. Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

    PubMed Central

    Weber, Tillmann; Renzland, Insa; Baur, Max; Mönks, Simon; Herrmann, Elke; Huppert, Verena; Nürnberg, Frank; Schönig, Kai; Bartsch, Dusan

    2012-01-01

    The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood. PMID:22693598

  14. An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells.

    PubMed Central

    Hosler, B A; Rogers, M B; Kozak, C A; Gudas, L J

    1993-01-01

    The message for the zinc finger gene Rex-1 (Zfp-42) is expressed in undifferentiated murine F9 teratocarcinoma cells and embryonic stem cells. Expression of Rex-1 is reduced at the transcriptional level when F9 cells are induced by the addition of retinoic acid (RA) to differentiate. We have isolated genomic DNA for the Rex-1 gene (Zfp-42), characterized the gene's structure, and mapped the gene to mouse chromosome 8. Promoter elements contributing to the regulation of the Rex-1 promoter in F9 cells have been identified. A region required for Rex-1 promoter activity in F9 stem cells contains an octamer motif (ATTTGCAT) which is a binding site for octamer transcription factor members of the POU domain family of DNA-binding proteins. Rex-1 reporter plasmids including this octamer site also exhibited reduced expression in F9 cells treated with RA. Thus, the octamer motif is a regulatory element required for the activity of the Rex-1 promoter in F9 stem cells, and this motif contributes to the negative regulation by RA of the transcription of the Rex-1 gene. As an initial confirmation of the in vivo relevance of the isolated fragment, a larger Rex-1 promoter fragment, also containing the octamer site, was able to promote expression of the bacterial lacZ gene in mouse embryos at the morula stage. Images PMID:8474450

  15. AB158. Report of a Gardner’s syndrome case with an APC gene mutation

    PubMed Central

    Ngo, Phuoc; Nguyen, Suong; Bui, Lam; Nguyen, Tron; Huynh, Khai; Bui, Minh; Do, Thuy

    2015-01-01

    Background Gardner’s syndrome is an autosomal dominant disorder with complete penetrance, caused by mutations in the adenomatous polyposis coli gene(APC gene). Gardner’s syndrome is characterized by intestinal polyposis, osteomas and dental abnormalities. APC mutations are mostly point mutations, causing a truncated and dysfunctional APC protein. Detection of mutations in APC gene from a patient with Gardner’s syndrome. Methods A 19-year-old female patient with typical symptoms of Gardner’s syndrome was sent from the Hospital of Odonto-Stomatology, Ho Chi Minh City for detection of APC gene mutations. We performed APC gene sequencing and used bioinformatics tools to detect APC gene mutations and predict the effects of mutations. Results We detected the p.Gln1517ArgfsX6 mutation in APC gene and analyzed the effects of this mutation on functions of the APC protein. Conclusions We reported a p.Gln1517ArgfsX6 mutation in APC gene from a patient with Gardner’s syndrome.

  16. Robust marking of photoreceptor cells and pinealocytes with several reporters under control of the Crx gene.

    PubMed

    Samson, Maria; Emerson, Mark M; Cepko, Constance L

    2009-12-01

    Crx is a member of the Otx family of homeobox genes with expression restricted to vertebrate retinal photoreceptor and bipolar cells as well as the pinealocytes of the pineal organ. To facilitate the visualization of Crx-expressing cells, we generated transgenic mice expressing several reporters under the control of the Crx regulatory sequences present within a bacterial artificial chromosome (BAC). These mice expand the transgenic mouse collection, which uses photoreceptor regulatory elements for reporter gene expression by providing a broader repertoire of reporter genes. In addition, because Crx is expressed very soon after a cell fated to be a photoreceptor cell becomes postmitotic, they provide a means for early identification of immature photoreceptor cells. PMID:19882727

  17. In situ gene transfer and suicide gene therapy of gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine in dogs.

    PubMed

    Matsukura, N; Hoshino, A; Igarashi, T; Hasegawa, H; Okino, T; Onda, M; Iijima, O; Akiyama, K; Goto, T; Takubo, K; Suzuki, S; Shimada, T

    1999-09-01

    Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV-TK) gene was introduced in situ into cancer tissues in the stomach of three dogs, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus-producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus-producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAGlacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV-TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV-TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAGlacZ gene transfer, but was found after high-titer Ad.CAGHSV-TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage. PMID:10551335

  18. First Case Report of EX3del4765 Mutation in PAH Gene in Asian Population

    PubMed Central

    Soltani, Ziba; Karami, Fatemeh; Yassaee, Vahidreza; Hashemi Gorji, Feyzollah; Talebzadeh, Mahdieh; Miryounesi, Mohammad

    2016-01-01

    Introduction: Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine metabolism, which is caused by mutation in phenylalanine hydroxylase (PAH) gene. Most of the PAH mutations are missense mutations (67%), which are followed by small or large deletions (13%). Case Presentation: We reported a patient with classic PKU and his parents harboring a large deletion in exon 3 (EX3del4765) of PAH gene. This is the first case report of EX3del4765 in Asian patients with PKU. Conclusions: This finding may help improve early detection, differential diagnosis, genetic counseling, and even treatment of patients with PKU.

  19. Genetics and molecular biology of methanogen genes. Final report

    SciTech Connect

    Konisky, J.

    1997-10-07

    Adenylate kinase has been isolated from four related methanogenic members of the Archaea. For each the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 C for Methanococcus voltage, 70 C for Methanococcus thermolithotrophicus, 80 C for Methanococcus igneus and 80--90 C for Methanococcus jannaschii. The enzymes were sensitive to the adenylate kinase inhibitor, Ap{sub 5}A [P{sup 1}, P{sup 5}-di(adenosine-5{prime}) pentaphosphate], a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography. The enzymes had an estimated molecular weight (approximately 23--25 kDa) in the range common for adenylate kinases. Each of the enzymes had a region of amino acid sequence close to its N-terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases. However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases including an enzyme isolated from the Archeon, Sulfolobus acidocaldarius. If verified as a nucleotide binding domain, the methanogen sequence would represent a novel nucleotide binding motif. There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.

  20. Gene transcription and electromagnetic fields. Final progress report

    SciTech Connect

    Henderson, A.S.

    1992-12-31

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

  1. Conditional Gene Targeting in Mouse Pancreatic β-Cells

    PubMed Central

    Wicksteed, Barton; Brissova, Marcela; Yan, Wenbo; Opland, Darren M.; Plank, Jennifer L.; Reinert, Rachel B.; Dickson, Lorna M.; Tamarina, Natalia A.; Philipson, Louis H.; Shostak, Alena; Bernal-Mizrachi, Ernesto; Elghazi, Lynda; Roe, Michael W.; Labosky, Patricia A.; Myers, Martin G.; Gannon, Maureen; Powers, Alvin C.; Dempsey, Peter J.

    2010-01-01

    OBJECTIVE Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain. CONCLUSIONS Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells. PMID:20802254

  2. FOXP2 gene deletion and infant feeding difficulties: a case report

    PubMed Central

    Zimmerman, Emily; Maron, Jill L.

    2016-01-01

    Forkhead box protein P2 (FOXP2) is a well-studied gene known to play an essential role in normal speech development. Deletions in the gene have been shown to result in developmental speech disorders and regulatory disruption of downstream gene targets associated with common forms of language impairments. Despite similarities in motor planning and execution between speech development and oral feeding competence, there have been no reports to date linking deletions within the FOXP2 gene to oral feeding impairments in the newborn. The patient was a nondysmorphic, appropriately and symmetrically grown male infant born at 35-wk gestational age. He had a prolonged neonatal intensive care unit stay because of persistent oral feeding incoordination requiring gastrostomy tube placement. Cardiac and neurological imagings were within normal limits. A microarray analysis found an ∼9-kb loss within chromosome band 7q3.1 that contains exon 2 of FOXP2, demonstrating a single copy of this region instead of the normal two copies per diploid gene. This case study expands our current understanding of the role FOXP2 exerts on motor planning and coordination necessary for both oral feeding success and speech–language development. This case report has important consequences for future diagnosis and treatment for infants with FOXP2 deletions, mutations, and varying levels of gene expression. PMID:27148578

  3. FOXP2 gene deletion and infant feeding difficulties: a case report.

    PubMed

    Zimmerman, Emily; Maron, Jill L

    2016-01-01

    Forkhead box protein P2 (FOXP2) is a well-studied gene known to play an essential role in normal speech development. Deletions in the gene have been shown to result in developmental speech disorders and regulatory disruption of downstream gene targets associated with common forms of language impairments. Despite similarities in motor planning and execution between speech development and oral feeding competence, there have been no reports to date linking deletions within the FOXP2 gene to oral feeding impairments in the newborn. The patient was a nondysmorphic, appropriately and symmetrically grown male infant born at 35-wk gestational age. He had a prolonged neonatal intensive care unit stay because of persistent oral feeding incoordination requiring gastrostomy tube placement. Cardiac and neurological imagings were within normal limits. A microarray analysis found an ∼9-kb loss within chromosome band 7q3.1 that contains exon 2 of FOXP2, demonstrating a single copy of this region instead of the normal two copies per diploid gene. This case study expands our current understanding of the role FOXP2 exerts on motor planning and coordination necessary for both oral feeding success and speech-language development. This case report has important consequences for future diagnosis and treatment for infants with FOXP2 deletions, mutations, and varying levels of gene expression. PMID:27148578

  4. The Ice Nucleation Gene from Pseudomonas syringae as a Sensitive Gene Reporter for Promoter Analysis in Zymomonas mobilis

    PubMed Central

    Drainas, C.; Vartholomatos, G.; Panopoulos, N. J.

    1995-01-01

    The expression of the ice nucleation gene inaZ from Pseudomonas syringae in Zymomonas mobilis strains under the control of three different promoters was investigated to establish the utility of the gene as a reporter and examine the possible use of the organism as a source of ice nuclei for biotechnological applications. A promoterless version of the inaZ gene was placed under the control of three different promoters: P(infpdc) (pyruvate decarboxylase), a homologous strong promoter from Z. mobilis; P(infbla) ((beta)-lactamase) of plasmid pBR325; and P(infhrpR), the promoter of hrpR, a regulatory gene from P. syringae pv. phaseolicola. The apparent strengths of all three promoters, measured by quantifying the ice nucleation activity at -9 deg C, were lower in Z. mobilis than in Escherichia coli. The levels of ice nucleation activity expressed under the P(infpdc) promoter were significantly higher than those obtained with the two heterologous promoters in Z. mobilis. Plasmid pCG4521 (RK2 replicon) gave much lower levels of ice nucleation activity when propagated in strain uvs-51, a plasmid instability mutant of Z. mobilis, compared with the wild-type strain. The ice nucleation activity in Z. mobilis cultures showed unusual partitioning in that the culture supernatants obtained after low-speed centrifugation contained the majority of ice nuclei. Analysis of the ice nucleation spectra revealed that the cell pellets contained both "warm" and "cold" nuclei, while the culture supernatant contained primarily cold nuclei, suggesting that the cold nucleus activity may be extracellular. However, all nucleation activity was retained by 0.22-(mu)m-pore-size filters. PMID:16534909

  5. A novel delta-endotoxin gene cryIM from Bacillus thuringiensis ssp. wuhanensis.

    PubMed

    Shevelev, A B; Kogan YaN; Bushueva, A M; Voronina, E J; Rebrikov, D V; Novikova, S I; Chestukhina, G G; Kuvshinov, V; Pehu, E; Stepanov, V M

    1997-03-10

    A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp. wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034]. Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator. Nevertheless, its product was not previously found in the crystals of the host strain [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034] which shows its weak or absent natural expression. The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other delta-endotoxins of the CryI class. An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation. Artificial expression of the cloned gene in E. coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product. PMID:9119053

  6. Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori?

    PubMed Central

    Boneca, Ivo G.; Ecobichon, Chantal; Chaput, Catherine; Mathieu, Aurlie; Guadagnini, Stphanie; Prvost, Marie-Christine; Colland, Frdric; Labigne, Agns; de Reuse, Hilde

    2008-01-01

    The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring ?-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-?-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds. PMID:18245237

  7. Post-transcriptional regulation of sex determination in Caenorhabditis elegans: widespread expression of the sex-determining gene fem-1 in both sexes.

    PubMed Central

    Gaudet, J; VanderElst, I; Spence, A M

    1996-01-01

    The fem-1 gene of C. elegans is one of three genes required for all aspects of male development in the nematode. Current models of sex determination propose that the products of the fem genes act in a novel signal-transduction pathway and that their activity is regulated primarily at the post-translational level in somatic tissues. We analyzed the expression of fem-1 to determine whether it revealed any additional levels of regulation. Both XX hermaphrodites and XO males express fem-1 at approximately constant levels throughout development. Somatic tissues in hermaphrodites adopt female fates, but they nonetheless express fem-1 mRNA and FEM-1 protein, suggesting that the regulation of fem-1 activity is post-transcriptional and probably post-translational. A compact promoter directs functional expression of fem-1 transgenes, as assayed by their masculinizing activity in fem-1 mutants. Activity also requires any two or more introns, suggesting that splicing may enhance fem-1 expression. The minimal noncoding sequences required for activity of fem-1 transgenes suffice to direct expression of a fem-1::lacZ reporter gene in all somatic tissues in both sexes. Many fem-1 transgenes, including those that rescue male somatic development in fem-1 mutants, paradoxically feminize the germline. We suggest that they do so by interfering with the germline expression of the endogenous fem-1 gene. Images PMID:8862524

  8. Targeted gene expression without a tissue-specific promoter: Creating mosaic embryos using laser-induced single-cell heat shock

    PubMed Central

    Halfon, Marc S.; Kose, Hiroyuki; Chiba, Akira; Keshishian, Haig

    1997-01-01

    We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of β-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice. PMID:9177204

  9. Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

    NASA Technical Reports Server (NTRS)

    Halfon, M. S.; Kose, H.; Chiba, A.; Keshishian, H.

    1997-01-01

    We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

  10. DNA elements of the type 1 adenylyl cyclase gene locus enhance reporter gene expression in neurons and pinealocytes.

    PubMed

    Chan, G C; Lernmark, U; Xia, Z; Storm, D R

    2001-06-01

    The Ca2+-stimulated type 1 adenylyl cyclase (AC1) contributes to several forms of synaptic plasticity and is the only known neurospecific adenylyl cyclase. Furthermore, the protein and mRNA levels of AC1 undergo a circadian oscillation in the pineal gland, and AC1 may play a pivotal role in regulating nocturnal melatonin synthesis. To better understand the expression of AC1, we isolated mouse genomic DNA clones of AC1. The transcription and translation start regions of mouse AC1 share extensive homologies with the bovine counterpart. The upstream proximal region has potential binding sites for transcription factors, including the steroid receptor family, the E-box factors, and Sp1. A 280-bp fragment that contains the transcription start site directed reporter gene expression in cultured cortical neurons and pinealocytes functioning as a basal neuro- and pineal-directed promoter. Interestingly, pinealocyte expression of the reporter gene was inhibited by increases in cAMP. This cAMP sensitivity may explain why AC1 mRNA in the pineal is low at night when cAMP is elevated and high during the day when cAMP signals drop. An adjacent 330-bp fragment interacted specifically with nuclear factor(s) that we designate binary E-box factor (BEF). Methylation interference and DNase I footprinting identified the BEF-binding site sequence as 5'-CCAAGGTCACGTGGC-3'. When linked to the basal tissue-directed promoter, this 15-bp sequence further enhanced reporter expression in neurons and pinealocytes. We propose that this 15-bp sequence may contribute to increased expression of AC1 in neurons and pinealocytes relative to other cells. PMID:11422446

  11. Use of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coli.

    PubMed

    Thakker, Chandresh; Lin, Kevin; Martini-Stoica, Heidi; Bennett, George N

    2016-01-01

    Various methods have been developed for gene disruption in bacteria; however, extra in vitro manipulation steps or the residual presence of a scar in the host chromosome limits the use of such methods. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The ? Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. We demonstrated lacZ gene point mutation repair, two precise disruptions of the lacZ gene and constructed a library of lacZ variants having variable ?-galactosidase activity by changing its ribosome binding site sequences using the ISHp608 system. This technique can be used in E. coli genome modification and could be extended for use in other bacteria. PMID:26282057

  12. Embryonic activation of the myoD gene is regulated by a highly conserved distal control element.

    PubMed

    Goldhamer, D J; Brunk, B P; Faerman, A; King, A; Shani, M; Emerson, C P

    1995-03-01

    MyoD belongs to a small family of basic helix-loop-helix transcription factors implicated in skeletal muscle lineage determination and differentiation. Previously, we identified a transcriptional enhancer that regulates the embryonic expression of the human myoD gene. This enhancer had been localized to a 4 kb fragment located 18 to 22 kb upstream of the myoD transcriptional start site. We now present a molecular characterization of this enhancer. Transgenic and transfection analyses localize the myoD enhancer to a core sequence of 258 bp. In transgenic mice, this enhancer directs expression of a lacZ reporter gene to skeletal muscle compartments in a spatiotemporal pattern indistinguishable from the normal myoD expression domain, and distinct from expression patterns reported for the other myogenic factors. In contrast to the myoD promoter, the myoD enhancer shows striking conservation between humans and mice both in its sequence and its distal position. Furthermore, a myoD enhancer/heterologous promoter construct exhibits muscle-specific expression in transgenic mice, demonstrating that the myoD promoter is dispensable for myoD activation. With the exception of E-boxes, the myoD enhancer has no apparent sequence similarity with regulatory regions of other characterized muscle-specific structural or regulatory genes. Mutation of these E-boxes, however, does not affect the pattern of lacZ transgene expression, suggesting that myoD activation in the embryo is E-box-independent. DNase I protection assays reveal multiple nuclear protein binding sites in the core enhancer, although none are strictly muscle-specific. Interestingly, extracts from myoblasts and 10T1/2 fibroblasts yield identical protection profiles, indicating a similar complement of enhancer-binding factors in muscle and this non-muscle cell type. However, a clear difference exists between myoblasts and 10T1/2 cells (and other non-muscle cell types) in the chromatin structure of the chromosomal myoD core enhancer, suggesting that the myoD enhancer is repressed by epigenetic mechanisms in 10T1/2 cells. These data indicate that myoD activation is regulated at multiple levels by mechanisms that are distinct from those controlling other characterized muscle-specific genes. PMID:7720572

  13. MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch

    PubMed Central

    Westmeyer, Gil G.; Emer, Elena G.; Lintelmann, Jutta; Jasanoff, Alan

    2014-01-01

    SUMMARY The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with magnetic resonance imaging (MRI) could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by post mortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets. PMID:24613020

  14. Analysis of reported SCO2 gene mutations affecting cytochrome c oxidase activity in various diseases

    PubMed Central

    Chadha, Radhika; Shah, Ritika; Mani, Shalini

    2014-01-01

    A large number of mutations have been reported in SCO2 (synthesis of cytochrome c oxidase) gene in association with COX deficiency reported in different diseases such as cardioencephalomyopathy, cardiomyopathy and Leigh syndrome. However, very few of these mutations have been functionally analyzed.SCO2 gene encodes for an essential assembly factor for the formation of cytochrome c oxidase (COX). It is a nuclear encoded protein that helps in transfer of copper ions to COX. This study is an attempt to understand the possible effect of these mutations on the structure and function of SCO2 protein, by using different in silico tools. As per Human Gene Mutation Database, total 11 non synonymous variations have been reported in SCO2 gene. Among these 11 variations, only E140K and R171W are functionally proven to cause COX deficiency. They have been used as controls in this study. The remaining variations were further analyzed using ClustalW, SIFT, PolyPhen-2, GOR4, MuPro and Panther softwares. As compared to the results of the controls, most of these variations were predicted to affect the structure of SCO2 protein and hence, may cause COX dysfunction. Thus, we hypothesize that these variations have the potential to result in a disease phenotype and should be investigated by subsequent functional analyses. This will help in an appropriate diagnosis and management of the wide spectrum of COX deficiency diseases. PMID:25097374

  15. Multi-wavelength photoacoustic imaging of inducible tyrosinase reporter gene expression in xenograft tumors

    PubMed Central

    Paproski, Robert J.; Heinmiller, Andrew; Wachowicz, Keith; Zemp, Roger J.

    2014-01-01

    Photoacoustic imaging is an emerging hybrid imaging technology capable of breaking through resolution limits of pure optical imaging technologies imposed by optical-scattering to provide fine-resolution optical contrast information in deep tissues. We demonstrate the ability of multi-wavelength photoacoustic imaging to estimate relative gene expression distributions using an inducible expression system and co-register images with hemoglobin oxygen saturation estimates and micro-ultrasound data. Tyrosinase, the rate-limiting enzyme in melanin production, is used as a reporter gene owing to its strong optical absorption and enzymatic amplification mechanism. Tetracycline-inducible melanin expression is turned on via doxycycline treatment in vivo. Serial multi-wavelength imaging reveals very low estimated melanin expression in tumors prior to doxycycline treatment or in tumors with no tyrosinase gene present, but strong signals after melanin induction in tumors tagged with the tyrosinase reporter. The combination of new inducible reporters and high-resolution photoacoustic and micro-ultrasound technology is poised to bring a new dimension to the study of gene expression in vivo. PMID:24936769

  16. Recurrent anencephaly: a case report and examination of the VANGL1 and FOXN1 genes.

    PubMed

    Sergi, Consolato; Gekas, Jean; Kamnasaran, Deepak

    2013-07-01

    We report a new and rare case of recurrent anencephaly in a family with no other apparent abnormalities. The karyotypes of the family and all affected subjects were normal. Thorough mutational analyses of VANGL1 of chromosome 1p13.1 and FOXN1 of chromosome 17q11-q12, genes that are associated with phenotypes of the anencephaly spectrum, unfortunately did not disclose any DNA variations in an affected fetus of this family. The etiology of recurrent anencephaly in this family is therefore due to mutations in genes yet to be discovered, perhaps of the planar cell polarity pathway, or to possible environmental gestational factors during development. PMID:23301910

  17. Molecular characterization of a maize regulatory gene. Progress report, July 1989--March 1990

    SciTech Connect

    Wessler, S.

    1990-12-31

    This progress report contains information concerning the characterization of the Maize regulatory gene. The findings of this research program have immediate significance. Firstly, it provides support for the notion that R proteins, produced by the regulatory gene, are functionally equivalent. Secondly, the success of these experiments provides a simple transient assay for either natural or constructed R protein mutations. The relative ease of this assay coupled with overnight results are important prerequisites to the proposed experiments involving a structure-function analysis of the R protein.

  18. Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

    PubMed Central

    Ferkol, T; Perales, J C; Eckman, E; Kaetzel, C S; Hanson, R W; Davis, P B

    1995-01-01

    Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung. Images PMID:7860731

  19. Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures.

    PubMed Central

    Barthels, N; van der Lee, F M; Klap, J; Goddijn, O J; Karimi, M; Puzio, P; Grundler, F M; Ohl, S A; Lindsey, K; Robertson, L; Robertson, W M; Van Montagu, M; Gheysen, G; Sijmons, P C

    1997-01-01

    In the quest for plant regulatory sequences capable of driving nematode-triggered effector gene expression in feeding structures, we show that promoter tagging is a valuable tool. A large collection of transgenic Arabidopsis plants was generated. They were transformed with a beta-glucuronidase gene functioning as a promoter tag. Three T-DNA constructs, pGV1047, p delta gusBin19, and pMOG553, were used. Early responses to nematode invasion were of primary interest. Six lines exhibiting beta-glucuronidase activity in syncytia induced by the beet cyst nematode were studied. Reporter gene activation was also identified in galls induced by root knot and ectoparasitic nematodes. Time-course studies revealed that all six tags were differentially activated during the development of the feeding structure. T-DNA-flanking regions responsible for the observed responses after nematode infection were isolated and characterized for promoter activity. PMID:9437858

  20. Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

    PubMed

    Bej, A K; Steffan, R J; DiCesare, J; Haff, L; Atlas, R M

    1990-02-01

    Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies. PMID:2306085

  1. Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

    PubMed Central

    2010-01-01

    Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. Results We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. Conclusions From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species. PMID:20184756

  2. Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli

    PubMed Central

    Sperandio, Vanessa; Mellies, Jay L.; Nguyen, William; Shin, Sooan; Kaper, James B.

    1999-01-01

    Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora. PMID:10611361

  3. [Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system].

    PubMed

    Chen, Wei; Chen, Chong; Zhang, Huan-Xin; Cao, Jiang; Sang, Wei; Wu, Qing-Yun; Zhao, Kai; Zang, Yu; Zeng, Ling-Yu; Xu, Kai-Lin

    2012-06-01

    This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter. PMID:22739193

  4. Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis.

    PubMed

    Yang, Jingni; Peng, Qi; Chen, Zhen; Deng, Chao; Shu, Changlong; Zhang, Jie; Huang, Dafang; Song, Fuping

    2013-06-01

    In Bacillus thuringiensis, a novel N-acetylmuramoyl-L-alanine amidase gene (named cwlB) was detected, and the CwlB protein was purified and characterized. Reverse transcription-PCR (RT-PCR) results indicated that cwlB and an upstream gene (named cwlA) formed one transcriptional unit. 5' rapid amplification of cDNA ends (5'-RACE)-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter, P(cwlA), which is located upstream from the cwlA gene and that the transcription start site is a single 5'-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon. Moreover, the activity of P(cwlA) was controlled by σ(K). Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis. PMID:23603740

  5. Autosomal Recessive Nonsyndromic Hearing Loss: A Case Report with a Mutation in TRIOBP Gene

    PubMed Central

    Fardaei, Majid; Sarrafzadeh, Shaghayegh; Ghafouri-Fard, Soudeh; Miryounesi, Mohammad

    2015-01-01

    Hearing loss (HL) is the most common sensory defect. Various genetic as well as environmental factors have been shown to contribute in it. More than 100 loci have been recognized to cause autosomal recessive nonsyndromic hearing loss (ARNSHL). Here, we report a 6-year old female patient with bilateral pre-lingual HL in whom a mutation has been identified in TRIOBP gene (c.6362C>T, S2121L). In silico analysis has shown that this variant is possibly pathogenic. Although several mutations have been detected in this gene in various populations, this is the first report identifying TRIOBP mutation in Iranian population. Consequently, the results of the present study may be of importance in genetic counseling. PMID:27014650

  6. Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection

    SciTech Connect

    Roberto, F.F.; Barnes, J.M.; Bruhn, D.F.

    2002-03-28

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as B-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  7. Tbx18 targets dermal condensates for labeling, isolation and gene ablation during embryonic hair follicle formation

    PubMed Central

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2012-01-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. While functional analysis of hair follicle epithelial stem cells by gene targeting is well-established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking due to the absence of cell type-specific drivers. Here we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18LacZ, Tbx18H2BGFP and Tbx18Cre knock-in mouse models we demonstrate LacZ/GFP expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. Since Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we utilize tamoxifen-inducible Cre expressing mice, Tbx18MerCreMer, to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target embryonic DP precursors for labeling, isolation and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis. PMID:22992803

  8. Smooth Muscle Calponin: An Unconventional CArG-Dependent Gene that Antagonizes Neointimal Formation

    PubMed Central

    Long, Xiaochun; Slivano, Orazio J.; Cowan, Sarah L.; Georger, Mary A.; Lee, Ting-Hein; Miano, Joseph M.

    2011-01-01

    Objective Smooth muscle calponin (CNN1) contains multiple, conserved intronic CArG elements that bind serum response factor (SRF) and display enhancer activity in vitro. The objectives here were to evaluate these CArG elements for activity in transgenic mice and determine the effect of human CNN1 on injury-induced vascular remodeling. Methods and Results Mice carrying a lacZ reporter under control of intronic CArG elements in the human CNN1 gene failed to show smooth muscle cell (SMC)-restricted activity. However, deletion of the orthologous sequences in mice abolished endogenous Cnn1 promoter activity suggesting their necessity for in vivo Cnn1 expression. Mice carrying a 38-kilobase bacterial artificial chromosome (BAC) harboring the human CNN1 gene displayed SMC- restricted expression of the corresponding CNN1 protein as measured by immunohistochemistry and Western blotting. Extensive BAC recombineering studies revealed the absolute necessity of a single intronic CArG element for correct SMC-restricted expression of human CNN1. Over-expressing human CNN1 suppressed neointimal formation following arterial injury. Mice with an identical BAC carrying mutations in CArG elements that inhibit human CNN1 expression, showed outward remodeling and neointimal formation. Conclusions A single intronic CArG element is necessary but insufficient for proper CNN1 expression in vivo. CNN1 over-expression antagonizes arterial injury-induced neointimal formation. PMID:21817093

  9. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    SciTech Connect

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  10. Final Report [Function of the Arabidopsis TIR1 gene in auxin response

    SciTech Connect

    Estelle, Mark

    2000-12-18

    During this grant period substantial progress was made in the characterization of the TIR1 gene in Arabidopsis. Studies showed that the TIR1 protein is part of a protein complex that includes AtCUL1, ASK1 and RBX1. This complex, called SCF-TIR1, functions in the ubiquitin-mediated protein degradation pathway. Our work is the first report of an SCF complex in a plant system. The results of our studies are described in more detail in the report together with a publication resulting from this study.

  11. Components of the ESCRT Pathway, DFG16, and YGR122w Are Required for Rim101 To Act as a Corepressor with Nrg1 at the Negative Regulatory Element of the DIT1 Gene of Saccharomyces cerevisiae

    PubMed Central

    Rothfels, Karen; Tanny, Jason C.; Molnar, Enik; Friesen, Helena; Commisso, Cosimo; Segall, Jacqueline

    2005-01-01

    The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression. PMID:16024810

  12. Influence of transcriptional and translational control sequences on the expression of foreign genes in Caulobacter crescentus.

    PubMed Central

    Yap, W H; Thanabalu, T; Porter, A G

    1994-01-01

    The influence of expression control sequences (ECSs; promoters and ribosome-binding sites [RBSs]), transcriptional terminators, and gene orientation on the expression of the Escherichia coli lacZ gene in the gram-negative microorganisms Caulobacter crescentus and E. coli was investigated. A series of broad-host-range expression vectors, based on the RK2 plasmid derivative pRK248, were constructed. The ECSs included the tac promoter, the promoter for the surface layer protein of C. crescentus, and promoters from a number of gram-positive bacteria together with their associated RBSs. In addition, synthetic ECSs were constructed by using different combinations of promoters and RBSs. lacZ expression was found to be dependent on the nature of the promoter and RBS and, to a lesser extent, on the presence of a transcriptional terminator and the orientation of the promoter-lacZ construct in pRK248. The relative efficiencies of the various ECSs in driving lacZ expression differed markedly in C. crescentus and E. coli. In C. crescentus, the ECS ptac1 (tac promoter and consensus RBS for C. crescentus mRNAs) appeared to be the most efficient, producing 12-fold-higher activity than did pSL (promoter for the surface layer protein of C. crescentus and its putative RBS). pSL was not transcribed in E. coli, whereas various promoters from gram-positive microorganisms were transcribed in both C. crescentus and E. coli. A number of ECSs were also used to drive mosquitocidal toxin gene expression in C. crescentus, and a correlation between toxin expression and lacZ expression was observed. PMID:8169208

  13. Rational design of a triple reporter gene for multimodality molecular imaging.

    PubMed

    Hsieh, Ya-Ju; Hwu, Luen; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Wang, Hsin-Ell; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

    2014-01-01

    Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

  14. The Epstein-Barr virus immediate-early gene product, BMLF1, acts in trans by a posttranscriptional mechanism which is reporter gene dependent.

    PubMed Central

    Kenney, S; Kamine, J; Holley-Guthrie, E; Mar, E C; Lin, J C; Markovitz, D; Pagano, J

    1989-01-01

    In DNA cotransfection experiments, the Epstein-Barr virus immediate-early gene product, BMLF1, stimulated the chloramphenicol acetyltransferase (CAT) activity of both latent and productive EBV promoters linked to CAT. This BMLF1-induced increase in CAT activity was out of proportion to the effect on CAT mRNA, suggesting a posttranscriptional mechanism. Furthermore, when growth hormone was used as a reporter gene instead of CAT, BMLF1 no longer functioned. Thus, the BMLF1 effect was reporter-gene dependent. The effect of the BMLF1 gene product does not then appear to be directed at promoter activation, but instead may function to increase the level of an as yet unidentified protein(s) required for Epstein-Barr virus infection. Images PMID:2548002

  15. The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions.

    PubMed Central

    Nghiem, Y; Cabrera, M; Cupples, C G; Miller, J H

    1988-01-01

    We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions. Images PMID:3128795

  16. Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.

    PubMed

    Kopylova-Sviridova, T N; Krauzova, V I; Timiryasova, T M; Gorelova, T V; Shuppe, N G; Fodor, I

    1992-11-01

    Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses. PMID:1475909

  17. The human lysozyme promoter directs reporter gene expression to activated myelomonocytic cells in transgenic mice.

    PubMed Central

    Clarke, S; Greaves, D R; Chung, L P; Tree, P; Gordon, S

    1996-01-01

    The 5' region of the human lysozyme gene from -3500 to +25 was fused to a chloramphenicol acetyltransferase (CAT) reporter gene and three transgenic founder mice were obtained. All three transgenic lines showed the same pattern of CAT enzyme expression in adult mouse tissues that was consistent with the targeting of elicited, activated macrophages in tissues and developing and elicited granulocytes. In normal mice high CAT enzyme activity was found in the spleen, lung, and thymus, tissues rich in phagocytically active cells, but not in many other tissues, such as the gut and muscle, which contain resident macrophages. Cultured resident peritoneal macrophages and cells elicited 18 hr (granulocytes) and 4 days (macrophages) after injection of sterile thioglycollate broth expressed CAT activity. Bacillus Calmette-Guérin infection of transgenic mice resulted in CAT enzyme expression in the liver, which contained macrophage-rich granulomas, whereas the liver of uninfected mice did not have any detectable CAT enzyme activity. Although the Paneth cells of the small intestine in both human and mouse produce lysozyme, the CAT gene, under the control of the human lysozyme promoter, was not expressed in the mouse small intestine. These results indicate that the human lysozyme promoter region may be used to direct expression of genes to activated mouse myeloid cells. Images Fig. 1 PMID:8643649

  18. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  19. The MADS-box gene srfA is expressed in a complex pattern under the control of alternative promoters and is essential for different aspects of Dictyostelium development.

    PubMed

    Escalante, R; Vicente, J J; Moreno, N; Sastre, L

    2001-07-15

    srfA displays a complex temporal and cell type-specific pattern of expression in Dictyostelium and is expressed by most of its cell types at some stage of their development. This complexity is achieved by the use of alternative promoters. The promoter activity of the proximal region was found to be restricted to a subset of prestalk cells. Little or no associated expression was observed in the lower cup and basal disc during culmination. The middle promoter region was preferentially active in prestalk cells under usual conditions of filter development. Interestingly, during slug migration, the activity of this promoter in posterior prespore cells was strongly induced. The distal region displayed a dual pattern of expression. Thus, before culmination, this region drove lacZ expression in a few cells scattered along the entire structure. However, intense lacZ staining was found in the spores by the end of culmination. We have previously reported that srfA expression is essential for spore differentiation (R. Escalante and L. Sastre, Development 125, 3801-3808). Our novel finding of the expression of the gene in prestalk cells before culmination suggested that it might play additional roles in Dictyostelium development. The study of knockout strains revealed that srfA is also required for proper slug migration. Spore differentiation and slug migration defects were rescued by reexpression of srfA in the null mutant background, under the appropriate promoter control. The expression of srfA under the activity of the distal promoter region was able to rescue spore differentiation but not slug migration. Conversely, reexpression under the control of the middle promoter rescued slug morphogenesis and migration. Our results demonstrate that the correct spatial and temporal pattern of expression of srfA is essential for the different functions that this transcription factor plays in development. PMID:11437439

  20. Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an Artificial Mitochondrial Reporter DNA Vector.

    PubMed

    Yasuzaki, Yukari; Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi

    2015-12-01

    For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the in vivo mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology. PMID:26567847

  1. Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes

    PubMed Central

    Moroz, Maxim A.; Zhang, Hanwen; Lee, Jason; Moroz, Ekaterina; Zurita, Juan; Shenker, Larissa; Serganova, Inna; Blasberg, Ronald; Ponomarev, Vladimir

    2015-01-01

    Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodiumiodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 × 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (back-ground) was measured. Results The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 × 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 × 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2′-18F-fluoro-5-ethyl-1-β-D-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 × 105 T cells, respectively. 18F-FEAU was a more efficient probe (higher uptake, lower background) than 124I-1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK. Conclusion A comparison of different reporter gene–reporter probe systems for imaging of T cell number was performed, and the hNET/18F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35–40 × 103 T cells at the site of T cell injection in the animal model. PMID:26025962

  2. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1990--June 30, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5` UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  3. A single-vector EYFP reporter gene assay for G protein-coupled receptors.

    PubMed

    Hald, Helle; Wu, Boqian; Bouakaz, Lamine; Meldal, Morten

    2015-05-01

    We here present an improved and simplified assay to study signal transduction of the Gs class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any Gs protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2). PMID:25681566

  4. Gene-Environment Interactions in Cancer Epidemiology: A National Cancer Institute Think Tank Report

    PubMed Central

    Hutter, Carolyn M.; Mechanic, Leah E.; Chatterjee, Nilanjan; Kraft, Peter; Gillander, Elizabeth M.

    2014-01-01

    Cancer risk is determined by a complex interplay of genetic and environmental factors. Genome-wide association studies (GWAS) have identified hundreds of common (minor allele frequency [MAF]>0.05) and less common (0.01genes and environment, including gene-environment interactions, into epidemiologic studies of cancer. To help address these questions, and to better inform research priorities and allocation of resources, the National Cancer Institute sponsored a “Gene-Environment Think Tank” on January 10th–011th, 2012. The objective of the Think Tank was to facilitate discussions on: 1) the state of the science; 2) the goals of gene-environment interaction studies in cancer epidemiology; and 3) opportunities for developing novel study designs and analysis tools. This report summarizes the Think Tank discussion, with a focus on contemporary approaches to the analysis of gene-environment interactions. Selecting the appropriate methods requires first identifying the relevant scientific question and rationale, with an important distinction made between analyses aiming to characterize the joint effects of putative or established genetic and environmental factors and analyses aiming to discover novel risk factors or novel interaction effects. Other discussion items include measurement error, statistical power, significance and replication. Additional designs, exposure assessments, and analytical approaches need to be considered as we move from the current small number of success stories to a fuller understanding of the interplay of genetic and environmental factors. PMID:24123198

  5. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

    ABSTRACT Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation. PMID:26787828

  6. In vivo Tracking of Dendritic Cell using MRI Reporter Gene, Ferritin.

    PubMed

    Kim, Hoe Suk; Woo, Jisu; Lee, Jae Hoon; Joo, Hyun Jung; Choi, YoonSeok; Kim, Hyeonjin; Moon, Woo Kyung; Kim, Seung Ja

    2015-01-01

    The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R2*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R2*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R2* in both in vivo and ex vivo T2*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines. PMID:25993535

  7. Characterization of two trpE genes encoding anthranilate synthase {alpha}-subunit in Azospirillum brasilense

    SciTech Connect

    Ge Shimei; Xie Baoen; Chen Sanfeng . E-mail: chensf@cau.edu.cn

    2006-03-10

    The previous report from our laboratory has recently identified a new trpE gene (termed trpE {sub 2}) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE {sub 1}(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is First report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE {sub 1}(G) while these sequence features did not exist in front of trpE {sub 2}. The {beta}-galactosidase activity of an A. brasilense strain carrying a trpE {sub 2}-lacZ fusion remained constant at different tryptophan concentrations, but the {beta}-galactosidase activity of the same strain carrying a trpE {sub 1}(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE {sub 1}(G) is regulated at the transcriptional level by attenuation while trpE {sub 2} is constantly expressed. The anthranilate synthase assays with trpE {sub 1}(G){sup -} and trpE {sub 2} {sup -} mutants demonstrated that TrpE{sub 1}(G) fusion protein is feedback inhibited by tryptophan while TrpE{sub 2} protein is not. We also found that both trpE {sub 1}(G) and trpE {sub 2} gene products were involved in IAA synthesis.

  8. Search for major genes with progeny test data to accelerate the development of genetically superior loblolly pine. Technical progress report

    SciTech Connect

    2000-02-15

    This report details the progress of the three tasks of this project. The tasks are: (1) develop genetic models and analytical methods; (2) molecular confirmation of major gene segregation; and (3) develop strategies for marker-assisted breeding.

  9. Brief Report: Aggression and Stereotypic Behavior in Males with Fragile X Syndrome-- Moderating Secondary Genes in a "Single Gene" Disorder

    ERIC Educational Resources Information Center

    Hessl, David; Tassone, Flora; Cordeiro, Lisa; Koldewyn, Kami; McCormick, Carolyn; Green, Cherie; Wegelin, Jacob; Yuhas, Jennifer; Hagerman, Randi J.

    2008-01-01

    Although fragile X syndrome (FXS) is a single gene disorder with a well-described phenotype, it is not known why some individuals develop more significant maladaptive behaviors such as aggression or autistic symptoms. Here, we studied two candidate genes known to affect mood and aggression, the serotonin transporter (5-HTTLPR) and monoamine…

  10. Detection of transformed cells in crown gall tumors using the GUS reporter gene and correlation of GUS stained cells with T-DNA gene activity

    SciTech Connect

    Black, R.C. ); Labriola, J.; Binns, A.N. )

    1990-05-01

    Crown gall tumors are a mixture of transformed hormone producing cells and normal cells. Until now it has not been possible to directly visualize these cell types in situ. We have constructed strains of Agrobacterium tumefaciens that carry the 35S-{beta}-glucuronidase (GUS) reporter gene in either wild type or mutant Ti plasmids. Using histochemical staining for GUS activity, blue (GUS positive) sectors are observed in tumor sections. In order to demonstrate that the blue sectors actually represent cells expressing other T-DNA genes, we have looked for T-DNA gene encoded enzyme activity in the stained and unstained sectors. The blue sectors accumulate octopine (a product of the octopine synthase gene on the T-DNA) while the white (GUS negative) sectors do not. We conclude that the use of the GUS reporter gene provides a sensitive and reliable method for visualizing transformation events in plant tissues. A comparison of the proportion of transformed and nontransformed cells in wild type tumors vs. tumors deficient in auxin or cytokinin encoding genes will be discussed.

  11. Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene.

    PubMed

    Tuominen, H; Puech, L; Regan, S; Fink, S; Olsson, O; Sundberg, B

    2000-06-01

    The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth. PMID:10859183

  12. Use of Reporter Genes for Optical Measurements of Neoplastic Disease In vivo1

    PubMed Central

    Contag, Christopher H; Jenkins, Darlene; Contag, Pamela R; Negrin, Robert S

    2000-01-01

    Abstract Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies. PMID:10933067

  13. The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells.

    PubMed

    Pakula, Tiina M; Laxell, Marjukka; Huuskonen, Anne; Uusitalo, Jaana; Saloheimo, Markku; Penttilä, Merja

    2003-11-01

    To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca(2+)-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter. PMID:12941955

  14. Assembly of pseudorabies virus genome-based transfer vehicle carrying major antigen sites of S gene of transmissible gastroenteritis virus: potential perspective for developing live vector vaccines.

    PubMed

    Yin, Jiechao; Ren, Xiaofeng; Tian, Zhijun; Li, Yijing

    2007-03-01

    Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed. PMID:16731004

  15. Genetic analysis of the regulation of TCH gene expression, Final Report

    SciTech Connect

    Braam, Janet

    2008-10-28

    The Arabidopsis TCH genes, originally isolated as a consequence of their upregulation in response to the mechanical stimulus of touch, are also upregulated by a variety of seemingly disparate environmental and hormonal stimuli. To gain insight into the complexities of TCH gene regulation, a number of approaches were taken. Regulatory elements responsible for regulation were identified and characteristics of the regulation were evaluated. Reporter genes were used to monitor expression localization and dynamics. Microarray analyses of genome-wide expression behavior indicated that touch-inducible gene expression is more widespread than generally appreciated. Identification of all touch-regulated genes shed light on the types of cellular processes that may be altered in response to mechanical stress perturbations. Expression of the TCH2 gene, also called CML24, encoding a calmodulin (CaM)-like (CML) protein, was evaluated. CML24 shares over 40% amino acid sequence identity with CaM, has 4 EF hands and undergoes a Ca2+-dependent change in migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs and is induced from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress, in regions undergoing growth, in vascular tissues and various floral organs and in stomata, trichomes and hydathodes. CML24 underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4 and MgCl2. These data present evidence that CML24 encodes a potential Ca2+ sensor that may function to enable responses to ABA, day length and presence of various salts. Further investigation of CML24 function and regulation led to the finding that CML24 has a critical role in nitric oxide regulation. Distinct tilling mutant alleles demonstrated that CML24 can act as a switch in the response to day length perception. Because of potential redundancy with the related CML23 gene, CML23 T-DNA insertion mutants were identified and characterized. Together, CML23 and CML24 impact the autonomous regulatory pathway of the transition to flowering. Nitric oxide levels are elevated in cml23/cml24 double mutants. Therefore, CML23 and CML24 are potential calcium sensors regulate nitric oxide accumulation. In collaboration with Drs. McCann and Carpita, fourier transform infrared spectroscopy (FTIR) was used to assess, verify and classify wall architectural changes that occur as a result of single XTH insertion mutations. Thirty-four homozygous mutant lines of Arabidopsis representing 21 members of the xyloglucan endotransglucosylase/hydrolase gene family provided a set of mutants to characterize. Kohonen networks classified cell wall architectures of xth mutant lines and previously characterized cell wall mutants. The xth mutants were found to have chemical changes in their cell walls not detectable as phenotypic growth and development changes, consistent with the existence of feed-back loops that modify wall composition in response to a life-long deficiency of a cell wall enzyme. To gain insight into the potential physiological relevance of the distinct members of the XTH family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data revealed that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs showed XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes suggesting that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics.

  16. Molecular biological evaluation of bioactive glass microspheres and adjunct bone morphogenetic protein 2 gene transfer in the enhancement of new bone formation.

    PubMed

    Välimäki, Ville-Valtteri; Yrjans, Jessica J; Vuorio, Eero I; Aro, Hannu T

    2005-01-01

    Bioactive glass is a promising osteoconductive silica-based biomaterial for guidance of new bone growth. On the basis of several in vitro studies, the material appears able to promote osteoblast functions. In our in vivo study, the osteopromotive effect of bioactive glass microspheres seemed to surpass the osteoinductive action of direct adenovirus-mediated human bone morphogenetic protein 2 (BMP-2) gene transfer in a noncritical size bone defect model. The current study was initiated to elucidate the molecular mechanism behind bioactive glass action with or without adjunct BMP-2 gene transfer. A standardized bone defect of the rat tibia was filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (RAdBMP-2). Control defects were left empty or filled with bioactive glass microspheres with injection of adenovirus carrying the lacZ reporter gene or saline. Quantitative polymerase chain reaction confirmed the expression of the transferred human BMP-2 gene at the defect area at 4 days, but not in intact reference tissues. Bone matrix components (collagens I, II, and III, osteocalcin, osteonectin, and osteopontin) and resorption markers (cathepsin K and MMP-9), determined by Northern analysis, showed a completely different pattern of gene expression in defects filled with bioactive glass compared with control defects left to heal without filling. Bioactive glass induced a long-lasting production of bone matrix with concurrent upregulation of osteoclastic markers, a sign of high bone turnover. Combining RAdBMP-2 gene transfer with bioactive glass decelerated the high turnover, but did not influence the balance of synthesis and resorption. This molecular analysis confirmed not only the highly osteopromotive effect of bioactive glass microspheres, but also the accelerated rate of new bone resorption on its surface. At least in noncritical size defects this impact of bioactive glass seems to saturate new bone formation on its surface and thereby overshadow the effect of BMP-2 gene transfer. PMID:15869418

  17. Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage

    PubMed Central

    Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W.; Burt, David W.; Kaiser, Pete; Hume, David A.; Sang, Helen M.

    2014-01-01

    We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. PMID:25063453

  18. Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo.

    PubMed Central

    Rocha, M.; Hexel, K.; Bucur, M.; Schirrmacher, V.; Umansky, V.

    1996-01-01

    We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy. Images Figure 3 PMID:8883407

  19. The Role of Gcr1p in the Transcriptional Activation of Glycolytic Genes in Yeast Saccharomyces Cerevisiae

    PubMed Central

    Uemura, H.; Koshio, M.; Inoue, Y.; Lopez, M. C.; Baker, H. V.

    1997-01-01

    To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity. Likewise, a lone CT-box did not show any UAS activity. By contrast, oligonucleotides containing tandem CT-boxes but no RPG-box conferred strong promoter activity. This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5' noncoding region. However, it was dependent on both GCR1 and GCR2. These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p. Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box. Five mutants had single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box. These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target. PMID:9335590

  20. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    SciTech Connect

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  1. Sporadic Cerebral Cavernous Malformations: Report of Further Mutations of CCM Genes in 40 Italian Patients

    PubMed Central

    D'Angelo, Rosalia; Alafaci, Concetta; Scimone, Concetta; Ruggeri, Alessia; Salpietro, Francesco Maria; Bramanti, Placido; Tomasello, Francesco; Sidoti, Antonina

    2013-01-01

    Cerebral cavernous malformations (CCMs) are vascular lesions characterized by abnormally enlarged capillary cavities, affecting the central nervous system. CCMs can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (K-Rev interaction trapped 1 (KRIT1)), CCM2 (MGC4607), and CCM3 (PDCD10). CCMs occur as a single or multiple malformations that can lead to seizures, focal neurological deficits, hemorrhagic stroke, and headache. However, patients are frequently asymptomatic. In our previous mutation screening, performed in a cohort of 95 Italian patients, both sporadic and familial, we have identified several mutations in CCM genes, three of which in three distinct sporadic patients. In this study, representing further molecular screening of the three CCM genes, in a south Italian cohort of CCM patients enrolled by us in the last three years, we report the identification of other four new mutations in 40 sporadic patients with either single or multiple CCM. PMID:24058906

  2. A Novel Homozygous Mutation in the Transient Receptor Potential Melastatin 6 Gene: A Case Report

    PubMed Central

    Altıncık, Ayça; Schlingmann, Karl Peter; Tosun, Mahya Sultan

    2016-01-01

    Hereditary hypomagnesemia with secondary hypocalcemia (HSH) is a rare autosomal recessive disease caused by mutations in the transient receptor potential melastatin 6 (TRPM6) gene. Affected individuals present in early infancy with seizures caused by the severe hypocalcemia and hypomagnesemia. By presenting this case report, we also aimed to highlight the need for molecular genetic analysis in inbred or familial cases with hypomagnesemia. A Turkish inbred girl, now aged six years, had presented to another hospital at age two months with seizures diagnosed to be due to hypomagnesemia. She was on magnesium replacement therapy when she was admitted to our clinic with complaints of chronic diarrhea at age 3.6 years. During her follow-up in our clinic, she showed an age-appropriate physical and neurological development. In molecular genetic analysis, a novel homozygous frame-shift mutation (c.3447delT>p.F1149fs) was identified in the TRPM6 gene. This mutation leads to a truncation of the TRPM6 protein, thereby complete loss of function. We present the clinical follow-up findings of a pediatric HSH case due to a novel mutation in the TRPM6 gene and highlight the need for molecular genetic analysis in inbred or familial cases with hypomagnesemia. PMID:26759217

  3. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

    PubMed Central

    Xu, Huifen; Zhou, Cuilan; Zhang, Andy K.; Li, Wen; Zhang, Jia; Li, Kai

    2015-01-01

    Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay. PMID:26167508

  4. Genes and gene expression: Localization, damage and control -- A multi-level and interdisciplinary study. Progress report, February 1, 1992--January 31, 1993

    SciTech Connect

    Ts`o, P.O.P.

    1992-08-01

    This progress report describes gains made in three projects entitled (1) 3-Dimensional nuclear topography of genes and chromosomes in interphase nuclei, (2) Sequence specific identification and perturbation of the genomic DNA in living cells by nonionic oligonucleotide analogs (Matagen), and Resolution and isolation of specific DNA restriction fragments.(DT)

  5. One of Two hemN Genes in Bradyrhizobium japonicum Is Functional during Anaerobic Growth and in Symbiosis

    PubMed Central

    Fischer, Hans-Martin; Velasco, Leonardo; Delgado, Maria J.; Bedmar, Eulogio J.; Schären, Simon; Zingg, Daniel; Göttfert, Michael; Hennecke, Hauke

    2001-01-01

    Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472–1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN1) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN2), which was then cloned by using hemN1 as a probe. The hemN2 gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN1 and HemN2, respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (≥20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK2. In addition, maximal anaerobic hemN1 expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN1 mutant, strains lacking a functional hemN2 gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN2, but not hemN1, encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN2 only. PMID:11157943

  6. MEGDEL Syndrome in a Child From Palestine: Report of a Novel Mutation in SERAC1 Gene.

    PubMed

    Dweikat, Imad M; Abdelrazeq, Samer; Ayesh, Suhail; Jundi, Tawfeeq

    2015-07-01

    We report the first Palestinian child manifesting with 3-methylglutaconic aciduria psychomotor delay, muscle hypotonia, sensori-neural deafness, and Leigh-like lesions on brain magnetic resonance imaging (MRI), a clinical phenotype that is characteristic of MEGDEL syndrome. MEGDEL syndrome was recently found to be caused by mutations in SERAC1, encoding a protein essential for mitochondrial function, phospholipid remodeling, and intracellular cholesterol trafficking. We identified a novel homozygous mutation in SERAC1 gene (c.1018delT) that generates frame shift and premature termination of protein translation. Plasma and cerebrospinal fluid lactate, plasma alanine, and respiratory chain complexes in fresh muscle were normal. This report further expands the genetic spectrum of MEGDEL syndrome and adds to the evidence that it is associated with variable patterns of respiratory chain abnormalities. PMID:25051967

  7. Blastic plasmacytoid dendritic cell neoplasm with leukemic manifestation and ETV6 gene rearrangement: A case report

    PubMed Central

    GAO, NA; WANG, XUE-XIA; SUN, JIAN-RONG; YU, WEN-ZHENG; GUO, NONG-JIAN

    2015-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare malignant tumor of the hemopoietic system that arises from plasmacytoid dendritic cell precursors with a highly aggressive course. BPDCN frequently involves the skin, lymph nodes, peripheral blood and bone marrow. BPDCN is known to develop leukemic dissemination as a feature of myelomonocytic leukemia in the late phase of the disease, which leads to a poorer prognosis. In the present study, a case of BPDCN with leukemic manifestation without cutaneous involvement was reported. In addition, ETS variant gene 6 (ETV6) gene rearrangement was detected in the patient. The patient relapsed soon after complete remisson and had no response to further treatment. To the best of our knowledge, this is the first reported case of BPDCN with ETV6 rearrangement. Following chemotherapy treatment, the patient suffered from severe headache in the complete remission stage; however, brain CT scans showed no significant abnormalities. Several lumbar punctures and intrathecal chemotherapy were performed, and the patient recovered gradually. Therefore, the patient was considered to suffer from central nervous system leukemia. In conclusion, implementation of lumbar punctures and preventive intrathecal chemotherapy are required in BPDCN patients with leukemic manifestation during the remission stage. PMID:25780395

  8. The use of the NIS reporter gene for optimizing oncolytic virotherapy

    PubMed Central

    Miller, Amber; Russell, Stephen J

    2016-01-01

    Introduction: Oncolytic viruses are experimental cancer therapies being translated to the clinic. They are unique in their ability to amplify within the body, therefore requiring careful monitoring of viral replication and biodistribution. Traditional monitoring strategies fail to recapitulate the dynamic nature of oncolytic virotherapy. Consequently, clinically relevant, noninvasive, high resolution strategies are needed to effectively track virotherapy in real time. Areas covered: The expression of the sodium iodide symporter (NIS) reporter gene is tightly coupled to viral genome replication and mediates radioisotope concentration, allowing noninvasive molecular nuclear imaging of active viral infection with high resolution. This provides insight into replication kinetics, biodistribution, the impact of vector design, administration, and dosing on therapeutic outcomes, and highlights the heterogeneity of spatial distribution and temporal evolution of infection. NIS-mediated imaging in clinical trials confirms the feasibility of this technology to noninvasively and longitudinally observe oncolytic virus infection, replication, and distribution. Expert opinion: NIS-mediated imaging provides detailed functional and molecular information on the evolution of oncolytic virus infection in living animals. The use of NIS reporter gene imaging has rapidly advanced to provide unparalleled insight into the spatial and temporal context of oncolytic infection which will be integral to optimization of oncolytic treatment strategies. PMID:26457362

  9. A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo*

    PubMed Central

    Laker, Rhianna C.; Xu, Peng; Ryall, Karen A.; Sujkowski, Alyson; Kenwood, Brandon M.; Chain, Kristopher H.; Zhang, Mei; Royal, Mary A.; Hoehn, Kyle L.; Driscoll, Monica; Adler, Paul N.; Wessells, Robert J.; Saucerman, Jeffrey J.; Yan, Zhen

    2014-01-01

    Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo. PMID:24644293

  10. Development of tyrosinase-based reporter genes for preclinical photoacoustic imaging of mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Märk, Julia; Ruschke, Karen; Dortay, Hakan; Schreiber, Isabelle; Sass, Andrea; Qazi, Taimoor; Pumberger, Matthias; Laufer, Jan

    2014-03-01

    The capability to image stem cells in vivo in small animal models over extended periods of time is important to furthering our understanding of the processes involved in tissue regeneration. Photoacoustic imaging is suited to this application as it can provide high resolution (tens of microns) absorption-based images of superficial tissues (cm depths). However, stem cells are rare, highly migratory, and can divide into more specialised cells. Genetic labelling strategies are therefore advantageous for their visualisation. In this study, methods for the transfection and viral transduction of mesenchymal stem cells with reporter genes for the co-expression of tyrosinase and a fluorescent protein (mCherry). Initial photoacoustic imaging experiments of tyrosinase expressing cells in small animal models of tissue regeneration were also conducted. Lentiviral transduction methods were shown to result in stable expression of tyrosinase and mCherry in mesenchymal stem cells. The results suggest that photoacoustic imaging using reporter genes is suitable for the study of stem cell driven tissue regeneration in small animals.

  11. Testotoxicosis: Report of Two Cases, One with a Novel Mutation in LHCGR Gene

    PubMed Central

    Özcabı, Bahar; Tahmiscioğlu Bucak, Feride; Ceylaner, Serdar; Özcan, Rahşan; Büyükünal, Cenk; Ercan, Oya; Tüysüz, Beyhan; Evliyaoğlu, Olcay

    2015-01-01

    Testotoxicosis is a rare disorder which presents as isosexual peripheral precocious puberty in males. Despite the pattern of autosomal dominant inheritance, sporadic cases also may occur. Due to activating mutation in luteinizing hormone (LH))/choriogonadotropin receptor (LHCGR) gene, early virilization and advancement in bone age are common with increased serum testosterone levels above adult ranges, despite low LH and follicular-stimulating hormone (FSH) levels. There are different treatment regimens, such as combination of bicalutamide (antiandrogen agent) and a third-generation aromatase inhibitor, that are reported to be well-tolerated and successful in slowing bone age advancement and preventing progression of virilization. We report here two patients who presented with peripheral precocious puberty and an activating mutation in the LHCGR gene: one with a family history and previously determined mutation and the other without family history and with a novel mutation (c.830G>T). Combination of bicalutamide+anastrozole was ineffective in slowing pubertal progression and bone age. Short-term results were better with ketoconazole. PMID:26831561

  12. FMR1 CGG repeat lengths mediate different regulation of reporter gene expression in comparative transient and locus specific integration assays.

    PubMed

    Sølvsten, Christina; Nielsen, Anders Lade

    2011-10-15

    The Fragile X mental retardation (FMR1) gene contains a polymorphic CGG trinucleotide repeat in the 5'-untranslated region. The repeat length in the normal population is between 5 and 54 repeats. A repeat length between 55 and 200 is defined as the pre-mutation repeat size. Elderly carriers of the pre-mutation can develop the progressive neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS the FMR1 mRNA levels are increased and it is hypothesized that FXTAS is caused by a RNA gain of function mechanism. Repeat lengths beyond 200 CGGs are defined as the full-mutation and causes Fragile X-syndrome which is the most common inherited form of mental retardation. The full-mutation results in the absence of the FMR1 mRNA and protein, FMRP, through abnormal CpG methylation and FMR1 gene silencing. In this report we have used the Flp-In T-REx system to generate locus directed stable cell lines harboring the FMR1 5'-UTR with varying CGG repeat lengths in front of a reporter gene. By this system the influence of various CGG repeat lengths for reporter gene expression can be comparatively examined in cell lines where the only genetic difference is CGG repeat lengths. In such cell lines we find that a full-mutation CGG repeat confers inhibition of reporter gene expression, whereas a pre-mutation CGG repeat did not increase reporter gene expression. In transient transfection assays using the same expression vectors the pre-mutation and full-mutation CGG repeats increased reporter gene expression. This study shows that locus directed integration of model FMR1 CGG transgenes could be a new basic tool to further elucidating the basic molecular mechanisms behind transcriptional deregulation of the FMR1 gene in fragile X-syndrome and FXTAS. PMID:21767618

  13. Characterization of the Cis-Regulatory Region of the Drosophila Homeotic Gene Sex Combs Reduced

    PubMed Central

    Gindhart-Jr., J. G.; King, A. N.; Kaufman, T. C.

    1995-01-01

    The Drosophila homeotic gene Sex combs reduced (Scr) controls the segmental identity of the labial and prothoracic segments in the embryo and adult. It encodes a sequence-specific transcription factor that controls, in concert with other gene products, differentiative pathways of tissues in which Scr is expressed. During embryogenesis, Scr accumulation is observed in a discrete spatiotemporal pattern that includes the labial and prothoracic ectoderm, the subesophageal ganglion of the ventral nerve cord and the visceral mesoderm of the anterior and posterior midgut. Previous analyses have demonstrated that breakpoint mutations located in a 75-kb interval, including the Scr transcription unit and 50 kb of upstream DNA, cause Scr misexpression during development, presumably because these mutations remove Scr cis-regulatory sequences from the proximity of the Scr promoter. To gain a better understanding of the regulatory interactions necessary for the control of Scr transcription during embryogenesis, we have begun a molecular analysis of the Scr regulatory interval. DNA fragments from this 75-kb region were subcloned into P-element vectors containing either an Scr-lacZ or hsp70-lacZ fusion gene, and patterns of reporter gene expression were assayed in transgenic embryos. Several fragments appear to contain Scr regulatory sequences, as they direct reporter gene expression in patterns similar to those normally observed for Scr, whereas other DNA fragments direct Scr reporter gene expression in developmentally interesting but non-Scr-like patterns during embryogenesis. Scr expression in some tissues appears to be controlled by multiple regulatory elements that are separated, in some cases, by more than 20 kb of intervening DNA. Interestingly, regulatory sequences that direct reporter gene expression in an Scr-like pattern in the anterior and posterior midgut are imbedded in the regulatory region of the segmentation gene fushi tarazu (ftz), which is normally located between 10 and 20 kb 5' of the Scr transcription start site. This analysis provides an entry point for the study of how Scr transcription is regulated at the molecular level. PMID:7713432

  14. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between <1 and 20 mm apart from each other, and imaged with the appropriate imaging modality. Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  15. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli.

    PubMed

    Paproski, Robert J; Li, Yan; Barber, Quinn; Lewis, John D; Campbell, Robert E; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9×dilution sample was 55, suggesting that ∼20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between <1 and 20 mm apart from each other, and imaged with the appropriate imaging modality. Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted. PMID:26502231

  16. An enhancer trap screen for ecdysone-inducible genes required for Drosophila adult leg morphogenesis.

    PubMed Central

    Gates, J; Thummel, C S

    2000-01-01

    Although extensive studies of Drosophila imaginal disc development have focused on proliferation and patterning, relatively little is known about how the patterned imaginal discs are transformed into adult structures during metamorphosis. Studies focused primarily on leg development have shown that this remarkable transformation is coordinated by pulses of the steroid hormone ecdysone and requires the function of ecdysone-inducible transcription factors as well as proteases and components of the contractile cytoskeleton and adherens junctions. Here, we describe a genetic screen aimed at expanding our understanding of the hormonal regulation of Drosophila adult leg morphogenesis. We screened 1300 lethal P-element enhancer trap insertions on the second chromosome for a series of sequential parameters including pupal lethality, defects in leg morphogenesis, and ecdysone-induced lacZ reporter gene expression. From this screen we identified four mutations, one of which corresponds to bancal, which encodes the Drosophila homolog of hnRNP K. We also identified vulcan, which encodes a protein that shares sequence similarity with a family of rat SAPAP proteins. Both bancal and vulcan are inducible by ecdysone, thus linking the hormone signal with leg morphogenesis. This screen provides new directions for understanding the hormonal regulation of leg development during Drosophila metamorphosis. PMID:11102372

  17. Knockin of Cre Gene at Ins2 Locus Reveals No Cre Activity in Mouse Hypothalamic Neurons.

    PubMed

    Li, Ling; Gao, Lin; Wang, Kejia; Ma, Xianhua; Chang, Xusheng; Shi, Jian-Hui; Zhang, Ye; Yin, Kai; Liu, Zhimin; Shi, Yuguang; Xie, Zhifang; Zhang, Weiping J

    2016-01-01

    The recombination efficiency and cell specificity of Cre driver lines are critical for exploring pancreatic β cell biology with the Cre/LoxP approach. Some commonly used Cre lines are based on the short Ins2 promoter fragment and show recombination activity in hypothalamic neurons; however, whether this stems from endogenous Ins2 promoter activity remains controversial. In this study, we generated Ins2-Cre knockin mice with a targeted insertion of IRES-Cre at the Ins2 locus and demonstrated with a cell lineage tracing study that the Ins2 gene is not transcriptionally active in the hypothalamus. The Ins2-Cre driver line displayed robust Cre expression and activity in pancreatic β cells without significant alterations in insulin expression. In the brain, Cre activity was mainly restricted to the choroid plexus, without significant recombination detected in the hippocampus or hypothalamus by the LacZ or fluorescent tdTomato reporters. Furthermore, Ins2-Cre mice exhibited normal glucose tolerance and insulin secretion upon glucose stimulation in vivo. In conclusion, this Ins2-Cre driver line allowed high-fidelity detection of endogenous Ins2 promoter activity in vivo, and the negative activity in the hypothalamus demonstrated that this system is a promising alternative tool for studying β cell biology. PMID:26830324

  18. Knockin of Cre Gene at Ins2 Locus Reveals No Cre Activity in Mouse Hypothalamic Neurons

    PubMed Central

    Li, Ling; Gao, Lin; Wang, Kejia; Ma, Xianhua; Chang, Xusheng; Shi, Jian-Hui; Zhang, Ye; Yin, Kai; Liu, Zhimin; Shi, Yuguang; Xie, Zhifang; Zhang, Weiping J.

    2016-01-01

    The recombination efficiency and cell specificity of Cre driver lines are critical for exploring pancreatic β cell biology with the Cre/LoxP approach. Some commonly used Cre lines are based on the short Ins2 promoter fragment and show recombination activity in hypothalamic neurons; however, whether this stems from endogenous Ins2 promoter activity remains controversial. In this study, we generated Ins2-Cre knockin mice with a targeted insertion of IRES-Cre at the Ins2 locus and demonstrated with a cell lineage tracing study that the Ins2 gene is not transcriptionally active in the hypothalamus. The Ins2-Cre driver line displayed robust Cre expression and activity in pancreatic β cells without significant alterations in insulin expression. In the brain, Cre activity was mainly restricted to the choroid plexus, without significant recombination detected in the hippocampus or hypothalamus by the LacZ or fluorescent tdTomato reporters. Furthermore, Ins2-Cre mice exhibited normal glucose tolerance and insulin secretion upon glucose stimulation in vivo. In conclusion, this Ins2-Cre driver line allowed high-fidelity detection of endogenous Ins2 promoter activity in vivo, and the negative activity in the hypothalamus demonstrated that this system is a promising alternative tool for studying β cell biology. PMID:26830324

  19. Epitope-Tagged Autotransporters as Single-Cell Reporters for Gene Expression by a Salmonella Typhimurium wbaP Mutant.

    PubMed

    Curkić, Ismeta; Schütz, Monika; Oberhettinger, Philipp; Diard, Médéric; Claassen, Manfred; Linke, Dirk; Hardt, Wolf-Dietrich

    2016-01-01

    Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells. PMID:27149272

  20. Epitope-Tagged Autotransporters as Single-Cell Reporters for Gene Expression by a Salmonella Typhimurium wbaP Mutant

    PubMed Central

    Curkić, Ismeta; Schütz, Monika; Oberhettinger, Philipp; Diard, Médéric; Claassen, Manfred; Linke, Dirk; Hardt, Wolf-Dietrich

    2016-01-01

    Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells. PMID:27149272

  1. Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location.

    PubMed

    Gabriel, Gabriele V M; Viviani, Vadim R

    2014-12-01

    Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes. PMID:25285909

  2. YY1 negatively regulates mouse myelin proteolipid protein (Plp1) gene expression in oligodendroglial cells.

    PubMed

    Zolova, Olga E; Wight, Patricia A

    2011-01-01

    YY1 (Yin and Yang 1) is a multifunctional, ubiquitously expressed, zinc finger protein that can act as a transcriptional activator, repressor, or initiator element binding protein. Previous studies have shown that YY1 modulates the activity of reporter genes driven by the myelin PLP (proteolipid protein) (PLP1/Plp1) promoter. However, it is known that Plp1 intron 1 DNA contains regulatory elements that are required for the dramatic increase in gene activity, coincident with the active myelination period of CNS (central nervous system) development. The intron in mouse contains multiple prospective YY1 target sites including one within a positive regulatory module called the ASE (anti-silencer/enhancer) element. Results presented here demonstrate that YY1 has a negative effect on the activity of a Plp1-lacZ fusion gene [PLP(+)Z] in an immature oligodendroglial cell line (Oli-neu) that is mediated through sequences present in Plp1 intron 1 DNA. Yet YY1 does not bind to its alleged site in the ASE (even though the protein is capable of recognizing a target site in the promoter), indicating that the down-regulation of PLP(+)Z activity by YY1 in Oli-neu cells does not occur through a direct interaction of YY1 with the ASE sequence. Previous studies with Yy1 conditional knockout mice have demonstrated that YY1 is essential for the differentiation of oligodendrocyte progenitors. Nevertheless, the current study suggests that YY1 functions as a repressor (not an activator) of Plp1 gene expression in immature oligodendrocytes. Perhaps YY1 functions to keep the levels of PLP in check in immature cells before vast quantities of the protein are needed in mature myelinating oligodendrocytes. PMID:21973168

  3. In vitro study for laser gene transfer in BHK-21 fibroblast cell line

    NASA Astrophysics Data System (ADS)

    Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

    2009-02-01

    Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated that, no ultradamages or changes for cell; membrane, organilles or any component of transfected fibroblast cell as a result of using laser microbeam compared with control cell.

  4. The Sodium Iodide Symporter (NIS) as an Imaging Reporter for Gene, Viral, and Cell-based Therapies

    PubMed Central

    Penheiter, Alan R; Russell, Stephen J; Carlson, Stephanie K

    2012-01-01

    Preclinical and clinical tomographic imaging systems increasingly are being utilized for non-invasive imaging of reporter gene products to reveal the distribution of molecular therapeutics within living subjects. Reporter gene and probe combinations can be employed to monitor vectors for gene, viral, and cell-based therapies. There are several reporter systems available; however, those employing radionuclides for positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) offer the highest sensitivity and the greatest promise for deep tissue imaging in humans. Within the category of radionuclide reporters, the thyroidal sodium iodide symporter (NIS) has emerged as one of the most promising for preclinical and translational research. NIS has been incorporated into a remarkable variety of viral and non-viral vectors in which its functionality is conveniently determined by in vitro iodide uptake assays prior to live animal imaging. This review on the NIS reporter will focus on 1) differences between endogenous NIS and heterologously-expressed NIS, 2) qualitative or comparative use of NIS as an imaging reporter in preclinical and translational gene therapy, oncolytic viral therapy, and cell trafficking research, and 3) use of NIS as an absolute quantitative reporter. PMID:22263922

  5. Conditional deletion of the relaxin receptor gene in cells of smooth muscle lineage affects lower reproductive tract in pregnant mice.

    PubMed

    Kaftanovskaya, Elena M; Huang, Zaohua; Lopez, Carolina; Conrad, Kirk; Agoulnik, Alexander I

    2015-04-01

    Relaxin hormone secreted into the circulation during pregnancy was discovered through its effects on pubic symphysis relaxation and parturition. Genetic inactivation of the relaxin gene or its cognate relaxin family peptide receptor 1 (RXFP1) in mice caused failure of parturition and mammary nipple enlargement, as well as increased collagen fiber density in the cervix and vagina. However, the relaxin effect on discrete cells and tissues has yet to be determined. Using transgenic mice with a knockin LacZ reporter in the Rxfp1 allele, we showed strong expression of this gene in vaginal and cervical stromal cells, as well as pubic ligament cells. We produced a floxed Rxfp1 allele that was used in combination with the Tagln-cre transgene to generate mice with a smooth muscle-specific gene knockout. In pregnant females, the ROSA26 reporter activated by Tagln-cre was detected in smooth muscle cells of the cervix, vagina, uterine artery, and in cells of the pubic symphysis. In late pregnant females with conditional gene ablation, the length of pubic symphysis was significantly reduced compared with wild-type or heterozygous Rxfp1(+/-) females. Denser collagen content was revealed by Masson trichrome staining in reproductive tract organs, uterine artery, and pubic symphysis. The cervical and vaginal epithelium was less developed than in heterozygous or wild-type females, although nipple size was normal and the dams were able to nurse their pups. In summary, our data indicate that relaxin/RXFP1 signaling in smooth muscle cells is important for normal collagen turnover and relaxation of the pubic symphysis during pregnancy. PMID:25715795

  6. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    PubMed Central

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-01-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5′ untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection. PMID:10792029

  7. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    NASA Astrophysics Data System (ADS)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  8. Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene.

    PubMed

    Van den Hoecke, Silvie; Verhelst, Judith; Saelens, Xavier

    2016-01-01

    Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the 'CCCGCC' motif in the GFP coding sequence. PMID:27193250

  9. [Multiple paragangliomas associated to a SDHB gene mutation: report of one case].

    PubMed

    Díaz, René E; Utreras, Carlos; Ascuí, Rodrigo; Hidalgo, Fernando; Véliz, Jesús; Wohllk, Nelson

    2011-11-01

    Paragangliomas are tumors arising from sympathetic and parasympathetic tissues. The classic associated syndromes are neurofibromatosis type 1, multiple endocrine neoplasia type 2 and von Hippel-Lindau. Germline mutations of succinate dehydrogenase subunits genes, are associated with familial paraganglioma syndromes 1,2,3 and 4. We report a 29-year-old woman with a family background of pheochromocytoma and history of paroxysmal headache, nausea, sweating, palpitations, associated with severe hypertension. The patient had elevated plasma noradrenalin and urinary normetanephrines. Imaging studies revealed three retroperitoneal extra-adrenal masses. The clinical and laboratory study of classic syndromes associated with paraganglioma was negative. The patient was operated and the pathological study of the surgical specimen was consistent with paragangliomas. The genetic study showed a mutation in the SDHB succinate dehydrogenase gen, Exon 2 of CCTCA c.300_304 (p.P56delYfsX5). PMID:22446654

  10. Rapid detection of common mutations of the FGFR3 gene causing thanatophoric dysplasia type I: two case reports.

    PubMed

    Yang, Yu; Liu, Ying-Na; Li, Dong-Zhi

    2012-06-01

    Thanatophoric dysplasia (TD) is a relatively common lethal skeletal dysplasia. These malformations result from the mutations in fibroblast growth factor receptor 3 (FGFR3) gene, which is located on the short arm of chromosome 4. Accurate diagnosis of fetal TD is important for patient counseling and to plan the management. A definite diagnosis can be established by molecular genetic analysis to find out the abnormal mutations in the FGFR3 gene. We reported on two cases of TD type I found by prenatal ultrasound and confirmed by molecular analysis of FGFR3 gene using high-resolution melting analysis. PMID:22414243

  11. [Construction and identification of luciferase reporter gene containing mouse T-bet promoter].

    PubMed

    Xu, Huijuan; Zhou, Shoubiao

    2014-11-01

    The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with Kpn I and Sfi I and DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T cells (P = 0.012 2) and Jurkat cells (P = 0.002 2) was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription. PMID:25985524

  12. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    PubMed

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-01

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations. PMID:12109482

  13. Bioluminescence Reporter Gene Imaging Characterize Human Embryonic Stem Cell-Derived Teratoma Formation

    PubMed Central

    Su, Weijun; Zhou, Manqian; Zheng, Yizhou; Fan, Yan; Han, Zhongchao; Kong, Deling; Wu, Joseph C.; Xiang, Rong; Li, Zongjin

    2011-01-01

    Human embryonic stem (hES) cells are capable of differentiation into virtually all cell types and hold tremendous potential as cell sources for regenerative therapies. However, teratoma formation can be the main obstacle for hES cells therapy. In order to understand the biology and physiology of hES cells teratoma formation, we investigated the angiogenic process within teratomas and characterized teratoma cells. In this study, hES cells transduced with double fusion reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) were injected into hind limbs of SCID mice and performed longitudinal bioluminescence imaging on these animals. To test angiogenic contribution of teratoma from host or hES cells, human and mouse endothelial cells marker CD31 was stained respectively. To further explore the characterization of teratoma derived cells, flow cytometry analysis was carried out and GFP+/SSEA-4+ cells were isolated and subcultured. Then, we re-injected the isolated GFP+/SSEA-4+ teratoma cells into SCID mice and observed by imaging. Our results show that the reporter gene imaging is an ideal technology for monitoring long-term stem cell viability, death, and proliferation. Teratomas contained vasculatures are from hES cells and host. hESCs derived teratomas express a high level of undifferentiated marker SSEA-4 and CD56, and subcultured GFP+/SSEA-4+ cells had similar expression pattern comparing to undifferentiated hES cells, except for a very high level of CD56 and a little lower expression of undifferentiated markers, such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Moreover, the SSEA-4+ teratoma cells can form teratomas in SCID mice, and this type teratomas grow at a lower rate compared to teratomas derived from hES cells, and are more differentiated. PMID:21328457

  14. Effects of the polyubiquitin gene Ubi. U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum.

    PubMed

    Plesse, B; Criqui, M C; Durr, A; Parmentier, Y; Fleck, J; Genschik, P

    2001-04-01

    We have previously shown by RNA gel blot analyses that the tobacco polyubiquitin-encoding gene Ubi.U4 is expressed in a complex pattern during plant development (Genschik et al., 1994). In order to study its tissue-specific expression, we cloned the fragment containing the -263 bp proximal promoter of the gene, the leader intron and the first ubiquitin monomer in front of the reporter GUS gene. Histochemical analyses for GUS activity during tobacco plant development revealed that the gene is expressed at variable amounts in many plant tissues with high levels in metabolically active and/or dividing cells and in the vascular tissues of the plant. We also analysed the expression pattern of constructs in which either the intron or the intron together with the first ubiquitin monomer were deleted. Our results indicate that the ubiquitin leader intron is not only a quantitative determinant of gene expression but may also influence the tissue-specific expression pattern. PMID:11430428

  15. The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

    PubMed Central

    Srikantha, T; Klapach, A; Lorenz, W W; Tsai, L K; Laughlin, L A; Gorman, J A; Soll, D R

    1996-01-01

    The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters. PMID:8550405

  16. Heterologous Expression of Mannanase and Developing a New Reporter Gene System in Lactobacillus casei and Escherichia coli

    PubMed Central

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie; She, Qunxin; Liang, Yunxiang; Chen, Zhengjun; Ge, Xiangyang

    2015-01-01

    Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli. PMID:26562012

  17. McArdle Disease: Update of Reported Mutations and Polymorphisms in the PYGM Gene.

    PubMed

    Nogales-Gadea, Gisela; Brull, Astrid; Santalla, Alfredo; Andreu, Antoni L; Arenas, Joaquin; Martín, Miguel A; Lucia, Alejandro; de Luna, Noemi; Pinós, Tomàs

    2015-07-01

    McArdle disease is an autosomal-recessive disorder caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (or "myophosphorylase"), which catalyzes the first step of glycogen catabolism, releasing glucose-1-phosphate from glycogen deposits. As a result, muscle metabolism is impaired, leading to different degrees of exercise intolerance. Patients range from asymptomatic to severely affected, including in some cases, limitations in activities of daily living. The PYGM gene codifies myophosphoylase and to date 147 pathogenic mutations and 39 polymorphisms have been reported. Exon 1 and 17 are mutational hot-spots in PYGM and 50% of the described mutations are missense. However, c.148C>T (commonly known as p.R50X) is the most frequent mutation in the majority of the studied populations. No genotype-phenotype correlation has been reported and no mutations have been described in the myophosphorylase domains affecting the phosphorylated Ser-15, the 280's loop, the pyridoxal 5'-phosphate, and the nucleoside inhibitor binding sites. A newly generated knock-in mouse model is now available, which renders the main clinical and molecular features of the disease. Well-established methods for diagnosing patients in laboratories around the world will shorten the frequent ∼20-year period stretching from first symptoms appearance to the genetic diagnosis. PMID:25914343

  18. Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.

    PubMed Central

    Nordlund, M E; Johansson, J O; von Pawel-Rammingen, U; Byström, A S

    2000-01-01

    The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2. PMID:10864043

  19. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos.

    PubMed

    Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

    2012-07-01

    To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

  20. Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

    PubMed Central

    Sinnott, M L; Smith, P J

    1978-01-01

    1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500. PMID:105721

  1. Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

    PubMed

    Sinnott, M L; Smith, P J

    1978-11-01

    1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500. PMID:105721

  2. The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression.

    PubMed

    Owen-Hughes, T A; Pavitt, G D; Santos, D S; Sidebotham, J M; Hulton, C S; Hinton, J C; Higgins, C F

    1992-10-16

    H-NS is an abundant structural component of bacterial chromatin and influences many cellular processes, including recombination, transposition, and transcription. We have studied the mechanism of action of H-NS at the osmotically regulated proU promoter. The interaction of H-NS with a curved DNA element located downstream of the proU promoter is required for normal regulation of expression. Heterologous curved sequences can replace the regulatory role of the proU curve. Hence, the luxAB and lacZ reporter genes, which differ in the presence or absence of a curve, can indicate very different patterns of transcription. H-NS interacts preferentially with these curved DNA elements in vitro. Furthermore, in vivo the interaction of H-NS with curved DNA participates in the control of plasmid linking number. The data suggest that H-NS-dependent changes in DNA topology play a role in the osmoregulation of proU expression. PMID:1423593

  3. Gene Expression Noise in Spatial Patterning: hunchback Promoter Structure Affects Noise Amplitude and Distribution in Drosophila Segmentation

    PubMed Central

    Holloway, David M.; Lopes, Francisco J. P.; da Fontoura Costa, Luciano; Travençolo, Bruno A. N.; Golyandina, Nina; Usevich, Konstantin; Spirov, Alexander V.

    2011-01-01

    Positional information in developing embryos is specified by spatial gradients of transcriptional regulators. One of the classic systems for studying this is the activation of the hunchback (hb) gene in early fruit fly (Drosophila) segmentation by the maternally-derived gradient of the Bicoid (Bcd) protein. Gene regulation is subject to intrinsic noise which can produce variable expression. This variability must be constrained in the highly reproducible and coordinated events of development. We identify means by which noise is controlled during gene expression by characterizing the dependence of hb mRNA and protein output noise on hb promoter structure and transcriptional dynamics. We use a stochastic model of the hb promoter in which the number and strength of Bcd and Hb (self-regulatory) binding sites can be varied. Model parameters are fit to data from WT embryos, the self-regulation mutant hb14F, and lacZ reporter constructs using different portions of the hb promoter. We have corroborated model noise predictions experimentally. The results indicate that WT (self-regulatory) Hb output noise is predominantly dependent on the transcription and translation dynamics of its own expression, rather than on Bcd fluctuations. The constructs and mutant, which lack self-regulation, indicate that the multiple Bcd binding sites in the hb promoter (and their strengths) also play a role in buffering noise. The model is robust to the variation in Bcd binding site number across a number of fly species. This study identifies particular ways in which promoter structure and regulatory dynamics reduce hb output noise. Insofar as many of these are common features of genes (e.g. multiple regulatory sites, cooperativity, self-feedback), the current results contribute to the general understanding of the reproducibility and determinacy of spatial patterning in early development. PMID:21304932

  4. Activity of the dietary antioxidant ergothioneine in a virus gene-based assay for inhibitors of HIV transcription.

    PubMed

    Xiao, Lianchun; Zhao, Lijun; Li, Ting; Hartle, Diane K; Aruoma, Okezie I; Taylor, Ethan Will

    2006-01-01

    The "Long Terminal Repeat" (LTR) of HIV-1 is the target of cellular transcription factors such as NF-kappaB, and serves as the promoter-enhancer for the viral genome when integrated in host DNA. Various LTR-reporter gene constructs have been used for in vitro studies of activators or inhibitors of HIV-1 transcription, e.g., to show that antioxidants such as lipoic acid and selenium inhibit NF-kappaB-dependent HIV-1 LTR activation. One such construct is the pHIVlacZ plasmid, with the HIV-1 LTR driving expression of the lacZ gene (encoding beta-galactosidase, beta-gal). Typically, for inhibitor screening, cells transfected with pHIVlacZ are activated using tumor necrosis factor-alpha (TNF-alpha), and the colorimetric o-nitrophenol assay is used to assess changes in beta-gal activity. A variant of this assay was developed as described here, in which LTR activation was induced by pro-fs, a novel HIV-1 gene product encoded via a -1 frameshift from the protease gene. Cotransfection of cells with pHIVlacZ along with a pro-fs construct produced a significant increase in beta-gal activity over controls. L-ergothioneine dose dependently inhibited both TNF-alpha-mediated and pro-fs-mediated increases in beta-gal activity, with an IC50 of about 6 mM. Thus antioxidant strategy involving ergothioneine derived from food plants might be of benefit in chronic immunodeficiency diseases. PMID:17012772

  5. Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression.

    PubMed

    Jayaraj, Jayaraman; Muthukrishnan, Subbaratnam; Liang, George H

    2004-07-01

    Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani. PMID:15381976

  6. In vivo gene delivery into ocular tissues by eye drops of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles.

    PubMed

    Liaw, J; Chang, S F; Hsiao, F C

    2001-07-01

    The primary objective of this study was to investigate the feasibility of using PEO-PPO-PEO non-ionic copolymeric micelles as a carrier for eye-drop gene delivery of plasmid DNA with lacZ gene in vivo. Using pyrene fluorescence probe methods, zeta potential, and dynamic light scattering test (DLS), the ability of micelle formation of these block copolymers with plasmid was studied. Gene expressions were visualized by both the quality of enzymatic color reaction using X-gal staining and by the quantification of the substrate chlorophenol red galactopyranoside (CPRG) in enucleated eyes on day 2 after gene transfer. In addition, microscopy to identify the types of cell showing uptake and expression of the transferred gene was used. We found that the block polymeric micelles were formed above 0.1% (w/v) of block copolymer with a size of 160 nm and a zeta potential of -4.4 mV. After 2 days of topically delivery three times a day, the most intense gene expression was observed on days 2 and 3. Reporter expression was detected around the iris, sclera, conjunctiva, and lateral rectus muscle of rabbit eyes and also in the intraocular tissues of nude mice upon in vivo topical application for 48 h with a DNA/polymeric micelle formulation. Furthermore, after two enhancement treatments, the transport mechanisms of the block copolymeric micelles were found through endocytosis in tissues by enhancement through the tight junction pathway. Thus, efficient and stable transfer of the functional gene could be achieved with PEO-PPO-PEO polymeric micelles through topical delivery in mice and rabbits. These in vivo experiments indicate the possible potential use of block copolymers for DNA transfer. PMID:11438834

  7. Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

    PubMed Central

    2015-01-01

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein–protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein–protein interactions within whole animals. PMID:25265085

  8. Asynchronous Bilateral Renal Infarction and Thrombophilia With Associated Gene Mutations in a 43-Year-Old Man: A Case Report.

    PubMed

    Zhou, Xu-Jie; Liu, Li-Jun; Chen, Min; Zhou, Fu-De

    2016-04-01

    Renal infarction (RI) is frequently misdiagnosed or diagnosed late because of its rarity and nonspecific clinical presentation, which may result in irreversible damage to the renal parenchyma or increase the risk of other embolic events affecting additional organs. Multiple causal mechanisms and cases of idiopathic RI have been reported, but the causal factors are not clear in most cases.Here, we report the case of a patient with heterochronic bilateral RI caused by thrombophilia. Although he had several risk factors for hypercoagulation disorders, two gene mutations-MTHFR 677 C>T and PLG 1858G>A-were identified by genome sequencing of the entire exome. The findings suggest the possibility of a synergistic relationship between the two gene mutations.Thus, screening for gene mutations may provide additional clues for clarifying the cause of RI and thrombophilia. PMID:27057875

  9. Effect of the silk protein sericin on the production of adenovirus-based gene-therapy vectors.

    PubMed

    Yanagihara, Kana; Terada, Satoshi; Miki, Masao; Sasaki, Masahiro; Yamada, Hideyuki

    2006-09-01

    Adenoviral vectors are extensively used as gene-delivery vehicles in gene therapy. They are usually produced by HEK-293 cell (human embryonic kidney-293 cell) culture, which requires specially formulated serum-free medium, the cost of which is considerable or by supplementation with FBS (fetal bovine serum). The risk of infectious diseases such as BSE (bovine spongiform encephalopathy) and endogenous retrovirus derived from cattle is a serious concern. The present study reports the use of sericin protein derived from silkworm (Bombyx mori) as an effective supplement instead of FBS. Without FBS, HEK-293 cells significantly proliferated in the presence of 0.025-0.4% sericin, especially at 0.1%, but the effect was inferior to that of FBS. When a lower titre [MOI (multiplicity of infection) 0.03] of adenoviral vector pAxCAiLacZ was used as the inoculum, HEK-293 cells in the presence of 0.1% sericin produced a nearly 3-fold higher vector titre than culture in the presence of 5% (v/v) FBS. However, when a higher vector titre (MOI 3.7) was used as the inoculum, HEK-293 cells in the presence of sericin produced a slightly higher vector titre than in the presence of FBS, which might suggest that HEK-293 cells produce a maximum amount when a higher vector titre is used as the inoculum. These increases in vector production with sericin were confirmed by LacZ (beta-galactosidase reporter gene) activity assay. Supplementation with sericin decreased lactate dehydrogenase activity, an indicator of cell death, suggesting that sericin improved cell survival; hence, prolonging the culture period might be one of the reasons for increased vector production. On the basis of these results, sericin peptide seems to be a potent and effective alternative supplement for production of adenoviral vectors without such risks as BSE and retrovirus. PMID:16674313

  10. Cox-2 gene overexpression in ureteral stump urothelial carcinoma following nephrectomy for renal cell carcinoma: a case report

    PubMed Central

    2012-01-01

    Introduction A primary ureteral stump tumor after a nephrectomy is rare; urothelial carcinoma of the ureteral stump after a nephrectomy for renal cell carcinoma is even rarer. A thorough review of the literature indicated that only seven cases have previously been reported. In this study, we report the first Taiwanese case of urothelial carcinoma of the ureteral stump after a nephrectomy. It is also the first female case in the literature. The relationship between inflammatory genes, medication history and ureteral stump carcinoma after a nephrectomy for renal cell carcinoma has not been reported. Case presentation A 72-year-old Asian Taiwanese women with chronic hepatitis C, liver cirrhosis and chronic kidney disease underwent a hand-assisted laparoscopic radical nephrectomy in 2001 due to renal cell carcinoma. Nine years later, she was diagnosed with ureteral stump urothelial carcinoma. Genetic and medication surveys were performed. Importantly, our patient had taken Chinese herbal drugs for more than 10 years and the inflammatory gene, Cox-2, was highly expressed in this patient. This is the first report to study the relationship between the Cox-2 gene and ureteral stump carcinoma after a nephrectomy for renal cell carcinoma. Conclusion Long-term multiple use of Chinese herbal drugs could be one of the important risk factors for developing urothelial cancer. Close functional coupling between Chinese herbal drugs, Cox-2 gene activation and urothelial cancer should be further investigated. PMID:22289336

  11. Bioluminescent whole-cell reporter gene assays as screening tools in the identification of antimicrobial natural product extracts.

    PubMed

    Nybond, Susanna; Karp, Matti; Yrjönen, Teijo; Tammela, Päivi

    2015-07-01

    We describe novel tools, bioluminescent whole-cell reporter gene assays, for facilitating the use of natural products in antimicrobial drug discovery. As proof-of-concept, a plant extract library was screened and follow-up experiments were carried out. Primary results can be obtained in 2-4h with high sensitivity, leading to significant improvements of the process. PMID:25937087

  12. Adenocarcinoma of the lung with concomitant ALK fusion gene and EGFR gene mutation: A case report and literature review

    PubMed Central

    FAN, TAO; SONG, YING-JIE; LIU, XIU-LI

    2016-01-01

    Targeted therapy is currently a very popular approach to cancer treatment. Personalized targeted therapy of non-small-cell lung cancer (NSCLC) is based on the mutation status of epithelial growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog, B-Raf proto-oncogene and anaplastic lymphoma kinase (ALK) fusion gene, with different mutations requiring different treatment. Herein, we present a rare case of adenocarcinoma of the lung harboring EGFR- and ALK-activating mutations simultaneously. The patient is currently on pemetrexed and carboplatin chemotherapy and faring well; follow-up will be continued. It is recommended that the biological behavior of cancers with coexisting genetic mutations and their clinicopathological characteristics are more closely investigated in future studies, in order to determine the sensitivity of such cancers to targeted drugs and select the optimal agent. PMID:26893862

  13. Preliminary Report of a Neurokinin-Like Receptor Gene Sequence for the Nemertean Paranemertes sp.

    PubMed

    Chung, Brian M; Stevens, Rainee C; Thomas, Chelsie L; Palmere, Laura N; Okazaki, Robert K

    2015-12-01

    Tachykinins (TKs) are a family of neurotransmitters that function as signaling molecules for such processes as maintaining homeostasis, regulating stress response, and modulating pain. TKs require the expression of at least one of three receptor subtypes: Neurokinin Receptor-1 (NKR-1), Neurokinin Receptor-2 (NKR-2), or Neurokinin Receptor-3 (NKR-3). We have isolated and cloned a portion of a gene coding for a tachykinin-like receptor from the nemertean Paranemertes sp. This 488-bp portion contains a short 101-bp segment that shares 85% similarity to the mouse substance-K receptor in Mus musculus and 83% similarity to the moth neuropeptide receptor A24 in Bombyx mori. Translated homology analysis aligning the coding sequence with the initial cytoplasmic carboxyl terminus of numerous G-protein coupled neuropeptide receptors also revealed 73% similarity to B. mori neuropeptide receptor A24. Our finding is the first report of a sequence amplified from Paranemertes sp. that may code for a small portion of a G-protein-coupled neuropeptide receptor with significant similarity to the TKR family, particularly the NKR-3 receptor isoform. This novel finding may open new avenues into exploring the role of tachykinin and its receptor in nemertean neurophysiology. PMID:26654039

  14. Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene.

    PubMed

    Qian, Yan; Wang, Xiao-Li; Lv, Zhan-Lu; Tysklind, Mats; Guo, Chen; Liang, Bao; Wu, Jia-Bing; Yang, Yong-Jian; Yang, Yi-Shu; Wang, Fei-Fei; Duan, Xiao-Li; Ma, Jin; Wei, Yong-Jie; Wang, Chun-Hui; Yang, Li-Xin; Zhang, Jin-Liang; Shi, Xiao-Ming; Wang, Xian-Liang

    2016-04-01

    The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747μM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents. PMID:26774982

  15. Reporter gene imaging identifies intratumoral infection voids as a critical barrier to systemic oncolytic virus efficacy

    PubMed Central

    Miller, Amber; Suksanpaisan, Lukkana; Naik, Shruthi; Nace, Rebecca; Federspiel, Mark; Peng, Kah Whye; Russell, Stephen J

    2014-01-01

    Systemically administered oncolytic viruses have the ability to cause tumor destruction through the expansion and coalescence of intratumoral infectious centers. Efficacy is therefore dependent upon both the density and intratumoral distribution of virus-infected cells achieved after initial virus infusion, and delivery methods are being developed to enhance these critical parameters. However, the three-dimensional (3D) mapping of intratumoral infectious centers is difficult using conventional immunohistochemical methodology, requiring painstaking 3D reconstruction of numerous sequential stained tumor sections, with no ability to study the temporal evolution of spreading infection in a single animal. We therefore developed a system using very high-resolution noninvasive in vivo micro single-photon emitted computed tomography/computed tomography (microSPECT/CT) imaging to determine the intratumoral distribution of thyroid radiotracers in tumors infected with an oncolytic virus encoding the thyroidal sodium–iodide symporter (NIS). This imaging system was used for longitudinal analysis of the density, distribution, and evolution of intratumoral infectious centers after systemic administration of oncolytic vesicular stomatitis virus in tumor-bearing mice and revealed heterogeneous delivery of virus particles both within and between tumors in animals receiving identical therapy. This study provides compelling validation of high resolution in vivo reporter gene mapping as a convenient method for serial monitoring of intratumoral virus spread that will be necessary to address critical barriers to systemic oncolytic virus efficacy such as intratumoral delivery.

  16. Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay.

    PubMed

    Connolly, Lisa; Cai, Kai; Van der Heiden, Edwige; Scippo, Marie-Louise; Muller, Marc; Tarbin, Jonathan; Elliott, Chris

    2009-04-01

    The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems. We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC(50) of 0.79 ngmL(-1) for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay. Cross-reactivity data for a range of 11beta-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques. PMID:19286046

  17. Assessing hormone receptor activities of pyrethroid insecticides and their metabolites in reporter gene assays.

    PubMed

    Du, Guizhen; Shen, Ouxi; Sun, Hong; Fei, Juan; Lu, Chuncheng; Song, Ling; Xia, Yankai; Wang, Shoulin; Wang, Xinru

    2010-07-01

    Pyrethroid insecticides, the most commonly used insecticides worldwide, are suspected endocrine-disrupting chemicals. But their interactions with hormone receptors are still unclear. The present study intended to evaluate and compare the hormone receptor (estrogen receptor [ER], androgen receptor [AR], and thyroid hormone receptor [TR]) activities of nine pyrethroids (cycloprothrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, etofenprox, fenvalerate, permethrin, and tetramethrin) and their metabolites (3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropne carboxylic acid [DCCA] and 3-phenoxybenzoic acid [3-PBA]) using receptor-mediated luciferase reporter gene assays. Of the 11 compounds tested, four showed very weak ER agonistic activities and six displayed antiestrogenic effects, among which cyhalothrin and DCCA possessed the most potent estrogenic and antiestrogenic activity respectively. Antagonistic effects to AR were found in 7 compounds, with cyfluthrin and deltamethrin exhibiting stronger AR antagonistic capacity. In the TR assay, all of tested chemicals except DCCA showed antagonistic effects. In this study, we provided evidence that a variety of pyrethroids and their metabolites might disrupt the function of multiple nuclear hormone receptors and thus have the potentials to affect the endocrine and the reproductive systems in humans. PMID:20410157

  18. Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Zemp, Roger J.

    2012-02-01

    We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

  19. Malignant perivascular epithelioid cell tumor (PEComa) of cervix with TFE3 gene rearrangement: a case report.

    PubMed

    Liu, Feifei; Zhang, Renya; Wang, Zi-Yu; Xia, Qiuyuan; Shen, Qin; Shi, Shanshan; Tu, Pin; Shi, Qunli; Zhou, Xiaojun; Rao, Qiu

    2014-01-01

    In this study, we reported the first PEComa arising within the cervix with TFE3 gene rearrangement and aggressive biological behavior. Morphologically, the tumor showed infiltrative growth into the surrounding parenchyma. The majority of tumor cells were arrayed in sheets, alveolar structures, or nests separated by delicate fibrovascular septa. There was marked intratumoral hemorrhage, necrosis, and stromal calcifications. The tumor cells had abundant clear cytoplasm, focally containing finely granular dark brown pigment, morphologically considered to be melanin. Immunohistochemically, the tumor cells demonstrated moderately (2+) or strongly (3+) positive staining for TFE3, HMB45, and Melan A but negative for CKpan, SMA, S100, PAX8, and PAX2. The presence of Ki-67 protein demonstrated a moderate proliferation rate, with a few Ki-67-positive nuclei. Using a recently developed TFE3 split FISH assay, the presence of TFE3 rearrangement was demonstrated. All these clinicopathologic features are suggestive of TFE3-rearranged PEComas of the cervix. Our results both expand the known characteristics of primary cervix PEComas and add to the data regarding TFE3 rearrangement-associated PEComas. PMID:25337301

  20. Conditional Deletion of the Relaxin Receptor Gene in Cells of Smooth Muscle Lineage Affects Lower Reproductive Tract in Pregnant Mice1

    PubMed Central

    Kaftanovskaya, Elena M.; Huang, Zaohua; Lopez, Carolina; Conrad, Kirk; Agoulnik, Alexander I.

    2015-01-01

    ABSTRACT Relaxin hormone secreted into the circulation during pregnancy was discovered through its effects on pubic symphysis relaxation and parturition. Genetic inactivation of the relaxin gene or its cognate relaxin family peptide receptor 1 (RXFP1) in mice caused failure of parturition and mammary nipple enlargement, as well as increased collagen fiber density in the cervix and vagina. However, the relaxin effect on discrete cells and tissues has yet to be determined. Using transgenic mice with a knockin LacZ reporter in the Rxfp1 allele, we showed strong expression of this gene in vaginal and cervical stromal cells, as well as pubic ligament cells. We produced a floxed Rxfp1 allele that was used in combination with the Tagln-cre transgene to generate mice with a smooth muscle-specific gene knockout. In pregnant females, the ROSA26 reporter activated by Tagln-cre was detected in smooth muscle cells of the cervix, vagina, uterine artery, and in cells of the pubic symphysis. In late pregnant females with conditional gene ablation, the length of pubic symphysis was significantly reduced compared with wild-type or heterozygous Rxfp1+/− females. Denser collagen content was revealed by Masson trichrome staining in reproductive tract organs, uterine artery, and pubic symphysis. The cervical and vaginal epithelium was less developed than in heterozygous or wild-type females, although nipple size was normal and the dams were able to nurse their pups. In summary, our data indicate that relaxin/RXFP1 signaling in smooth muscle cells is important for normal collagen turnover and relaxation of the pubic symphysis during pregnancy. PMID:25715795

  1. Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.

    PubMed Central

    Asselbergs, F A; Grossenbacher, R; Ortmann, R; Hengerer, B; McMaster, G K; Sutter, E; Widmer, R; Buxton, F

    1998-01-01

    Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts. PMID:9512559

  2. TYR as a multifunctional reporter gene regulated by the Tet-on system for multimodality imaging: an in vitro study

    PubMed Central

    Feng, Hongyan; Xia, Xiaotian; Li, Chongjiao; Song, Yiling; Qin, Chunxia; Zhang, Yongxue; Lan, Xiaoli

    2015-01-01

    The human tyrosinase gene TYR is a multifunctional reporter gene with potential use in photoacoustic imaging (PAI), positron emission tomography (PET), and magnetic resonance imaging (MRI). We sought to establish and evaluate a reporter gene system using TYR under the control of the Tet-on gene expression system (gene expression induced by doxycycline [Dox]) as a multimodality imaging agent. We transfected TYR into human breast cancer cells (MDA-MB-231), naming the resulting cell line 231-TYR. Using non-transfected MDA-MB-231 cells as a control, we verified successful expression of TYR by 231-TYR after incubation with Dox using western blot, cellular tyrosinase activity, Masson-Fontana silver staining, and a cell immunofluorescence study, while the control cells and 231-TYR cells without Dox exposure revealed no TYR expression. Detected by its absorbance at 405 nm, increasing concentrations of melanin correlated positively with Dox concentration and incubation time. TYR expression by Dox-induced transfected cells shortened MRI T1 and T2 relaxation times. Photoacoustic signals were easily detected in these cells. 18F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide (18F-5-FPN), which targets melanin, quickly accumulated in Dox-induced 231-TYR cells. These show that TYR induction of melanin production is regulated by the Tet-on system, and TYR-containing indicator cells may have utility in multimodality imaging. PMID:26483258

  3. Pine Gene Discovery Project - Final Report - 08/31/1997 - 02/28/2001

    SciTech Connect

    Whetten, R. W.; Sederoff, R. R.; Kinlaw, C.; Retzel, E.

    2001-04-30

    Integration of pines into the large scope of plant biology research depends on study of pines in parallel with study of annual plants, and on availability of research materials from pine to plant biologists interested in comparing pine with annual plant systems. The objectives of the Pine Gene Discovery Project were to obtain 10,000 partial DNA sequences of genes expressed in loblolly pine, to determine which of those pine genes were similar to known genes from other organisms, and to make the DNA sequences and isolated pine genes available to plant researchers to stimulate integration of pines into the wider scope of plant biology research. Those objectives have been completed, and the results are available to the public. Requests for pine genes have been received from a number of laboratories that would otherwise not have included pine in their research, indicating that progress is being made toward the goal of integrating pine research into the larger molecular biology research community.

  4. Transcriptional analysis of the 5' terminus of the flp fimbrial gene cluster from Actinobacillus actinomycetemcomitans.

    PubMed

    Haase, Elaine M; Stream, Joshua O; Scannapieco, Frank A

    2003-01-01

    Fresh isolates of the oral bacterial pathogen Actinobacillus actinomycetemcomitans exhibit a fimbriated, rough colony phenotype. Evidence suggests that the fimbrial subunit gene flp is part of a cluster of 14 genes (flp to tadG) thought to encode proteins involved in the synthesis, assembly and export of these fimbriae. To determine the transcriptional organization of the 5' terminus of this gene cluster, total RNA from rough and smooth phenotype variants of A. actinomycetemcomitans strain 283 were analysed by RT-PCR. Primers designed to amplify regions spanning gene junctions or multiple genes yielded amplicons at each individual gene junction from flp to tadD for both the rough and smooth variants. Semi-quantitative RT-PCR of the rcpA to tadZ amplicon revealed that significantly more mRNA was transcribed from the rough than the smooth variant. Longer amplicons encompassing flp to tadZ (3.9 kb) and tadA to tadD (2.1 kb) were also detected, but only from the rough variant. Rapid amplification of cDNA ends (RACE) was used to identify the 5' end of the mRNA containing flp. Antisense primers located within rcpC, orfB and flp-2 enabled amplification of a RACE product that was subsequently isolated and subcloned into pGEM-T. DNA sequencing indicated that the 5' end of the mRNA was located at a G or T nucleotide -102 to -101 nt upstream of flp. Corresponding sigma(70) consensus sequences were located at -10 (TATAAT) and -35 (TTGCAT) relative to the transcription start site. These data confirm that the flp gene cluster is an operon transcribed as a polycistronic message commencing from a G or T nucleotide located in the intergenic region upstream of flp. Promoter function of the flp upstream region was confirmed using a lacZ reporter gene construct transformed into Escherichia coli. RT-PCR analysis further suggests that although transcription does occur in both the rough and smooth variants, full-length transcripts are rapidly degraded or are significantly downregulated in the smooth variant. PMID:12576594

  5. Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI

    PubMed Central

    Bar-Shir, Amnon; Liu, Guanshu; Greenberg, Marc M; Bulte, Jeff W M; Gilad, Assaf A

    2013-01-01

    In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year. PMID:24177294

  6. The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.

    PubMed Central

    Foster-Hartnett, D; Kranz, R G

    1994-01-01

    The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region. Images PMID:8051036

  7. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. ); Schild, D. )

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  8. Fine regulation of cI857-controlled gene expression in continuous culture of recombinant Escherichia coli by temperature.

    PubMed Central

    Villaverde, A; Benito, A; Viaplana, E; Cubarsi, R

    1993-01-01

    The expression at different temperatures of the lacZ gene, which is controlled by the lambda pL and pR tandem promoters and the cI857 temperature-sensitive repressor, was studied in Escherichia coli continuous cultures. At temperatures between 30 and 42 degrees C, beta-galactosidase activity behaved according to an exponential equation. By inducing a culture at a temperature within this range, predefined, nearly constant submaximal levels of gene expression and recombinant product yield can be obtained. PMID:8250569

  9. The Bacillus thuringiensis PlcR-regulated gene inhA2 is necessary, but not sufficient, for virulence.

    PubMed

    Fedhila, Sinda; Gohar, Michel; Slamti, Leyla; Nel, Patricia; Lereclus, Didier

    2003-05-01

    We previously reported that Bacillus thuringiensis strain 407 Cry 32(-) secretes a zinc-requiring metalloprotease, InhA2, that is essential for virulence in orally infected insects. Analysis of the inhA2-lacZ transcriptional fusion showed that inhA2 expression is repressed in a PlcR(-) background. Using DNase I footprinting experiments, we demonstrated that PlcR activates inhA2 transcription directly by binding to a DNA sequence showing a one-residue mismatch with the previously reported PlcR box. It was previously reported that PlcR is essential for B. thuringiensis virulence in oral infection by contributing to the synergistic properties of the spores on the insecticidal activity of the Cry1C protein. We used complementation experiments to investigate whether the PlcR(-) phenotype was due to the absence of InhA2. The results indicated that overexpression of inhA2 in the (Delta)plcR strain did not restore the wild-type phenotype. However, virulence was fully restored in the (Delta)inhA2 complemented mutant. Thus, inhA2 is the first example of a PlcR-regulated gene found to be directly involved in virulence. However, it is not sufficient for pathogenicity when the other members of the PlcR regulon are lacking. This suggests that InhA2 may act in concert with other PlcR-regulated gene products to provide virulence. PMID:12700261

  10. The Bacillus thuringiensis PlcR-Regulated Gene inhA2 Is Necessary, but Not Sufficient, for Virulence

    PubMed Central

    Fedhila, Sinda; Gohar, Michel; Slamti, Leyla; Nel, Patricia; Lereclus, Didier

    2003-01-01

    We previously reported that Bacillus thuringiensis strain 407 Cry 32− secretes a zinc-requiring metalloprotease, InhA2, that is essential for virulence in orally infected insects. Analysis of the inhA2-lacZ transcriptional fusion showed that inhA2 expression is repressed in a PlcR− background. Using DNase I footprinting experiments, we demonstrated that PlcR activates inhA2 transcription directly by binding to a DNA sequence showing a one-residue mismatch with the previously reported PlcR box. It was previously reported that PlcR is essential for B. thuringiensis virulence in oral infection by contributing to the synergistic properties of the spores on the insecticidal activity of the Cry1C protein. We used complementation experiments to investigate whether the PlcR− phenotype was due to the absence of InhA2. The results indicated that overexpression of inhA2 in the ΔplcR strain did not restore the wild-type phenotype. However, virulence was fully restored in the ΔinhA2 complemented mutant. Thus, inhA2 is the first example of a PlcR-regulated gene found to be directly involved in virulence. However, it is not sufficient for pathogenicity when the other members of the PlcR regulon are lacking. This suggests that InhA2 may act in concert with other PlcR-regulated gene products to provide virulence. PMID:12700261

  11. Noninvasive visualization of microRNA-16 in the chemoresistance of gastric cancer using a dual reporter gene imaging system.

    PubMed

    Wang, Fu; Song, Xinxing; Li, Xiujuan; Xin, Jing; Wang, Shenxu; Yang, Weidong; Wang, Jing; Wu, Kaichun; Chen, Xiaoyuan; Liang, Jimin; Tian, Jie; Cao, Feng

    2013-01-01

    MicroRNAs (miRNAs) have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of (131)I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo (99m)Tc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16) and 5-fluorouracil (5-FU) activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic. PMID:23613938

  12. Molecular Screening of "MECP2" Gene in a Cohort of Lebanese Patients Suspected with Rett Syndrome: Report on a Mild Case with a Novel Indel Mutation

    ERIC Educational Resources Information Center

    Corbani, S.; Chouery, E.; Fayyad, J.; Fawaz, A.; El Tourjuman, O.; Badens, C.; Lacoste, C.; Delague, V.; Megarbane, A.

    2012-01-01

    Background: Rett syndrome (RTT), an X-linked, dominant, neurodevelopment disorder represents 10% of female subjects with profound intellectual disability. Mutations in the "MECP2" gene are responsible for up to 95% of the classical RTT cases, and nearly 500 different mutations distributed throughout the gene have been reported. Methods: We report

  13. Identification of a major cis-acting DNA element controlling the bidirectionally transcribed penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) of Aspergillus nidulans.

    PubMed

    Bergh, K T; Litzka, O; Brakhage, A A

    1996-07-01

    The beta-lactam antibiotic penicillin is produced as a secondary metabolite by some filamentous fungi. In this study, the molecular regulation of the Aspergillus (Emericella) nidulans penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) was analyzed. acvA and ipnA are divergently oriented and separated by an intergenic region of 872 bp. Translational fusions of acvA and ipnA with the two Escherichia coli reporter genes lacZ and uidA enabled us to measure the regulation of both genes simultaneously. A moving-window analysis of the 872-bp intergenic region indicated that the divergently oriented promoters are, at least in part, overlapping and share common regulatory elements. Removal of nucleotides -353 to -432 upstream of the acvA gene led to a 10-fold increase of acvA-uidA expression and simultaneously to a reduction of ipnA-lacZ expression to about 30%. Band shift assays and methyl interference analysis using partially purified protein extracts revealed that a CCAAT-containing DNA element within this region was specifically bound by a protein (complex), which we designated PENR1, for penicillin regulator. Deletion of 4 bp within the identified protein binding site caused the same contrary effects on acvA and ipnA expression as observed for all of the deletion clones which lacked nucleotides -353 to -432. The PENR1 binding site thus represents a major cis-acting DNA element. The intergenic regions of the corresponding genes of the beta-lactam-producing fungi Penicillium chrysogenum and Acremonium chrysogenum also diluted the complex formed between the A. nidulans probe and PENR1 in vitro, suggesting that these beta-lactam biosynthesis genes are regulated by analogous DNA elements and proteins. PMID:8682797

  14. A global investigation of gene deletion strains that affect premature stop codon bypass in yeast, Saccharomyces cerevisiae.

    PubMed

    Samanfar, Bahram; Tan, Le Hoa; Shostak, Kristina; Chalabian, Firoozeh; Wu, Zongbin; Alamgir, Md; Sunba, Noor; Burnside, Daniel; Omidi, Katayoun; Hooshyar, Mohsen; Galván Márquez, Imelda; Jessulat, Matthew; Smith, Myron L; Babu, Mohan; Azizi, Ali; Golshani, Ashkan

    2014-04-01

    Protein biosynthesis is an orderly process that requires a balance between rate and accuracy. To produce a functional product, the fidelity of this process has to be maintained from start to finish. In order to systematically identify genes that affect stop codon bypass, three expression plasmids, pUKC817, pUKC818 and pUKC819, were integrated into the yeast non-essential loss-of-function gene array (5000 strains). These plasmids contain three different premature stop codons (UAA, UGA and UAG, respectively) within the LacZ expression cassette. A fourth plasmid, pUKC815 that carries the native LacZ gene was used as a control. Transformed strains were subjected to large-scale β-galactosidase lift assay analysis to evaluate production of β-galactosidase for each gene deletion strain. In this way 84 potential candidate genes that affect stop codon bypass were identified. Three candidate genes, OLA1, BSC2, and YNL040W, were further investigated, and were found to be important for cytoplasmic protein biosynthesis. PMID:24535059

  15. A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.

    PubMed

    Xiao, Dan; Zhang, Weifeng; Li, Yan; Liu, Kuan; Zhao, Junli; Sun, Xiaohong; Shan, Linlin; Mao, Qinwen; Xia, Haibin

    2016-02-10

    Sox2 is an important transcriptional factor that has multiple functions in stem cell maintenance and tumorigenesis. To investigate the transcriptional regulation of the Sox2 gene, a luciferase knock-in reporter system was established in HEK293 cells by placing the luciferase gene in the genome under the control of the Sox2 gene promoter using a transcription activator-like effector nuclease (TALEN)-mediated genome editing technique. PCR and Southern blot results confirmed the site-specific integration of a single copy of the exogenous luciferase gene into the genome. To prove the reliability and sensitivity of this novel luciferase knock-in system, a CRISPR/Cas transcription activation system for the Sox2 gene was constructed and applied to the knock-in system. The results indicated that luciferase activity was directly correlated with the activity of the Sox2 endogenous promoter. This novel system will be a useful tool to study the transcriptional regulation of Sox2, and has great potential in medical and industrial applications. PMID:26721181

  16. The PR/SET Domain Zinc Finger Protein Prdm4 Regulates Gene Expression in Embryonic Stem Cells but Plays a Nonessential Role in the Developing Mouse Embryo

    PubMed Central

    Bogani, Debora; Morgan, Marc A. J.; Nelson, Andrew C.; Costello, Ita; McGouran, Joanna F.; Kessler, Benedikt M.

    2013-01-01

    Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal. PMID:23918801

  17. The PR/SET domain zinc finger protein Prdm4 regulates gene expression in embryonic stem cells but plays a nonessential role in the developing mouse embryo.

    PubMed

    Bogani, Debora; Morgan, Marc A J; Nelson, Andrew C; Costello, Ita; McGouran, Joanna F; Kessler, Benedikt M; Robertson, Elizabeth J; Bikoff, Elizabeth K

    2013-10-01

    Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal. PMID:23918801

  18. Transposon tagging of disease resistance genes. Final report, May 1, 1988--April 30, 1993

    SciTech Connect

    Michelmore, R.

    1994-09-01

    The goal of this project was to develop a transposon mutagenesis system for lettuce and to clone and characterize disease resistance genes by transposon tagging. The majority of studies were conducted with the Ac/Ds System. Researchers made and tested several constructs as well as utilized constructions shown to be functional in other plant species. Researchers demonstrated movement of Ac and DS in lettuce; however, they transposed at much lower frequencies in lettuce than in other plant species. Therefore, further manipulation of the system, particularly for flower specific expression of transposase, is required before a routine transposon system is available for lettuce. Populations of lettuce were generated and screened to test for the stability of resistance genes and several spontaneous mutations were isolated. Researchers also identified a resistance gene mutant in plants transformed with a Ds element and chimeric transposase gene. This is currently being characterized in detail.

  19. Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells

    PubMed Central

    Reavy, Brian; Dawson, Sheila; Canto, Tomas; MacFarlane, Stuart A

    2004-01-01

    Background RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. Results Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. Conclusion These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi. PMID:15331016

  20. Molecular characterization and developmental expression of the TFIIH factor p62 gene from Drosophila melanogaster: effects on the UV light sensitivity of a p62 mutant fly.

    PubMed

    Castro, Juan; Merino, Carlos; Zurita, Mario

    2002-05-30

    TFIIH is a multiprotein complex that has a central role in the RNA pol II mediated transcription, in DNA repair and in the control of the cell cycle. Mutations in some components of TFIIH are associated with three hereditary human syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). The p62 protein is a structural component of the TFIIH core and no syndromes have been identified up to date by mutations in this human gene. In this work we report the molecular and genetic characterization of the Drosophila melanogaster p62 gene (Dmp62). The Dmp62 gene product shows high identity with its human and mouse homologues. Using computer analysis we identified several common motifs in the p62 proteins from different organisms, suggesting that these motifs could be involved in possible protein-protein interactions within the TFIIH complex or with other transcription and DNA repair factors. The Dmp62 transcript is expressed at similar levels throughout development, although there is a significant increase of the transcript level during the late embryogenesis and in the adult male. The analysis of a Drosophila line with a P-element enhancer trap insertion at the Dmp62 5'-UTR that directs the lac-Z expression from the Dmp62 promoter, showed a high level of expression in the gut, the testis and the pericardial cells. A P-element that disrupts the Dmp62 gene (Dmp62mut) produces early embryo lethality in homozygous flies. Heterozygous Dmp62mut larvae are more sensitive to UV light irradiation, and those individuals that are able to develop into adults have severe abdominal cuticular damage after UV light irradiation. PMID:12509240

  1. [Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

    SciTech Connect

    Okita, T.W.

    1999-04-01

    Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.

  2. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the {beta}-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate {beta}-carboxy-cis,cis-muconate. {beta}-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for {beta}-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to {beta}-carboxy-cis,cis-muconate.

  3. Regulation of spo0H, an early sporulation gene in bacilli.

    PubMed Central

    Dubnau, E J; Cabane, K; Smith, I

    1987-01-01

    The construction of lacZ fusions in frame with the spo0H gene of Bacillus licheniformis enabled us to study the expression of this gene under various growth conditions and in various genetic backgrounds. spo0H was expressed during vegetative growth, but the levels increased during early stationary phase and then decreased several hours later. Expression of the gene was not repressed by glucose, but was induced by decoyinine, an inhibitor of guanine nucleotide biosynthesis, which can induce sporulation. Of those tested, the only spo0 gene required for the expression of spo0H was spo0A, and this requirement was eliminated by the abrB mutation, a partial suppressor of spo0A function. spo0H-lacZ expression was much higher in a strain with a deletion in the spo0H gene. Images PMID:3102457

  4. Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment.

    PubMed

    Reuther, Peter; Göpfert, Kristina; Dudek, Alexandra H; Heiner, Monika; Herold, Susanne; Schwemmle, Martin

    2015-01-01

    Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection. PMID:26068081

  5. Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment

    PubMed Central

    Reuther, Peter; Göpfert, Kristina; Dudek, Alexandra H.; Heiner, Monika; Herold, Susanne; Schwemmle, Martin

    2015-01-01

    Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection. PMID:26068081

  6. In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes

    PubMed Central

    Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan MW

    2006-01-01

    Background Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. Results The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. Conclusion We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable. PMID:16725020

  7. First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

    PubMed Central

    Habbal, Mohammad Zouheir; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F.

    2014-01-01

    Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin. PMID:25233259

  8. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    SciTech Connect

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  9. Seed-protein genes and the regulation of their expression. Progress report

    SciTech Connect

    Larkins, B.A.; Foard, D.E.

    1982-05-27

    Research has focused primarily on the isolation and identification of mRNAs directing the synthesis of major protease inhibitors of soybean seeds, the so called Bowman-Birk inhibitor and the family of related iso-inhibitors designated PI I-IV. A major objective of this research was to develop methods for detecting the primary translation products of the mRNA in a cell-free translation system, and use this procedure to identify the corresponding sequences among cDNA clones of the mRNAs. These clones are eventually to be used to characterize the expression of these genes during soybean embryo development, and to isolate and characterize the genes encoding these proteins in soybean and related legumes. We are also examining the potential for using Xenopus oocytes as a system for measuring transcription of plant genes. Research progress is summarized.

  10. Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992

    SciTech Connect

    Michelmore, R.

    1994-06-01

    Our goal is to clone genes in lettuce determining resistance to downy mildew. One approach involves the mobilization of transposons into resistance genes to mutate and tag the target gene. Because transposons have yet to be isolated and characterized from lettuce, the majority of our experiments have involved Ac from corn as this is increasingly the best characterized transposon. Over the past several years, various labs have contributed to a detailed understanding of the biology of Ac in corn and heterologous plant species. We have collaborated closely with several of these labs, exchanged materials and incorporated their advances into our analysis of transposition in lettuce. The original proposal described the development of a transposon mutagenesis system for lettuce and its subsequent use to tag disease resistance genes. The development phase involved characterization and manipulation of Ac transposition, identification of suitable whole plant selectable markers for the construction of chimeric non-autonomous elements, and investigation of the stability of resistance genes. Investigation of Ac transposition in lettuce has received the majority of our attention. Initially, we made a simple construct with wildtype Ac and introduced it into lettuce. No transposition was observed; although other labs demonstrated that the same construct was functional in tomato. We then focused on assaying for Ac transposition with constructs of increasing sophistication that had been demonstrated by others to be functional in other species. The latest constructs for transposon mutagenesis clearly demonstrated transposition in lettuce. This allowed us to generate seed stocks that we will start to screen for insertional inactivation of resistance genes this year.

  11. Inference of Quantitative Models of Bacterial Promoters from Time-Series Reporter Gene Data

    PubMed Central

    Stefan, Diana; Pinel, Corinne; Pinhal, Stphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for the dynamics of FliA-dependent promoters. PMID:25590141

  12. cis-Acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes

    SciTech Connect

    Hofmann, A.; Garfinkel, M.D.; Meyerowitz, E.M. )

    1991-06-01

    The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between {minus}211 and {minus}43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from {minus}133 to {minus}48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements.

  13. A novel SNP in 3' UTR of INS gene: A case report of neonatal diabetes mellitus.

    PubMed

    Bogari, Neda M; Rayes, Husni H; Mostafa, Fakri; Abdel-Latif, Azza M; Ramadan, Abeer; Al-Allaf, Faisal A; Taher, Mohiuddin M; Fawzy, Ahmed

    2015-09-01

    Neonatal diabetes mellitus (NDM) is a rare condition with a prevalence of 1 in 300,000 live births. We have found 3 known SNPs in 5'UTR and a novel SNP in 3' UTR in the INS gene. These SNPs were present in 9-month-old girl from Saudi Arabia and also present in the father and mother. The novel SNP we found is not present in 1000 Genome project or other databases. Further, the newly identified 3' UTR mutation in the INS gene may abolish the polyadenylation signal and result in severe RNA instability. PMID:26212367

  14. Molecular characterization of a maize regulatory gene. Annual progress report, November 1991--October 1992

    SciTech Connect

    Wessler, S.R.

    1994-05-01

    All aspects of this year`s work have converged on the central theme of post-transcriptional control of R gene expression. Unlike transcriptional control, relatively little is known about post-transcriptional regulation, especially in plants. We believe that three levels of post-transcriptional regulation have been identified: control of translation initiation as evidenced by the maize Lc gene; control of nuclear localization as evidenced by the Ds allele r-m9 of maize; and control of nuclear localization through alternative splicing of the rice R homolog.

  15. Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

    PubMed Central

    Leung, Alice; Katz, Anna B.; Lim, Foong-Yen; Habli, Mounira; Jones, Helen N.; Wilson, James M.; Crombleholme, Timothy M.

    2012-01-01

    Background Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls. Methodology/Principal Findings Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts. Conclusions/Significance Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis. PMID:22937069

  16. Aarskog-Scott syndrome: clinical update and report of nine novel mutations of the FGD1 gene.

    PubMed

    Orrico, A; Galli, L; Faivre, L; Clayton-Smith, J; Azzarello-Burri, S M; Hertz, J M; Jacquemont, S; Taurisano, R; Arroyo Carrera, I; Tarantino, E; Devriendt, K; Melis, D; Thelle, T; Meinhardt, U; Sorrentino, V

    2010-02-01

    Mutations in the FGD1 gene have been shown to cause Aarskog-Scott syndrome (AAS), or facio-digito-genital dysplasia (OMIM#305400), an X-linked disorder characterized by distinctive genital and skeletal developmental abnormalities with a broad spectrum of clinical phenotypes. To date, 20 distinct mutations have been reported, but little phenotypic data are available on patients with molecularly confirmed AAS. In the present study, we report on our experience of screening for mutations in the FGD1 gene in a cohort of 60 European patients with a clinically suspected diagnosis of AAS. We identified nine novel mutations in 11 patients (detection rate of 18.33%), including three missense mutations (p.R402Q; p.S558W; p.K748E), four truncating mutations (p.Y530X; p.R656X; c.806delC; c.1620delC), one in-frame deletion (c.2020_2022delGAG) and the first reported splice site mutation (c.1935+3A>C). A recurrent mutation (p.R656X) was detected in three independent families. We did not find any evidence for phenotype-genotype correlations between type and position of mutations and clinical features. In addition to the well-established phenotypic features of AAS, other clinical features are also reported and discussed. PMID:20082460

  17. Genetic studies of a thermoregulated gene in the psychrotrophic bacterium Pseudomonas fluorescens.

    PubMed

    Regeard, C; Mérieau, A; Leriche, F; Guespin-Michel, J F

    1999-09-01

    In the psychrotrophic bacterium Pseudomonas fluorescens, some genes are thermoregulated: they are maximally expressed at a particular temperature within the broad range of temperatures that allow growth of this bacterium. To study this regulation, random transcriptional insertion fusions were obtained by means of mini-Tn5lacZ1 or mini-Tn5luxAB transposition. One fusion was studied in which beta-galactosidase production was maximal at a low-growth temperature. The mutated gene (that we call xsf) was highly homologous to xseA from Escherichia coli (and from other bacteria) which encodes the large subunit of exonuclease VII. Genetic tools were constructed in order to analyse and manipulate this fusion: a plasmid derived from R68.45 was used for chromosome transfer and a replacement vector was constructed to allow in situ marker exchange of the mini-Tn5lacZ1 by an Hg(r) interposon. This vector was used to make double mutants and hence to study the effect of the insertion in xsf on the expression of other fusions. Six genes were thereby identified with a decreased expression in an xsf- background and with different characteristics of thermoregulation. PMID:10540908

  18. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

    PubMed Central

    Hu, Y; Cooper, T G; Kohlhaw, G B

    1995-01-01

    The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways. PMID:7799961

  19. [Distal hereditary motor neuropathy type II with mutation in heat shock protein 27 gene. A case report].

    PubMed

    Nishibayashi, Momoka; Kokubun, Norito; Nakamura, Arata; Hirata, Koichi; Yamamoto, Masahiko; Sobue, Gen

    2007-01-01

    A 48-year-old man was admitted to our hospital with a tendency to stumble during walking. The family history indicated that the father was diagnosed with Charcot-Marie-Tooth disease (CMT) at the age of 55 and his younger sister (aunt) had similar symptoms that were considered to reflect autosomal dominant inheritance. Examination showed no pes cavus or inverted champagne-bottle thighs. In addition, the patient walked with foot drop due to weakness and atrophy of the distal parts of the lower extremities. Sensory examination revealed no deficits or abnormalities. Nerve conduction study and needle electromyography indicated pure motor axonal neuropathy. The diagnosis of distal hereditary motor neuropathy (distal HMN) type II was made. Genetic analysis detected mutation in the heat shock protein 27 (HSP27) gene. A recent report indicated that mutations in the HSP27 gene cause both distal hereditary motor neuropathy and CMT2F. In Japan, there are only a few reports of distal hereditary motor neuropathy with mutation in the HSP27 gene. Distal HMN should be considered in the differential diagnosis of patients with CMT like distal amyotrophy. PMID:17491338

  20. Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome

    PubMed Central

    Wang, Shuwen; Zhao, Yuanjun; Leiby, Melanie A.; Zhu, Jiyue

    2009-01-01

    The endogenous human telomerase reverse transcriptase (hTERT) gene is repressed in somatic cells. To study the mechanisms of its repression, we developed a strategy of retrovirus-directed Cre recombinase-mediated BAC targeting, or RMBT, to generate single-copy integrations of BAC at pre-engineered chromosomal sites. This technique involved retroviral transduction of acceptor loci, containing an HSV thymidine kinase marker, and subsequent integration of BAC constructs into the acceptor sites, utilizing the loxP and lox511 sites present in the vector backbones. The BAC reporter, with a Renilla luciferase cassette inserted downstream of the hTERT promoter, was retrofitted with a puromycin marker. Through puromycin selection and ganciclovir counter-selection, a targeting efficiency of over 50% was achieved. We demonstrated that the activity and chromatin structures of the hTERT promoter in chromosomally integrated BAC reporter recapitulated its endogenous counterpart of the host cells. Therefore, we have established a genetically amendable platform to study chromatin and epigenetic regulation of the hTERT gene. The highly efficient and versatile RMBT technique has general applicability for studying largely unexplored chromatin-dependent mechanisms of promoter regulation of various genes. PMID:19528078

  1. Seed-protein genes and the regulation of their expression. Progress report

    SciTech Connect

    Larkins, B.A.; Hodges, T.K.; Foard, D.E.; Tomes, M.L.

    1981-08-01

    Research during the past year focused on two aspects of seed protein research. One objective explored the possibility of using Xenopus laevis oocytes as a potential system for studying expression of cloned storage protein genes. A second objective developed methods for isolating and partially purifying mRNAs directing the synthesis of the low molecular weight protease inhibitors of soybean seeds.

  2. Transgene-based anthocyanin hyper-pigmentation as a visual reporter of gene silencing in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    “Co-suppression” associated loss of flower pigmentation in transgenic petunia plants was one of the first clear indicators of the natural process of RNA-associated gene silencing in plants. We have been exploring the use of genetically engineered anthocyanin over-production in vegetative tissues as...

  3. Characterization of embryo-specific genes. Final report, April 1, 1987--March 31, 1992

    SciTech Connect

    Sung, R.

    1992-06-12

    The objective of the proposed research is to characterize the function and regulation of a set of embryonic genes which are expressed in the embryos, not in the plants. 22 cDNA clones were isolated from a cDNA library we constructed using mRNAS of -carrot somatic embryos. These cDNA clones identified mRNA species that are present in the somatic and zygotic embryos, but not in adult plants. The sequence of all 22cDNA clones were determined; genomic clones for three cDNA clones, DC8, DC59, and DC49 were isolated and gene sequences determined. DC8, DC49, and several other genes identified by the cDNA sequences belong to the category of late embryogenesis abundant protein genes, Lea. The function of these gens have not yet been determined, but they share common structural features, are regulated by ABA and are speculated to play a role in seed desiccation.

  4. Green Fluorescent Protein as a Reporter To Monitor Gene Expression and Food Colonization by Aspergillus flavus

    PubMed Central

    Du, Wanglei; Huang, Zhengyu; Flaherty, Joseph E.; Wells, Kevin; Payne, Gary A.

    1999-01-01

    Transformants of Aspergillus flavus containing the Aequorea victoria gfp gene fused to a viral promoter or the promoter region and 483 bp of the coding region of A. flavus aflR expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants. PMID:9925624

  5. INDUCTION OF AN ESTROGEN-RESPONSIVE REPORTER GENE IN RAINBOW TROUT HEPATOMA CELLS (RTH 149) AT 11 OR 18 DEGREES C

    EPA Science Inventory

    A reporter gene assay in a cultured rainbow trout cell line was used to determine the influence of temperature on the expression of an estrogen-responsive gene. Rainbow trout hepatoma cells (RTH 149) incubated at 11 or 18 degrees C were co-transfected with an estrogen-responsive ...

  6. Molecular Screening of "MECP2" Gene in a Cohort of Lebanese Patients Suspected with Rett Syndrome: Report on a Mild Case with a Novel Indel Mutation

    ERIC Educational Resources Information Center

    Corbani, S.; Chouery, E.; Fayyad, J.; Fawaz, A.; El Tourjuman, O.; Badens, C.; Lacoste, C.; Delague, V.; Megarbane, A.

    2012-01-01

    Background: Rett syndrome (RTT), an X-linked, dominant, neurodevelopment disorder represents 10% of female subjects with profound intellectual disability. Mutations in the "MECP2" gene are responsible for up to 95% of the classical RTT cases, and nearly 500 different mutations distributed throughout the gene have been reported. Methods: We report…

  7. Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.

    PubMed Central

    He, B; Shiau, A; Choi, K Y; Zalkin, H; Smith, J M

    1990-01-01

    Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression. Images PMID:2198266

  8. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    SciTech Connect

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  9. Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters

    PubMed Central

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-01-01

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time. PMID:24753407

  10. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-07-16

    We have found that soybean cotyledons could be cultured in vitro and that the storage proteins were formed essentially as on a plant. When methionine was added to the medium, the cotyledons grew faster, and the methionine content of the protein fraction was increased by over 20 percent. The high methionine content of the protein fraction was found to be due to a shift in the relative amounts of the two major storage proteins. The later effect was the result of methionine treatment suppressing the expression of one storage protein subunit gene. The goal was to determine the mechanism by which methionine is able to regulate the expression of the ..beta..-subunit gene.

  11. Gnathodiaphyseal dysplasia: report of a family with a novel mutation of the ANO5 gene.

    PubMed

    Duong, Hannah A; Le, Karen T; Soulema, Albert L; Yueh, Ronald H; Scheuner, Maren T; Holick, Michael F; Christensen, Russell; Tajima, Tracey L; Leung, Angela M; Mallya, Sanjay M

    2016-05-01

    Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant disorder characterized by florid osseous dysplasia of the jaws, bone fragility, and diaphyseal cortical thickening and bowing of long bones. We present a family with previously undiagnosed GDD. The disorder was identified by the characteristic gnathic and skeletal manifestations in the father. Clinical and radiologic examination of the patient's son also revealed the characteristic features of GDD. Gene sequencing revealed a novel mutation (c. 1067 G>A, p. Cys356 Tyr) in the ANO5 gene, which is causative for GDD. This mutation was predicted to be detrimental by computational analyses and by structural modeling of the protein. The implications for recognition and management of this disease are discussed. PMID:27068316

  12. A Gene Trap Knockout of the Tiam-1 Protein Results in Malformation of the Early Embryonic Brain

    PubMed Central

    Yoo, Sooyeon; Kim, Yujin; Lee, Haeryung; Park, Sungjeong; Park, Soochul

    2012-01-01

    Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice. PMID:22661025

  13. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  14. (Structure and expression of nuclear genes encoding rubisco activase): Progress report

    SciTech Connect

    Not Available

    1989-01-01

    Our first year's activities include: (1) completing a survey of the basic characteristics of activase gene expression in barley; and (2) isolating and structurally characterizing cDNA and genomic DNA sequences encoding activase from barley. Our goal was to determine whether activase mRNA and protein accumulation are coordinated with those of the rubisco subunits. We utilized the first leaves of barley as an experimental system for these studies because they can be used in two ways to study the expression of leaf genes: by following the naturally occurring differentiation of leaf cells, which occurs acropetally along the barley leaf; and by following the photomorphogenesis of etiolated barley seedlings. In the acropetal gradient of leaf cell differentiation, activase mRNA and mRNA and polypeptide expression is tightly coordinated with rubisco subunit mRNA and polypeptide expression. Although we have not measured their precise stoichiometry at each stage of leaf differentiation, activase protein is expressed at the level of about one polypeptide per rubisco holoenzyme in mature regions of the leaf. Coordination of the expression of activase mRNAs and polypeptides indicates that in the barley leaf gradient, activase gene expression is largely controlled at the level of transcription. However, translational controls may play a role in regulating activase expression on a short term basis.

  15. [Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report

    SciTech Connect

    Guerinot, M.L.

    1992-06-01

    We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

  16. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity. PMID:18661106

  17. Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

    PubMed Central

    2010-01-01

    Background The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1) as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi). Results Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ). This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ). Importantly, mites undergoing the immersion approach had greatly improved survival (75-80%) over 72 h, approaching that of mites not undergoing any treatment. Conclusions Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and immature stages of ticks. PMID:20712880

  18. Phenytoin toxicity in two-month-old Thai infant with CYP2C9 gene polymorphism--A case report.

    PubMed

    Veeravigrom, Montida; Jaroonvanichkul, Vorapol; Netbaramee, Wiracha; Phaisarn, Pichaya; Uyathanarat, Thanita

    2016-01-01

    Phenytoin is one of the most well established and most effective antiepileptic medications for the treatment of focal seizures. In our clinical practice, it has proven difficult to maintain therapeutic phenytoin levels in infants less than three months of age. Incidence of phenytoin toxicity in infants is very rare. The cytochrome P450 super family plays an important role in phenytoin metabolism, especially CYP2C9 and CYP2C19. In this case report, we profiled a two-month-old Thai infant who developed phenytoin toxicity resulting from CYP2C9 gene polymorphism. PMID:25998968

  19. A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.

    PubMed Central

    Van Spanning, R J; Wansell, C W; Reijnders, W N; Harms, N; Ras, J; Oltmann, L F; Stouthamer, A H

    1991-01-01

    A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes. Images FIG. 3 FIG. 4 PMID:1657872

  20. Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine.

    PubMed

    Leach, Derrik M; Zacal, Natalie J; Rainbow, Andrew J

    2013-09-01

    Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β-galactosidase (β-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass. PMID:23793457

  1. A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2: A case report

    PubMed Central

    SHI, YUNFANG; LI, XIAOZHOU; JU, DUAN; LI, YAN; ZHANG, XIULING; ZHANG, YING

    2016-01-01

    Waardenburg syndrome (WS) is an autosomal dominant disorder with varying degrees of sensorineural hearing loss, and accumulation of pigmentation in hair, skin and iris. There are four types of WS (WS1–4) with differing characteristics. Mutations in six genes [paired box gene 3 (PAX3), microphthalmia-associated transcription factor (MITF), endothelin 3 (END3), endothelin receptor type B (EDNRB), SRY (sex determining region Y)-box 10 (SOX10) and snail homolog 2 (SNAI2)] have been identified to be associated with the various types. This case report describes the investigation of genetic mutations in three patients with WS2 from a single family. Genomic DNA was extracted, and the six WS-related genes were sequenced using next-generation sequencing technology. In addition to mutations in PAX3, EDNRB and SOX10, a novel heterozygous MITF mutation, p.Δ315Arg (c.944_946delGAA) on exon 8 was identified. This is predicted to be a candidate disease-causing mutation that may affect the structure and function of the enzyme. PMID:27073475

  2. oct4-EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka.

    PubMed

    Froschauer, Alexander; Khatun, Mst Muslima; Sprott, David; Franz, Alexander; Rieger, Christiane; Pfennig, Frank; Gutzeit, Herwig O

    2013-01-01

    Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4-EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4-EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4-EGFP transgenic medaka in reproductive and stem cell biology. PMID:23139203

  3. Gastrointestinal stromal tumor of the uterus: a case report with genetic analyses of c-kit and PDGFRA genes.

    PubMed

    Terada, Tadashi

    2009-01-01

    The author reports a very rare case of a gastrointestinal stromal tumor in the uterus. A 74-year-old woman was admitted to our hospital because of pelvic pain. Imaging modalities showed a large tumor of the posterior aspect of the uterus, and enucleation was performed. The tumor was attached to the posterior uterus, similar to subserosal leiomyoma. No attachment to the gastrointestinal organs was recognized. The tumor was soft, tan, and measured 13 x 15 x 12 cm. The tumor consisted of cellular spindle cells with focal necrotic areas. Mitotic figures were noted in 3 of 50 high-power fields. The tumor cells were positive for KIT, CD34, platelet-derived growth factor receptor alpha, and vimentin, but negative for alpha-smooth muscle actin, S100 protein, p53 protein, HMB45, and desmin. Ki-67 labeling was 3%. Five normal uteruses used as controls showed KIT-positive Cajal-like mesenchymal cell scattering in the myometrium. Genetic analyses of the c-kit gene (exons 9, 11, 13, and 17) and platelet-derived growth factor receptor alpha gene (exons 12 and 18) revealed a point mutation at codon 559 (GTT-->GAT) of exon 11 of the c-kit gene. Other exons showed no abnormalities. This case shows that gastrointestinal stromal tumor may occur in the uterus. PMID:19047911

  4. TSC2/PKD1 contiguous gene syndrome: a report of 2 cases with emphasis on dermatopathologic findings.

    PubMed

    Kacerovska, Denisa; Vrtel, Radek; Michal, Michal; Vanecek, Tomas; Vodicka, Radek; Kreuzberg, Boris; Ricarova, Renata; Pizinger, Karel; Danis, Dusan; Reischig, Tomas; Kazakov, Dmitry V

    2009-08-01

    The association of tuberous sclerosis complex (TSC) and autosomal dominant polycystic kidney disease (ADPKD), termed TSC2/PKD1 contiguous gene syndrome, is a result of molecular defect demonstrating by deletion disrupting TSC2 and PKD1. Dermatopathology of this syndrome has never been addressed. We report 2 sporadic cases form of TSC2/PKD1 contiguous gene syndrome, with emphasis on dermatopathologic findings. Both patients presented with a typical phenotype of TSC and early-onset renal polycystic requiring kidney transplantation in one of the patients. Of a total of 13 cutaneous lesions studied, there were 7 facial angiofibromas, 2 shagreen patches, 1 periungual fibroma, 1 hypopigmented macule, 1 epidermoid cyst, and 1 intradermal melanocytic nevus. The histological features were basically similar to those occurring in TSC, but some unusual features were identified. In both patients, deletions in the region of TSC2 and PKD1 were revealed performing by multiplex ligation probe amplification test. It is concluded that the histopathological features of skin lesions in this syndrome are similar to those encountered in TSC. Clinical awareness and appropriate molecular investigation of TSC2/PKD1 contiguous gene syndrome is necessary in all patients with a typical phenotype of TSC in infancy, adolescence, or adult age, because of severity of the renal alterations. PMID:19590422

  5. Risk assessment of gene flow from genetically engineered virus resistant cassava to wild relatives in Africa: an expert panel report.

    PubMed

    Hokanson, Karen E; Ellstrand, Norman C; Dixon, Alfred G O; Kulembeka, Heneriko P; Olsen, Kenneth M; Raybould, Alan

    2016-02-01

    The probability and consequences of gene flow to wild relatives is typically considered in the environmental risk assessment of genetically engineered crops. This is a report from a discussion by a group of experts who used a problem formulation approach to consider existing information for risk assessment of gene flow from cassava (Manihot esculenta) genetically engineered for virus resistance to the 'wild' (naturalized) relative M. glaziovii in East Africa. Two environmental harms were considered in this case: (1) loss of genetic diversity in the germplasm pool, and (2) loss of valued species, ecosystem resources, or crop yield and quality due to weediness or invasiveness of wild relatives. Based on existing information, it was concluded that gene flow will occur, but it is not likely that this will reduce the genetic diversity in the germplasm pool. There is little existing information about the impact of the virus in natural populations that could be used to inform a prediction about whether virus resistance would lead to an increase in reproduction or survival, hence abundance of M. glaziovii. However, an increase in the abundance of M. glaziovii should be manageable, and would not necessarily lead to the identified environmental harms. PMID:26667472

  6. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences.

    PubMed

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N; Bolli, Roberto

    2011-11-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., "immunization against infarction"). PMID:21785893

  7. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N.

    2013-01-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 2.1%, n = 12, vs. 44.7 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., immunization against infarction). PMID:21785893

  8. Molecular characterization of a maize regulatory gene. Annual progress report, March 1990--November 1991

    SciTech Connect

    Wessler, S.R.

    1991-12-01

    Based on initial bombardment studies we have previously concluded that promoter diversity was responsible for the diversity of naturally occurring R alleles. During this period we have found that R is controlled at the level of translation initiation and intron 1 is alternatively spliced. The experiments described in Sections 1 and 2 sought to quantify these effects and to determine whether they contribute to the tissue specific expression of select R alleles. This study was done because very little is understood about the post-transcriptional regulation of plant genes. Section 3 and 4 describe experiments designed to identify important structural components of the R protein.

  9. Fanconi anemia-D1 due to homozygosity for the BRCA2 gene Cypriot founder mutation: A case report

    PubMed Central

    LOIZIDOU, MARIA A.; HADJISAVVAS, ANDREAS; TANTELES, GEORGE A.; SPANOU-ARISTIDOU, ELENA; KYRIACOU, KYRIACOS; CHRISTOPHIDOU-ANASTASIADOU, VIOLETTA

    2016-01-01

    Fanconi anemia (FA) is a rare disorder characterized by multiple congenital malformations, progressive bone marrow failure and susceptibility to malignancies. Biallelic mutations in the breast cancer 2, early onset (BRCA2) gene are responsible for the FA-D1 subgroup, which accounts for ~3% of all the FA cases. Patients with biallelic BRCA2 mutations generally display a more severe phenotype, with earlier onset and increased incidence of leukaemia and other solid tumors, than other patients with FA. In the present report, the first Cypriot patient with FA-D1 is described, which is the fifth case of a homozygote for the same null allele reported thus far, and the third known case of neuroblastoma in association with FA-D1. PMID:26834852

  10. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    PubMed Central

    Lee, Sin Hang

    2016-01-01

    Lyme disease (LD), the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA) gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. PMID:27186082

  11. A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection.

    PubMed

    Monteiro, Freddy; Genin, Stphane; van Dijk, Irene; Valls, Marc

    2012-08-01

    Although much is known about the signals that trigger transcription of virulence genes in plant pathogens, their prevalence and timing during infection are still unknown. In this work, we address these questions by analysing expression of the main pathogenicity determinants in the bacterial pathogen Ralstonia solanacearum. We set up a quantitative, non-invasive luminescent reporter to monitor in planta transcription from single promoters in the bacterial chromosome. We show that the new reporter provides a real-time measure of promoter output in vivo - either after re-isolation of pathogens from infected plants or directly in situ - and confirm that the promoter controlling exopolysaccharide (EPS) synthesis is active in bacteria growing in the xylem. We also provide evidence that hrpB, the master regulator of type III secretion system (T3SS) genes, is transcribed in symptomatic plants. Quantitative RT-PCR assays demonstrate that hrpB and type III effector transcripts are abundant at late stages of plant infection, suggesting that their function is required throughout disease. Our results challenge the widespread view in R. solanacearum pathogenicity that the T3SS, and thus injection of effector proteins, is only active to manipulate plant defences at the first stages of infection, and that its expression is turned down when bacteria reach high cell densities and EPS synthesis starts. PMID:22609750

  12. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    SciTech Connect

    Zacharewski, T.

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  13. Suicidal gene therapy in an NF-κB-controlled tumor environment as monitored by a secreted blood reporter

    PubMed Central

    Badr, CE; Niers, JM; Morse, D; Koelen, JA; Vandertop, P; Noske, D; Wurdinger, T; Zalloua, PA; Tannous, BA

    2013-01-01

    The nuclear factor-κB (NF-κB) is known to be activated in many cancer types including lung, ovarian, astrocytomas, melanoma, prostate as well as glioblastoma, and has been shown to correlate with disease progression. We have cloned a novel NF-κB-based reporter system (five tandem repeats of NF-κB responsive genomic element (NF; 14bp each)) to drive the expression cassette for both a fusion between the yeast cytosine deaminase and uracil phosphoribosyltransferase (CU) as a therapeutic gene and the secreted Gaussia luciferase (Gluc) as a blood reporter, separated by an internal ribosomal entry site (NF-CU-IGluc). We showed that malignant tumor cells have high expression of Gluc, which correlates to high activation of NF-κB. When NF-κB was further activated by tumor necrosis factor-α in these cells, we observed up to 10-fold increase in Gluc levels and therefore transgene expression in human glioma cells served to greatly enhance the sensitization of these cells to the prodrug, 5-fluorocytosine both in cultured cells and in vivo subcutaneous tumor xenograft model. This inducible system provides a tool to enhance the expression of imaging and therapeutic genes for cancer therapy. PMID:21150937

  14. Gene-enzyme telationships in somatic cells and their organismal derivatives in higher plants. Progress report

    SciTech Connect

    Jensen, R. A.

    1980-04-21

    Progress is reported in the following subject areas: (1) chemistry of the arogenate molecule; (2) plant enzymology at the organismal level; (3) isolation of regulatory mutants in tobacco; and (4) stability of the haploid state in Nicotiana sylvestris.

  15. Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes.

    PubMed

    Jindal, Sunita; Longchar, Bendangchuchang; Singh, Alka; Gupta, Vikrant

    2015-12-01

    Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants. PMID:26340695

  16. Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes

    PubMed Central

    Jindal, Sunita; Longchar, Bendangchuchang; Singh, Alka; Gupta, Vikrant

    2015-01-01

    Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants. PMID:26340695

  17. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    SciTech Connect

    Jirtle, Randy L.

    2012-10-11

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A{sup vy}) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure (<10 cGy) during early gestation. This information is particularly important to ascertain given the increased use of CT scans in disease diagnosis, increased number of people predicted to live and work in space, and the present concern about radiological terrorism. We showed for the first time that LDIR significantly increased DNA methylation at the A{sup vy} locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in animals and humans needs to be defined.

  18. Differential gene expression in Neurospora crassa cell types: Ribosomal RNA genes. Comprehensive final terminal report of the overall activities for past 20 years

    SciTech Connect

    Dutta, S.K.

    1988-03-01

    This paper summarizes the accomplishments over the past 20 years of DOE support to the author`s research program. Highlights include molecular analysis of genome of Neurospora crassa, RNase sensitive RNA-dependent DNA polymerase, gene amplification of rRNA genes, molecular cloning of r-DNA`s, restriction fragment length polymorphism of rDNA in N. crassa., and ribosomal RNA processing genes of N. crassa.

  19. Mouse Pop1 is required for muscle regeneration in adult skeletal muscle.

    PubMed

    Andrée, Birgit; Fleige, Anne; Arnold, Hans-Henning; Brand, Thomas

    2002-03-01

    Popeye (Pop) genes are a novel gene family encoding putative transmembrane proteins predominantly present in striated and smooth muscle cells. In this study, a null mutation of Pop1 was generated by replacing the first coding exon of the Pop1 gene with the lacZ reporter gene. Homozygous mice lacking Pop1 were fertile and had a normal life span without any apparent phenotype. LacZ staining of tissues of heterozygous and homozygous Pop1-LacZ mice revealed strong expression in embryonic and fetal hearts. Pop1-LacZ was also expressed in the myotome and in myogenic progenitor cells within the limb and in smooth muscle cells of various organs. In the heart, Pop1-LacZ activity was downregulated postnatally in heterozygous mice but not in homozygous mice. Administration of the beta-adrenergic agonist isoproterenol led to a rapid increase in Pop1-LacZ activity in heterozygotes without induction at the transcriptional level, suggesting stabilization of the protein. No difference, however, was observed between homozygous and heterozygous mice in the ability to develop cardiac hypertrophy in response to isoproterenol. The capacity to regenerate skeletal muscle was tested after cardiotoxin injection into the hind limbs of hetero- and homozygous mice. In activated satellite cells of both genotypes, rapid activation of Pop1-LacZ expression was observed. In heterozygous animals, LacZ activity was only transiently elevated in muscle precursor cells undergoing fusion and in newly formed myotubes. In homozygotes, persistence of LacZ expression and a retarded ability to regenerate skeletal muscle were apparent, suggesting that Pop1 plays a role in muscle regeneration. PMID:11839816

  20. Regulation of nod factor sulphation genes in Rhizobium tropici CIAT899.

    PubMed

    Manyani, H; Sousa, C; Soria Díaz, M E; Gil-Serrano, A; Megías, M

    2001-06-01

    Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ beta-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end. PMID:11467733

  1. RNA interference screening of interferon-stimulated genes with antiviral activities against classical swine fever virus using a reporter virus.

    PubMed

    Wang, Xiao; Li, Yongfeng; Li, Lian-Feng; Shen, Liang; Zhang, Lingkai; Yu, Jiahui; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2016-04-01

    Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is a highly contagious and often fatal disease of pigs, which leads to significant economic losses in many countries. Viral infection can induce the production of interferons (IFNs), giving rise to the transcription of hundreds of IFN-stimulated genes (ISGs) to exert antiviral effects. Although numerous ISGs have been identified to possess antiviral activities against different viruses, rare anti-CSFV ISGs have been reported to date. In this study, to screen anti-CSFV ISGs, twenty-one ISGs reported previously were individually knocked down using small interfering RNAs (siRNAs) followed by infection with a reporter CSFV expressing Renilla luciferase (Rluc). As a result, four novel anti-CSFV ISGs were identified, including natural-resistance-associated macrophage protein 1 (NRAMP1), cytosolic 5'-nucleotidase III A (NT5C3A), chemokine C-X-C motif ligand 10 (CXCL10), and 2'-5'-oligoadenylate synthetase 1 (OAS1), which were further verified to exhibit antiviral activities against wild-type CSFV. We conclude that the reporter virus is a useful tool for efficient screening anti-CSFV ISGs. PMID:26868874

  2. Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

    PubMed

    Cevenini, L; Calabretta, M M; Calabria, D; Roda, A; Michelini, E

    2016-01-01

    : The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples. PMID:25898810

  3. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    SciTech Connect

    Guldberg, P.; Henriksen, K.F.; Guettler, F.

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  4. Phenylalanine hydroxylase gene mutations in the United States: report from the Maternal PKU Collaborative Study.

    PubMed Central

    Guldberg, P.; Levy, H. L.; Hanley, W. B.; Koch, R.; Matalon, R.; Rouse, B. M.; Trefz, F.; de la Cruz, F.; Henriksen, K. F.; Güttler, F.

    1996-01-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g-->a, and Y414C, accounting for 18.7%, 7.8%, and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies < or = 1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment of mutations has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. Images Figure 1 PMID:8659548

  5. Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

    PubMed

    Kim, Young-Hwa; Youn, Hyewon; Na, Juri; Hong, Kee-Jong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene. PMID:25553100

  6. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation

    PubMed Central

    Mendenhall, Alexander R.; Tedesco, Patricia M.; Sands, Bryan; Johnson, Thomas E.; Brent, Roger

    2015-01-01

    In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, “classical” multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms. PMID:25946008

  7. Sol-gel-derived materials for production of pin-printed reporter gene living-cell microarrays.

    PubMed

    Ge, Xin; Eleftheriou, Nikolas M; Dahoumane, Si Amar; Brennan, John D

    2013-12-17

    We report the fabrication of three-dimensional living-cell microarrays via pin-printing of soft sol-gel-derived silica materials containing bacterial cells. Bacterial cells entrapped in the silica-glycerol microarray spots can express reporter genes and produce strong fluorescence signals. The signals responded to the presence and concentration of inducers or repressors as expected, indicating that the entrapped cells remained metabolically active. Microscopic imaging of individual microarray spots at different culture times suggests that the entrapped cells can grow and divide, phenomena further confirmed by experiments in bulk sol-gel materials that demonstrated the increases of entrapped cell density and fluorescence during incubation in culture media. The cell microarrays can also be printed into 96-well glass bottom microtiter plates in a multiplexed manner, and the fluorescence signals generated were able to quantitatively and selectively respond to the concentration of inducers, thus demonstrating the potential for multitarget biosensing and high-throughput/high-content cell-based screening. The signal levels of bacterial cells in silica were significantly higher than those in alginate arrays, presumably due to viability of the entrapped cells in silica sol-gels. Microarray stability assays proved that the entrapped cells retained their physiological activity after storage for four weeks. Given that a large number of fluorescent and luminescent protein-based cell assays have been developed, the reporter gene living-cell microarrays demonstrated in this paper are expected to be applicable to a wide variety of research areas ranging from bioanalysis and chemical biology to drug discovery and probing of cell-material interactions. PMID:24279880

  8. Mu-lac insertion-directed mutagenesis in a pectate lyase gene of Erwinia chrysanthemi.

    PubMed Central

    Diolez, A; Coleno, A

    1985-01-01

    The pelC gene, which encodes one of the five major pectate lyase (PL) isoenzymes in Erwinia chrysanthemi 3937, designated PLc, was subcloned from a hybrid lambda phage into a pBR322 derivative and mutagenized with a mini-Mu-lacZ transposable element able to form fusions to the lacZ gene. One plasmid (pAD1) which had an inactivated pelC gene and a Lac+ phenotype was selected in Escherichia coli. This plasmid was introduced into Erwinia chrysanthemi, and the pelC::mini-Mu insertion was substituted for the chromosomal allele by homologous recombination. This strain lacks the PLc isoenzyme. This Erwinia chrysanthemi strain has a Lac+ phenotype that is inducible by polygalacturonate, as are the wild-type PL activities. Images PMID:2993251

  9. Regulatable promoters for use in gene therapy applications: modification of the 5'-flanking region of the CFTR gene with multiple cAMP response elements to support basal, low-level gene expression that can be upregulated by exogenous agents that raise intracellular levels of cAMP.

    PubMed

    Suzuki, M; Singh, R N; Crystal, R G

    1996-10-01

    This study focuses on the design, construction, and evaluation of a chimeric promoter for gene therapy applications where it is desirable to have low-level basal expression of the newly transferred gene, which can be induced to higher levels of expression by the administration of pharmacologic agents that can be safely used locally and/or systemically in humans. To achieve this, a chimeric promoter was constructed using fragments of the 5'-flanking region of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, and multiple tandem repeats of the consensus sequence and flanking elements of the cAMP response element (CRE), promoter sequences that support increased transcription in response to elevations in intracellular cAMP levels. Preliminary studies using plasmid vectors demonstrated that: (i) the 5'-flanking sequences from the CFTR gene have low promoter activity in the human airway epithelial cell lines; (ii) chimeras using -718 bp fragment from the 5'-flanking sequence of CFTR gene as the base, with the addition of 4-10 units of a 25-bp sequence containing the CRE consensus sequence, were all inducible by a rise in intracellular cAMP, with the chimera having eight CRE repeats the most responsive; and (iii) a CF126(CRE8) chjimera, consisting of the -126 bp fragment from the 5'-flanking region of CFTR gene together with eight CRE repeats, yielded low-level basal activity but maximal upregulation by cAMP, resulting in expression of the reporter gene that was 51-58% of an RSV-LTR control. On the basis of these observations, replication-deficient adenoviral vectors containing the CF126(CRE8) chimera and the luciferase reporter gene [AdCF126(CRE8).Luc] or the Escherichia coli lacZ (beta-Gal) reporter gene [AdCF126(CRE8). beta gal] were constructed. In several human airway epithelial cell lines, the AdCF126 (CRE). Luc vector provided low basal activity, but was significantly upregulated by agents that increase cAMP levels. Intranasal administration of the beta-Gal-expressing AdCF126(CRE8) beta gal vector into C57B1/6 mice demonstrated cAMP-induced upregulation of the reporter gene in airway epithelial cells. Quantification of the inducibility of the basal promoter activity in the airway epithelium using the AdCF126(CRE8). Luc vector demonstrated an 11-fold upregulation of the basal promoter activity in the lung with the administration of a phosphodiesterase inhibitor and a cAMP analog. These observations demonstrate the feasibility of using a chimeric promoter comprised of a minimal fragment of the CFTR 5'-flanking region, together with added multiple CRE, to control genes delivered in vivo. Importantly, because there are many drugs used in humans that raise cAMP, the concept of using a cAMP-regulatable promoter may also be a useful approach to enhance the safety, efficacy, and feasibility of a variety of human gene therapy strategies. PMID:8894680

  10. Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model.

    PubMed

    Gorelick, Daniel A; Pinto, Caroline Lucia; Hao, Ruixin; Bondesson, Maria

    2016-01-01

    In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative dose-response information on estrogenic activity of chemical compounds. PMID:26585145

  11. Targeted Delivery of Gold Nanoparticle Contrast Agents for Reporting Gene Detection by Magnetic Resonance Imaging†

    PubMed Central

    Vistain, Luke F.; Rotz, Matthew W.; Rathore, Richa; Preslar, Adam T.; Meade, Thomas J.

    2015-01-01

    Detection of protein expression by MRI requires a high payload of Gd(III) per protein binding event. Presented here is a targeted AuDNA nanoparticle capable of delivering several hundred Gd(III) chelates to the HaloTag reporter protein. Incubating this particle with HaloTag-expressing cells produced a 9.4 contrast-to-noise ratio compared to non-expressing cells. PMID:26505558

  12. Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

    NASA Astrophysics Data System (ADS)

    Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

    2012-03-01

    B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

  13. Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

    PubMed Central

    Lao, Nga T.; Maloney, Alan P.; Atkins, John F.; Kavanagh, Tony A.

    2009-01-01

    Background Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context. Methodology We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP. Conclusions We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context. PMID:19816579

  14. Enhancement of glycoprotein hormone alpha subunit promoter reporter gene activity in co-transfection studies--a cautionary reminder.

    PubMed

    Fuhrer, D; Han, S; Ludgate, M

    2008-11-01

    Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. IN VITRO models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to -346, but eliminated in the promoter truncated upstream of -244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between -244 and -346. Enhancement was maintained with the addition of a second constitutive reporter (bos beta-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos beta-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site. PMID:18537081

  15. An IRE-Like AGC Kinase Gene, MtIRE, Has Unique Expression in the Invasion Zone of Developing Root Nodules in Medicago truncatula1[OA

    PubMed Central

    Pislariu, Catalina I.; Dickstein, Rebecca

    2007-01-01

    The AGC protein kinase family (cAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases C) have important roles regulating growth and development in animals and fungi. They are activated via lipid second messengers by 3-phosphoinositide-dependent protein kinase coupling lipid signals to phosphorylation of the AGC kinases. These phosphorylate downstream signal transduction protein targets. AGC kinases are becoming better studied in plants, especially in Arabidopsis (Arabidopsis thaliana), where specific AGC kinases have been shown to have key roles in regulating growth signal pathways. We report here the isolation and characterization of the first AGC kinase gene identified in Medicago truncatula, MtIRE. It was cloned by homology with the Arabidopsis INCOMPLETE ROOT HAIR ELONGATION (IRE) gene. Semiquantitative reverse transcription-polymerase chain reaction analysis shows that, unlike its Arabidopsis counterpart, MtIRE is not expressed in uninoculated roots, but is expressed in root systems that have been inoculated with Sinorhizobium meliloti and are developing root nodules. MtIRE expression is also found in flowers. Expression analysis of a time course of nodule development and of nodulating root systems of many Medicago nodulation mutants shows MtIRE expression correlates with infected cell maturation during nodule development. During the course of these experiments, nine Medicago nodulation mutants, including sli and dnf1 to 7 mutants, were evaluated for the first time for their microscopic nodule phenotype using S. meliloti constitutively expressing lacZ. Spatial localization of a pMtIRE-gusA transgene in transformed roots of composite plants showed that MtIRE expression is confined to the proximal part of the invasion zone, zone II, found in indeterminate nodules. This suggests MtIRE is useful as an expression marker for this region of the invasion zone. PMID:17237187

  16. Forebrain-specific promoter/enhancer D6 derived from the mouse Dach1 gene controls expression in neural stem cells.

    PubMed

    Machon, O; van den Bout, C J; Backman, M; Rsok, ; Caubit, X; Fromm, S H; Geronimo, B; Krauss, S

    2002-01-01

    Drosophila dachshund is involved in development of eye and limbs and in the development of mushroom bodies, a brain structure required for learning and memory in flies. Its mouse homologue mDach1 is expressed in various embryonic tissues, including limbs, the eye, the dorsal spinal cord and the forebrain. We have isolated a forebrain-specific 2.5-kb enhancer element termed D6 from the mouse mDach1 gene and created D6-LacZ and D6-green fluorescent protein (GFP) reporter gene mouse lines. In embryonic stages, the D6 enhancer activity is first detected at embryonic day 10.5 in scattered cells of the outbuldging cortical vesicles. By embryonic day 12.5, D6 activity expands throughout the developing neocortex and the hippocampus. In the adult mouse brain, D6 enhancer is active in neurons of the cortical plate, in the CA1 layer of the hippocampus and in cells of the subventricular zone and the ventricular ependymal zone. Adult mice also show D6 activity in the olfactory bulb and in the mamillary nucleus. Cultured D6-positive cells, which were derived from embryonic and postnatal brains, show characteristics of neural stem cells. They form primary and secondary neurospheres that differentiate into neurons and astrocytes as examined by cell-specific markers.Our results show that D6 enhancer exerts highly tissue-specific activity in the neurons of the neocortex and hippocampus and in neural stem cells. Moreover, the fluorescence cell sorting of D6-GFP cells from embryonic and postnatal stages allows specific selection of primary neural progenitors and their analysis. PMID:12088753

  17. Simultaneous evaluation of human CYP3A4 and ABCB1 induction by reporter assay in LS174T cells, stably expressing their reporter genes.

    PubMed

    Inami, Keita; Sasaki, Takamitsu; Kumagai, Takeshi; Nagata, Kiyoshi

    2015-04-01

    The bioavailability of orally administered therapies are often significantly limited in the human intestine by the metabolic activities of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). Predicting whether candidate compounds induce CYP3A4 and P-gp is a crucial stage in the drug development process, as drug-drug interactions may result in the induction of intestinal CYP3A4 and P-gp. However, the assay systems needed to evaluate both CYP3A4 and P-gp induction in the intestine are yet to be established. To address this urgent requirement, LS174T cells were used to create two stable cell lines expressing the CYP3A4 or ATP-binding cassette subfamily B member 1 (ABCB1, encoding P-gp) reporter genes. First, these stable cells were tested by treatment with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis RA) that induce CYP3A4 and P-gp in the intestines. All these compounds significantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously assess the induction of CYP3A4 and ABCB1, both stable cells were co-cultivated to measure their reporter activities. The mixed cells showed a significant increase in the CYP3A4 and ABCB1 reporter activities following treatment with 1,25(OH)2 D3, ATRA, and 9-cis RA. These activity levels were maintained after passaging more than 20 times and following multiple freeze-thaw cycles. These results demonstrate that our established cell lines can be used to evaluate simultaneously CYP3A4 and ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future drug candidates. PMID:25410880

  18. Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene

    PubMed Central

    Yoon, Sea Young; Gay-Antaki, Carlos; Ponde, Datta E; Poptani, Harish; Vite, Charles H; Wolfe, John H

    2014-01-01

    In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [18F]-fallypride analog of dopamine. The [18F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease. PMID:26015960

  19. Primary renal sclerosing epithelioid fibrosarcoma: report of 2 cases with EWSR1-CREB3L1 gene fusion.

    PubMed

    Argani, Pedram; Lewin, Jack R; Edmonds, Pamela; Netto, George J; Prieto-Granada, Carlos; Zhang, Lei; Jungbluth, Achim A; Antonescu, Cristina R

    2015-03-01

    We report the first 2 genetically confirmed cases of primary renal sclerosing epithelioid fibrosarcoma (SEF), occurring in a 17-year-old boy and a 61-year-old woman. In both cases, the tumors demonstrated the typical epithelioid clear cell morphology associated with extensive hyalinizing fibrosis, raising the differential diagnosis of solitary fibrous tumor, metanephric stromal tumor, and the sclerosing variant of clear cell sarcoma of the kidney. Both neoplasms demonstrated diffuse immunoreactivity for MUC4, a highly specific marker for SEF, and both demonstrated evidence of rearrangement of both the EWSR1 and CREB3L1 genes, which have recently been shown to be fused in this entity. Both neoplasms presented with metastatic disease. Primary renal SEF represents yet another translocation-associated sarcoma now shown to arise primarily in the kidney. PMID:25353281

  20. Radiation sensitivity of the gastrula-stage embryo: Chromosome aberrations and mutation induction in lacZ transgenic mice: The roles of DNA double-strand break repair systems.

    PubMed

    Jacquet, Paul; van Buul, Paul; van Duijn-Goedhart, Annemarie; Reynaud, Karine; Buset, Jasmine; Neefs, Mieke; Michaux, Arlette; Monsieurs, Pieter; de Boer, Peter; Baatout, Sarah

    2015-10-01

    At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/- embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including some stage-specific aspects that are not as yet understood. PMID:26433259

  1. Mechanism Profiling of Hepatotoxicity Caused by Oxidative Stress Using Antioxidant Response Element Reporter Gene Assay Models and Big Data

    PubMed Central

    Kim, Marlene Thai; Huang, Ruili; Sedykh, Alexander; Wang, Wenyi; Xia, Menghang; Zhu, Hao

    2015-01-01

    Background: Hepatotoxicity accounts for a substantial number of drugs being withdrawn from the market. Using traditional animal models to detect hepatotoxicity is expensive and time-consuming. Alternative in vitro methods, in particular cell-based high-throughput screening (HTS) studies, have provided the research community with a large amount of data from toxicity assays. Among the various assays used to screen potential toxicants is the antioxidant response element beta lactamase reporter gene assay (ARE-bla), which identifies chemicals that have the potential to induce oxidative stress and was used to test > 10,000 compounds from the Tox21 program. Objective: The ARE-bla computational model and HTS data from a big data source (PubChem) were used to profile environmental and pharmaceutical compounds with hepatotoxicity data. Methods: Quantitative structure–activity relationship (QSAR) models were developed based on ARE-bla data. The models predicted the potential oxidative stress response for known liver toxicants when no ARE-bla data were available. Liver toxicants were used as probe compounds to search PubChem Bioassay and generate a response profile, which contained thousands of bioassays (> 10 million data points). By ranking the in vitro–in vivo correlations (IVIVCs), the most relevant bioassay(s) related to hepatotoxicity were identified. Results: The liver toxicants profile contained the ARE-bla and relevant PubChem assays. Potential toxicophores for well-known toxicants were created by identifying chemical features that existed only in compounds with high IVIVCs. Conclusion: Profiling chemical IVIVCs created an opportunity to fully explore the source-to-outcome continuum of modern experimental toxicology using cheminformatics approaches and big data sources. Citation: Kim MT, Huang R, Sedykh A, Wang W, Xia M, Zhu H. 2016. Mechanism profiling of hepatotoxicity caused by oxidative stress using antioxidant response element reporter gene assay models and big data. Environ Health Perspect 124:634–641; http://dx.doi.org/10.1289/ehp.1509763 PMID:26383846

  2. Food flavonoid aryl hydrocarbon receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter gene assays.

    PubMed

    Van der Heiden, Edwige; Bechoux, Nathalie; Muller, Marc; Sergent, Thérèse; Schneider, Yves-Jacques; Larondelle, Yvan; Maghuin-Rogister, Guy; Scippo, Marie-Louise

    2009-04-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxin-responsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX((R)). Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cells were compared for species-dependent activities. We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24h. PMID:19286049

  3. Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains.

    PubMed

    Buckley, Anthony M; Jukes, Caitlin; Candlish, Denise; Irvine, June J; Spencer, Janice; Fagan, Robert P; Roe, Andrew J; Christie, John M; Fairweather, Neil F; Douce, Gillian R

    2016-01-01

    The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment. PMID:26996606

  4. Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains

    PubMed Central

    Buckley, Anthony M.; Jukes, Caitlin; Candlish, Denise; Irvine, June J.; Spencer, Janice; Fagan, Robert P.; Roe, Andrew J.; Christie, John M.; Fairweather, Neil F.; Douce, Gillian R.

    2016-01-01

    The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment. PMID:26996606

  5. Novel Mutation of the GNE Gene Presenting Atypical Mild Clinical Feature: A Korean Case Report.

    PubMed

    Choi, Young-Ah; Park, Sung-Hye; Yi, Youbin; Kim, Keewon

    2015-06-01

    Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is caused by mutations in GNE, a key enzyme in sialic acid biosynthesis. Here, we reported a case of GNE that presented with atypical mild clinical feature and slow progression. A 48-year-old female had a complaint of left foot drop since the age of 46 years. Electromyography (EMG) and muscle biopsy from left tibialis anterior muscle were compatible with myopathy. Genetic analysis led to the identification of c.1714G>C/c.527A>T compound heterozygous mutation, which is the second most frequent mutation in Japan as far as we know. Previous research has revealed that c.1714G>C/c.527A>T compound heterozygous mutation is a mild mutation as the onset of the disease is much later than the usual age of onset of GNE myopathy and the clinical course is slowly progressive. This was the first case report in Korea of the clinicopathological characteristics of GNE myopathy with GNE (c.1714G>C/c.527A>T compound heterozygous) mutation. PMID:26161358

  6. AB130. Pseudoaldosteronism due to mutation of SCNN1A gene: a case report

    PubMed Central

    Can, Ngoc Thi Bich; Vu, Dung Chi; Bui, Thao Phuong; Nguyen, Khanh Ngoc; Zennaro, Maria-Christina; Wudy, Stefan A.

    2015-01-01

    Background Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disease characterized by resistance to the actions of aldosterone. It was first described in 1958 by Cheek and Perry, and common clinical manifestations include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in the neonatal period. Objective To describe clinical characteristics, laboratory features and management of one Vietnamese patient with pseudohypoaldosteron. Methods Clinical features, biochemical finding, mutation analysis and management in a 1 month-old-boy was studied. Based on analysis of this patient’s clinical symptoms associated with biochemical examination, the urinary steroid metabolomics analysis was performed using gas chromatography spectrometry and mutation analysis of SCNN1A was performed using PCR & direct sequencing. Results Patient is the first child normal delivery with the gestation age of 41 weeks, birth weight of 3,200 g, and onset of the disease at 7 days of age. He presented with lost weight, dehydration without vomit, diarrhea or hyperpigmentation. He was admitted with the features of cyanosis, allorhythmic, electrolyte imbalance with sodium of 119 mmoL/L, potassium of 7.4 mmoL/L. Investigation show pH 7.26, PCO2 34 mmHg, PO2 110 mmHg, HCO3- 18 mmoL/L, BE -10, plasma 17OHP level: 2.4 ng/mL, testosterone level: 1.94 nmoL/L, Cortisol 8am: 2,662.8 pmoL/L, Ure 7.4 mmoL/L, Creatinine 44.2 µmoL/L, Glucose 4.8 mmoL/L. The urine steroid metabolomics analysis showed extensive excretion of aldosterone ID-ISTD1 of 1,157.41 µg/L. Novel homozygous mutation (c.1668C > A; p.S556R) of SCNN1A gene was identified in the proband. He was treated with florinef of 0.1 mg/kg/day for electrolyte balance. He had complication of intestinal perforation and died due to infection. In conclusions, PHA1 causes severe hyponatremia, metabolic acidosis, and life-threatening hyperkalemia, with normal 17-a-hydroxyprogesterone levels and high excretion of aldosterone levels.

  7. Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein▿ †

    PubMed Central

    Cheng, Zhihui; Wang, Xueqi; Rikihisa, Yasuko

    2008-01-01

    The type IV secretion (T4S) system is critical for the virulence of several pathogens. In the rickettsial pathogen Ehrlichia chaffeensis, the virBD genes are split into two operons, the virB3-virB6 (preceded by sodB) and virB8-virD4 operons. Between these two operons, there are duplications of virB4, virB8, and virB9. In this study we found that transcription of all five loci was downregulated prior to the release of E. chaffeensis from host THP-1 cells and was upregulated at the initiation of exponential growth. Electrophoretic mobility shift assays revealed an E. chaffeensis-encoded protein that specifically bound to the promoter regions upstream of the virBD loci. The protein was purified from the bacterial lysate by affinity chromatography using a biotinylated promoter region upstream of sodB. Mass spectrometry identified the protein as an E. chaffeensis 12.3-kDa hypothetical protein, which was designated EcxR. Recombinant EcxR bound to the promoter regions upstream of five individual virBD loci. EcxR also activated transcription of all five virBD loci in lacZ reporter constructs. The expression of ecxR was positively autoregulated by EcxR. These results suggest that the five virBD loci are coordinately regulated by EcxR to allow developmental stage-specific expression of the T4S system in E. chaffeensis. PMID:18192398

  8. A novel hybrid baculovirus-adeno-associated viral vector-mediated radionuclide reporter gene imaging system for stem cells transplantation monitoring.

    PubMed

    Pan, Yu; Yin, Hongyan; Lv, Jing; Ju, Huijun; Zhou, Xiang; Zhang, Yifan

    2015-02-01

    Hybrid baculovirus-adeno-associated virus (BV-AAV) containing enhanced green fluorescent protein (eGFP) reporter gene or human sodium-iodide symporter (hNIS) reporter gene flanked by inverted terminal repeats (ITRs) derived from AAV (BV-CMV-eGFP-ITR and BV-CMV-hNIS-ITR) were constructed and used to investigate the feasibility of using hybrid BV-AAV transgenic vector to mediate hNIS reporter gene imaging for monitoring bone marrow-derived mesenchymal stem cells (BM-MSCs) transplantation therapy as a novel biotechnological platform in radionuclide reporter gene imaging. The results showed that the infection efficiency of BV-CMV-eGFP-ITR in BM-MSCs reached 84.25 ± 1.38%, and there were no obvious adverse effects on BM-MSCs. The (125)I(-) and (99m)TcO₄(-) uptake assays showed that the radionuclide accumulation induced by BA-AAV-mediated hNIS was highly efficient in infected BM-MSCs. Furthermore, there was a robust correlation between the infected BM-MSCs cell number and the (125)I(-) accumulation amount (R(2) = 0.9026). The micro-SPECT/CT imaging showed that BV-CMV-hNIS-ITR-infected BM-MSCs accumulated radioiodine efficiently in vivo, exhibiting obvious radiotracer accumulation in transplantation sites. Further quantitative analysis revealed that 30 min might be the optimal imaging time point. Moreover, the revealed high target/individual organ background ratios also supported the feasibility of BV-AAV-mediated hNIS reporter gene imaging for monitoring BM-MSCs transplantation in most of commonly used transplantation sites, thus highlighting this promise biotechnological platform in radionuclide reporter gene imaging for stem cell transplantation therapy. PMID:25345809

  9. A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli

    PubMed Central

    Gonzalez de Valdivia, Ernesto I.; Isaksson, Leif A.

    2004-01-01

    The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A′ model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool. PMID:15459289

  10. A Novel, Photosynthesis-Associated Thioredoxin-Like Gene: Final Technical Report

    SciTech Connect

    Collier, Jackie, L

    2005-09-13

    Many aspects of the biosynthesis and physiological regulation of the photosynthetic apparatus of plants, algae and cyanobacteria remain to be understood, and are likely to involve yet-unidentified proteins that carry out oxidation/reduction (redox) reactions. TxlA from Synechococcus sp. strain PCC 7942 and its homologues from other cyanobacteria and plants, including Sll1980 from the cyanobacterium Synechocystis sp. strain PCC 6803, are likely to be among these proteins. In fact, the homologue of TxlA in the plant Arabidopsis thaliana, HCF164, may be required for synthesis of the cytochrome b6f complex that transfers electrons between the two photosynthetic reaction centers. TxlAs share an N-terminal hydrophobic domain, a central thioredoxin-like domain, and a unique C-terminal hydrophilic domain. Plant and algal TxlAs are nuclear-encoded and have an additional N-terminal domain that targets them to the chloroplast. We have found that the common N-terminal domain of TxlA anchors it to a membrane, probably the thylakoid (photosynthetic) membrane (where HCF164 is also localized, with its thioredoxin-like domain in the thylakoid lumen). We have also found that the thioredoxin-like domain is likely to assume the conformation typical of thioredoxins and possesses thioredoxin-like redox activity in vitro, and that the C-terminal domain is important to the structure and function of the thioredoxin-like domain both in vivo and in vitro. These data show that TxlAs have the cellular location and enzymatic activity expected of a protein involved in the biosynthesis of redox components or redox regulation of the photosynthetic apparatus. We were unable to inactivate the thioredoxin-like domain of TxlA in either PCC 7942 or PCC 6803, under either photosynthetic or heterotrophic growth conditions. We also found that expression of antisense txlA mRNA from an IPTG-regulated promoter in PCC 7942 was lethal, most likely because it effectively inactivated txlA by ''RNA silencing''. These results are consistent with a role for TxlA in the synthesis of the cytochrome b6f complex, which is required for both photosynthetic and respiratory electron transport in cyanobacteria. In contrast, our PCC 7942 mutants in which the C-terminal domain of TxlA was removed are viable and appear to have normal cytochrome content, but have a subtle pigmentation phenotype (increased content of phycocyanin relative to chlorophyll) that depends on both light and CO2 availability. We have also found that PCC 6803 Sll1980 inactivation mutant merodiploids have a similar pigmentation phenotype to the PCC 7942 C-terminal truncation mutants when grown photoautotrophically. In addition, when grown heterotrophically the PCC 6803 Sll1980 inactivation mutant merodiploids remain green instead of turning a golden color like the wild-type, and they are more sensitive to the b6f complex inhibitor DBMIB than is wild type PCC 6803. That the PCC 6803 Sll1980 inactivation mutant merodiploids have these phenotypes despite the fact that they still contain normal copies of the sll1980 gene suggests that the presence of truncated Sll1980 protein interferes with the function of normal Sll1980 protein. Together, these physiological data suggest that TxlA has an essential redox role in cyanobacteria, perhaps a biosynthetic one, and may also have a nonessential regulatory role reflected in the phenotypes of the PCC 7942 C-terminal truncation mutants and the PCC 6803 Sll1980 inactivation mutant merodiploids.

  11. Validating tyrosinase homologue MelA as a photoacoustic reporter gene for imaging Escherichia coli

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert; Zemp, Roger

    2015-03-01

    Antibiotic drug resistance is a major worldwide issue. Development of new therapies against pathogenic bacteria requires appropriate research tools for replicating and characterizing infections. Previously fluorescence and bioluminescence modalities have been used to image infectious burden in animal models but scattering significantly limits imaging depth and resolution. We hypothesize that photoacoustic imaging, which has improved depth-toresolution ratio, could be useful for visualizing MelA-expressing bacteria since MelA is a bacterial tyrosinase homologue involved in melanin production. Using an inducible expression system, E. coli expressing MelA were visibly black in liquid culture. Phosphate buffered saline (PBS), MelA-expressing bacteria (at different dilutions in PBS), and chicken embryo blood were injected in plastic tubes which were imaged using a VisualSonics Vevo LAZR system. Photoacoustic imaging at 6 different wavelengths (680, 700, 750, 800, 850 and 900nm) enabled spectral de-mixing to distinguish melanin signals from blood. The signal to noise ratio of 9x diluted MelA bacteria was 55, suggesting that ~20 bacteria cells could be detected with our system. When MelA bacteria were injected as a 100 μL bolus into a chicken embryo, photoacoustic signals from deoxy- and oxy- hemoglobin as well as MelA-expressing bacteria could be separated and overlaid on an ultrasound image, allowing visualization of the bacterial location. Photoacoustic imaging may be a useful tool for visualizing bacterial infections and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  12. In vivo imaging of orthotopic prostate cancer with far-red gene reporter fluorescence tomography and in vivo and ex vivo validation

    PubMed Central

    Darne, Chinmay D.; Tan, I-Chih; Wu, Grace; Wilganowski, Nathaniel; Robinson, Holly; Azhdarinia, Ali; Zhu, Banghe; Rasmussen, John C.; Sevick-Muraca, Eva M.

    2013-01-01

    Abstract. Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150  kDa) molecules. These results represent the first tomography of a living animal using far-red gene reporters. PMID:23797877

  13. A New Pyrimidine-Specific Reporter Gene: A Mutated Human Deoxycytidine Kinase Suitable for PET During Treatment with Acycloguanosine-Based Cytotoxic Drugs

    PubMed Central

    Likar, Yury; Zurita, Juan; Dobrenkov, Konstantin; Shenker, Larissa; Cai, Shangde; Neschadim, Anton; Medin, Jeffrey A.; Sadelain, Michel; Hricak, Hedvig; Ponomarev, Vladimir

    2015-01-01

    In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET. Methods Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2′-deoxy-2′-fluoroarabinofuranosylcytosine, 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs. Results Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals (18F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)–transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers (18F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine). Conclusion The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with 18F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells. PMID:20810757

  14. Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle.

    PubMed

    Wallace, Lindsay M; Moreo, Andrew; Clark, K Reed; Harper, Scott Q

    2013-01-01

    Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.Molecular Therapy - Nucleic Acids (2013) 2, e86; doi:10.1038/mtna.2013.16; published online 16 April 2013. PMID:23591809

  15. High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

    PubMed Central

    2010-01-01

    Background Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. Results A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. Conclusions Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community. PMID:20687964

  16. Genes and Gene Therapy

    MedlinePlus

    ... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  17. Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy.

    PubMed

    Baril, Patrick; Martin-Duque, Pilar; Vassaux, Georges

    2010-02-01

    Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer ((123)I(-), (124)I(-), (99m)TcO4(-)), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells. PMID:19814733

  18. Evolution of regulatory genes governing biodegradation in acinetobacter calcoaceticus. Final report, 15 July 1991-31 December 1994

    SciTech Connect

    Ornston, L.N.

    1995-02-22

    The Acinetobacter calcoaceticus pca-qui-pob supraoperonic gene cluster encodes bacterial enzymes that metabolize aromatic and hydroaromatic compounds in the environment. Our investigation is directed to understanding how mutation, gene rearrangement and selection contributed to evolution of the transcriptional controls exercised over genes in the cluster. The complete nucleotide sequence of the 18 kbp gene cluster has been determined, and genetic manipulations have been used to explore mechanisms contributing to expression of the genes. The results reveal that structural gene expression is governed by complex interactions between the products of different regulatory genes some of which share common ancestry. Additional controls appear to be exercised by compartmentation of some catabolic enzymes outside the inner cell membrane. Recombination appears to have made a major contribution to the evolution of existing control mechanisms, and their maintenance may be influence by continuing recombination. Contributions of recombination to mutation and repair are under investigation as are specific molecular mechanisms underlying transcriptional controls.

  19. Search for major genes with progeny test data to accelerate the development of genetically superior loblolly pines. Quarterly report [July - September 1999

    SciTech Connect

    Li, Bailian

    1999-08-16

    This quarterly report describes the progress made on the 3 research tasks of the project. The 3 research tasks are: (1) Develop genetic models and analytical methods; (2) Molecular confirmation of major gene segregation; and (3) Develop strategies for marker-assisted breeding.

  20. Brief Report: High Frequency of Biochemical Markers for Mitochondrial Dysfunction in Autism: No Association with the Mitochondrial Aspartate/Glutamate Carrier "SLC25A12" Gene

    ERIC Educational Resources Information Center

    Correia, Catarina; Coutinho, Ana M.; Diogo, Luisa; Grazina, Manuela; Marques, Carla; Miguel, Teresa; Ataide, Assuncao; Almeida, Joana; Borges, Luis; Oliveira, Catarina; Oliveira, Guiomar; Vicente, Astrid M.

    2006-01-01

    In the present study we confirm the previously reported high frequency of biochemical markers of mitochondrial dysfunction, namely hyperlactacidemia and increased lactate/pyruvate ratio, in a significant fraction of 210 autistic patients. We further examine the involvement of the mitochondrial aspartate/glutamate carrier gene ("SLC25A12") in

  1. Spontaneous coronary artery dissection in a young man with a factor v leiden gene mutation: a case report and review of the literature.

    PubMed

    Khan, Tahir; Danyi, Peter; Topaz, On; Ali, Asghar; Jovin, Ion S

    2013-12-01

    Spontaneous coronary artery dissection is a rare but increasingly recognized cause of acute myocardial ischemia in young adults, especially in women. We report a case of spontaneous coronary dissection in a young healthy man who was also a carrier of the factor V Leiden gene mutation. PMID:24436622

  2. Brief Report: High Frequency of Biochemical Markers for Mitochondrial Dysfunction in Autism: No Association with the Mitochondrial Aspartate/Glutamate Carrier "SLC25A12" Gene

    ERIC Educational Resources Information Center

    Correia, Catarina; Coutinho, Ana M.; Diogo, Luisa; Grazina, Manuela; Marques, Carla; Miguel, Teresa; Ataide, Assuncao; Almeida, Joana; Borges, Luis; Oliveira, Catarina; Oliveira, Guiomar; Vicente, Astrid M.

    2006-01-01

    In the present study we confirm the previously reported high frequency of biochemical markers of mitochondrial dysfunction, namely hyperlactacidemia and increased lactate/pyruvate ratio, in a significant fraction of 210 autistic patients. We further examine the involvement of the mitochondrial aspartate/glutamate carrier gene ("SLC25A12") in…

  3. The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis.

    PubMed

    Tanabe, Noriaki; Tamoi, Masahiro; Shigeoka, Shigeru

    2015-08-10

    Sweet potato is an important crop because of its high yield and biomass production. We herein investigated the potential of the promoter activity of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) from sweet potato (Ipomoea batatas) in order to develop the high expression system of exogenous DNA in Arabidopsis. We isolated two different cDNAs (IbRbcS1 and IbRbcS2) encoding RbcS from sweet potato. Their predicted amino acid sequences were well conserved with the mature RbcS protein of other plants. The tissue-specific expression patterns of these two genes revealed that expression of IbRbcS1 was specific to green tissue, whereas that of IbRbcS2 was non-photosynthetic tissues such as roots and tubers. These results suggested that IbRbcS1 was predominantly expressed in the green tissue-specific of sweet potato over IbRbcS2. Therefore, the IbRbcS1 promoter was transformed into Arabidopsis along with β-glucuronidase (GUS) as a reporter gene. GUS staining and semi-quantitative RT-PCR showed that the IbRbcS1 promoter conferred the expression of the GUS reporter gene in green tissue-specific and light-inducible manners. Furthermore, qPCR showed that the expression levels of GUS reporter gene in IbRbcS1 pro:GUS were same as those in CaMV 35S pro:GUS plants. These results suggest that the IbRbcS1 promoter is a potentially strong foreign gene expression system for genetic transformation in plants. PMID:25958348

  4. [Organization and regulation of the genes for nitrogen fixation in Rhodopseudomonas capsulata]. Progress report, [June 5, 1989--June 4, 1991

    SciTech Connect

    Not Available

    1991-12-31

    In prior support periods we identified, cloned and sequenced three genes involved in the regulation of nif gene expression in Rhodobacter capsulatus. These were called nifRI, nifR2 and nifR4; they turn out to be homologue of the ntrC, ntrB and ntrA genes of enterobacteria. We subsequently found that mutations in an additional gene, nifR5. render R. capsulatus nif genes constitutive with respect to ammonia. The nifR5 gene was shown to be similar to glnB of enteric bacteria, encoding the regulatory protein PII, and furthering the intersection of the glutamine synthetase adenylylation cascade with the control of nif gene transcription. In pursuit of the mechanism of 0{sub 2} control of nif gene expression, we constructed and analyzed the topology of a small plasmid in R. capsulatus as a function of 0{sub 2} concentration. We also cloned and obtained partial sequence data for two genes encoding the B subunit of DNA gyrase. The nucleotide sequence of the rpoB gene encoding RNA polymerase was nearly completed. A method for isolation of genes expressed differentially, developed for cyanobacteria, was applied successfully to R. capsulatus. Several genes that depend on nifR4 for their transcription were isolated. A transcription start site for a nifA gene was identified and the promoter sequence was analyzed. A physical map of the R calsulatus SB1003 chromosome was prepared, based on pulsed-field electrophoresis of XbaI and AseI fragments and hybridization with a gridded cosmid library, using a device that permits 864 cosmids to be hybridized at one time with a labeled chromosomal fragment.

  5. [Clinicopathological Study of Pilomyxoid-Spectrum Astrocytomas:An Analysis of the BRAF Gene. Report of Two Cases].

    PubMed

    Ito, Tamio; Sato, Kenichi; Oikawa, Mitsuteru; Sugio, Hironori; Asanome, Taku; Ozaki, Yoshimaru; Nakamura, Hirohiko; Tanaka, Shinya; Tsuda, Masumi; Nagashima, Kazuo

    2015-09-01

    In contrast to pilocytic astrocytomas(PAs), pilomyxoid astrocytomas(PMAs)demonstrate monophasic piloid cells with angiocentric distribution and a more aggressive clinical course. Recently, several reports have described combined histological features of both subtypes;accordingly, these were termed intermediate pilomyxoid tumors(IPTs). The KIAA1549-BRAF fusion gene has been found in approximately 70% of PAs, but is reportedly rare in PMAs. We describe a clinicopathological study of two patients with pilomyxoid-spectrum astrocytoma(PMSA). Case 1 was of a 29-year-old man who presented with a generalized seizure. Gadolinium-magnetic resonance imaging(Gd-MRI)demonstrated a less enhanced tumor in the left temporal lobe. Case 2 was of a 9-year-old boy who presented with headache. Gd-MRI revealed an irregularly enhanced tumor in the left cerebellum. In Case 1, the tumor showed monomorphous bipolar cells in a myxoid background and angiocentric arrangement;therefore, the diagnosis was PMA. In Case 2, part of the tumor had a myxoid, angiocentric pattern characteristic of PMA;the other part had a biphasic pattern characteristic of PA. PMA and PA were mixed in a 7:3 ratio;therefore, IPT was diagnosed. No BRAF V600E mutations were found by immunohistochemistry and sequencing in either case. Three major KIAA1549-BRAF fusion subtypes were analyzed by quantitative reverse transcription polymerase chain reaction(RT-PCR)and sequencing. No fusions were found in Case 1. However, K16-B9 fusion was identified in Case 2, and this fusion was more prevalent in the PA component than in the PMA component. In summary, no BRAF V600E mutations were found in PMSAs, but KIAA1549-BRAF fusion was identified in IPT, particularly in the PA component. PMID:26321697

  6. Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).

    PubMed

    Zaniboni, Andrea; Merlo, Barbara; Zannoni, Augusta; Bernardini, Chiara; Lavitrano, Marialuisa; Forni, Monica; Mari, Gaetano; Bacci, Maria Laura

    2013-02-01

    Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene. Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein. PMID:23312469

  7. Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk.

    PubMed

    Wielogorska, E; Elliott, C T; Danaher, M; Connolly, L

    2014-07-01

    Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer. Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds. A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid-liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36 pg g(-)(1) EEQ and has been successfully applied in testing a range of milk samples. PMID:24769019

  8. Pyridoxal 5ꞌ-phosphate-responsive epilepsy with novel mutations in the PNPO gene: a case report.

    PubMed

    Veeravigrom, M; Damrongphol, P; Ittiwut, R; Ittiwut, C; Suphapeetiporn, K; Shotelersuk, V

    2015-01-01

    Pyridoxal 5'-phosphate (PLP)-responsive epilepsy is a rare autosomal recessive epileptic disorder caused by deficiency of pyridox(am)-ne 5'-phosphate oxidase (PNPO). Neonatal onset seizures in PLP responsive epilepsy are usually resistant to common anticonvulsants and pyridoxine, but respond to PLP. Various PNPO mutations are associated with this disorder. In this report, we have described a case of a female baby with neonatal onset seizures responding to PLP. Exome sequencing revealed that the patient was compound heterozygous for pathogenic mutations [c.546+1G>A (IVS5+1 G>A) and c.620delG (p.G207VfsX215)] in the PNPO gene. The c.546+1G>A was inherited from the mother while the c.620delG was inherited from the father. Both mutations were absent in 122 unrelated Thai controls. The results of this study indicated the presence of two newly identified mutations in this Thai patient with PLP-responsive epilepsy for the first time, expanding the mutational spectrum of PNPO. PMID:26535729

  9. Validation and application of reporter gene assays for the determination of estrogenic and androgenic endocrine disruptor activity in sport supplements.

    PubMed

    Plotan, Monika; Elliott, Christopher T; Oplatowska, Michalina; Connolly, Lisa

    2012-07-01

    Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCα) and detection capabilities (CCβ) were established for both the estrogen and androgen RGAs. All samples were compliant with CCα and CCβ in both bioassays. Recovery rates were 96 % for 17β-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at -20 °C. Specificity, good repeatability (coefficients of variation (CV), 12-25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs. PMID:22411538

  10. Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems.

    PubMed

    Satoh, Kanako; Nonaka, Ryouichi; Ohyama, Ken-ichi; Nagai, Fumiko; Ogata, Akio; Iida, Mitsuru

    2008-03-01

    Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system. PMID:18310895

  11. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection a Technical Report

    PubMed Central

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nr, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  12. Kullback-Leibler information for ordering genes using sperm typing and radiation-hybrid mapping. Technical report

    SciTech Connect

    Chernoff, H.

    1991-10-01

    Two technologies applicable to gene mapping are those of sperm typing and radiation hybrid mapping. Sperm typing makes use of the polymerase chain reaction, a biochemical technique which allows enormous amplification (production of multiple copies) of small, selected DNA fragments from a single chromosome. A sample of sperm from a single donor is analyzed to see which alleles (distinct forms of the various genes) are present in the individual sperms. The frequencies with which the various possibilities occur can be used to supply estimates of the ordering and of the recombination probabilities among the genes for which that donor is heterozygous (having different alleles of the same gene.)

  13. Organ-specific gene expression in maize: The P-wr allele. Final report, August 15, 1993--August 14, 1996

    SciTech Connect

    Peterson, T.A.

    1997-06-01

    The ultimate aim of our work is to understand how a regulatory gene produces a specific pattern of gene expression during plant development. Our model is the P-wr gene of maize, which produces a distinctive pattern of pigmentation of maize floral organs. We are investigating this system using a combination of classical genetic and molecular approaches. Mechanisms of organ-specific gene expression are a subject of intense research interest, as it is the operation of these mechanisms during eukaryotic development which determine the characteristics of each organism Allele-specific expression has been characterized in only a few other plant genes. In maize, organ-specific pigmentation regulated by the R, B, and Pl genes is achieved by differential transcription of functionally conserved protein coding sequences. Our studies point to a strikingly different mechanism of organ-specific gene expression, involving post-transcriptional regulation of the regulatory P gene. The novel pigmentation pattern of the P-wr allele is associated with differences in the encoded protein. Furthermore, the P-wr gene itself is present as a unique tandemly amplified structure, which may affect its transcriptional regulation.

  14. Differentiation of LA-N-5 neuroblastoma cells into cholinergic neurons: methods for differentiation, immunohistochemistry and reporter gene introduction.

    PubMed

    Hill, D P; Robertson, K A

    1998-03-01

    The use of model systems derived from cell lines has been a valuable tool in understanding the molecules and cellular processes that govern differentiation processes (T.R. Breitman, S.E. Selonick, S.J. Collins, Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid, Proc. Natl. Acad. Sci. USA 77 (1980) 2936-2940 [2]; N. Gomez, S. Traverse, P. Cohen, Identification of a MAP kinase in phaeochromocytoma (PC12) cells, FEBS Lett. 314 (1992) 461-465 [4]). The use of such systems provides an inexpensive, quick and simple way to identify and test molecules that can be further studied in more complex in vivo experiments. Some cell lines such as embryonic stem cells can be induced to differentiate in vitro, however, the differentiation is difficult to control and most often leads to the generation of a wide variety of cell types. Cell lines derived from sources committed to a restricted cell fate provide an opportunity to examine cell growth and differentiation within a specific cell type (G.M. Keller, In vitro differentiation of embryonic stem cells, Curr. Opin. Cell Biol. 7 (1995) 862-869 [10]). In this article we describe a simple system for the differentiation of the human neuroblastoma cell line LA-N-5 into cholinergic neurons using all-trans retinoic acid (G. Han, B. Chang, M.J. Connor, N. Sidell, Enhanced potency of 9-cis versus all-trans retinoic acid to induce the differentiation of human neuroblastoma cells, Differentiation, 59 (1995) 61-69 [5]; D.P. Hill, K.R. Robertson, Characterization of the cholinergic neuronal differentiation of the human neuroblastoma cell line LA-N-5 after treatment with retinoic acid, Dev. Brain Res. 102 (1997) 53-67 [6]; J.A. Robson, N. Sidell, Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid, Neuroscience 14 (1985) 1149-1162 [12]; N. Sidell, C.A. Lucas, G.W. Kreutzberg, Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line, Exp. Cell Res. 155 (1984) 305-309 [14]). These cells provide a setting for the study of cholinergic neuronal differentiation and of the factors that influence that process. We also discuss procedures that can be used to study gene expression in LA-N-5 cells by immunohistochemistry and reporter gene analysis. PMID:9507116

  15. Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences.

    PubMed

    Li, Qianhong; Guo, Yiru; Wu, Wen-Jian; Ou, Qinghui; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Chen, Ning; Dawn, Buddhadeb; Luo, Li; O'Brien, Erin; Bolli, Roberto

    2011-11-01

    The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with inducible nitric oxide synthase (iNOS) is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS), which enables long-lasting transgene expression. The ability of rAAV/iNOS to direct the expression of functional iNOS protein was confirmed in COS-7 cells before in vivo gene transfer. Mice received injections in the anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+3-fold vs. the LacZ group, n = 6; P < 0.05) and iNOS activity (+4.4-fold vs. the LacZ group, n = 6; P < 0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5 ± 2.2%, n = 12, vs. 41.7 ± 2.9%, n = 10, in the LacZ group; P < 0.05). The infarct-sparing effect of iNOS gene therapy at 1 year was as powerful as that observed 24 h after ischemic preconditioning (six 4-min O/4-min R cycles) (19.3 ± 2.3%, n = 11; P < 0.05). Importantly, compared with the LacZ group (n = 11), iNOS gene transfer (n = 10) had no effect on LV dimensions or function for up to 1 year (at 1 year: FS 34.5 ± 2.0 vs. 34.6 ± 2.6%, EF 57.0 ± 2.0 vs. 59.7 ± 2.9%, LVEDD 4.3 ± 0.1 vs. 4.2 ± 0.2 mm, LVESD 2.8 ± 0.1 vs. 2.9 ± 0.2 mm) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year), cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy. PMID:21779912

  16. Analysis of LuxR Regulon Gene Expression during Quorum Sensing in Vibrio fischeri▿

    PubMed Central

    Qin, Nan; Callahan, Sean M.; Dunlap, Paul V.; Stevens, Ann M.

    2007-01-01

    The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-l-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position −40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth. PMID:17400743

  17. Analysis of LuxR regulon gene expression during quorum sensing in Vibrio fischeri.

    PubMed

    Qin, Nan; Callahan, Sean M; Dunlap, Paul V; Stevens, Ann M

    2007-06-01

    The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-l-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position -40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth. PMID:17400743

  18. Report of outbreaks of classical scrapie in Dorper sheep and associated prion protein gene polymorphisms in affected flocks.

    PubMed

    de Andrade, Caroline Pinto; de Oliveira, Eduardo Conceição; Leal, Juliano Souza; de Almeida, Laura Lopes; de Castro, Luiza Amaral; da Silva, Sergio Ceroni; Driemeier, David

    2015-08-01

    Scrapie is an infectious neurodegenerative disease affecting sheep and goats, related with conformational alteration of an isoform of the prion protein that leads to deposition and aggregation in the host's central nervous system. Occurrence of the natural disease can be influenced by host genetic factors, such as a single nucleotide polymorphism of the prion protein gene. This study reports three scrapie-affected Dorper flocks located on three different farms in Brazil. The objective of this study was to analyze these three flocks using scrapie diagnostics, combining histology, immunohistochemistry, genotyping, and western blot assays. For immunohistochemistry, 192 sheep were selected and 308 sheep blood samples were taken for genotyping. A total of 22 sheep were scrapie positive by immunohistochemistry. Of these, four presented clinical signs and had scrapie immunoreactivity at the obex in western blot assays. The sheep without clinical signs were positive in lymphoid organs, such as the third eyelid and rectal mucosa. The major genotypes found on the flocks were ARQ/ARQ, ARQ/ARR, and ARQ/VRQ for codons 136, 154, and 171. Most of the sheep were considered to be at moderate to high risk, based on risk groups for developing scrapie. Some blood samples were sequenced, and polymorphisms were identified in other codons, such as 127, 142, and 143. Our data demonstrate the importance of preclinical scrapie diagnosis in Brazilian sheep, as most of the affected sheep showed no clinical signs, and emphasize the relevance of genotyping other Dorper sheep to determine the genotypic profile of the breed. PMID:25995150

  19. Structure and molecular characterization of barley nudix hydrolase genes.

    PubMed

    Tanaka, Sayuri; Kihara, Makoto; Sugimoto, Manabu

    2015-01-01

    Putative nudix hydrolase (NUDX) genes, which encode amino acid sequences showing homology with those of Arabidopsis NUDXs and conserve nudix motif, were identified from barley. The 14 deduced barley NUDXs (HvNUDX1-14) were classified into established subfamilies, except for 8-oxo-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) pyrophosphohydrolase and mRNA decapping enzyme subfamilies, and three substrate-unknown subfamilies. Drought and UV-C stresses, respectively, up-regulated 7 and 4 HvNUDX genes, but some homologs of Arabidopsis NUDXs showed different responses to abiotic stress. HvNUDX12 gene, belonging to diadenosine tetraphosphates (Ap₄A) pyrophosphohydrolase subfamily gene and up-regulated by UV-C, was expressed in Escherichia coli cells. The recombinant protein showed 8-oxo-dGTP, Ap₄A, and guanosine-3',5'-tetraphosphate (ppGpp) pyrophosphohydrolase activities, and the suppression of the lacZ amber mutation in a mutT-deficient E. coli cells caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree. These results suggest that barley NUDXs have unique constitution and response of NUDX to abiotic stress. PMID:25379607

  20. Expression of regulatory nif genes in Rhodobacter capsulatus.

    PubMed Central

    Hübner, P; Willison, J C; Vignais, P M; Bickle, T A

    1991-01-01

    Translational fusions of the Escherichia coli lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nif gene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and nifR4 (the analog of ntrA in Klebsiella pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and nifR4 were not expressed under these conditions. The implications for the regulatory circuit of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph. PMID:1902215

  1. Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.

    PubMed Central

    Maciver, B; McHale, R H; Saul, D J; Bergquist, P L

    1994-01-01

    The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity. Images PMID:7993087

  2. Fundus albipunctatus: review of the literature and report of a novel RDH5 gene mutation affecting the invariant tyrosine (p.Tyr175Phe).

    PubMed

    Skorczyk-Werner, Anna; Pawłowski, Przemysław; Michalczuk, Marta; Warowicka, Alicja; Wawrocka, Anna; Wicher, Katarzyna; Bakunowicz-Łazarczyk, Alina; Krawczyński, Maciej R

    2015-08-01

    Fundus albipunctatus (FA) is a rare, congenital form of night blindness with rod system impairment, characterised by the presence of numerous small, white-yellow retinal lesions. FA belongs to a heterogenous group of so-called flecked retina syndromes. This disorder shows autosomal recessive inheritance and is caused mostly by mutations in the RDH5 gene. This gene encodes the enzyme that is a part of the visual cycle, the 11-cis retinol dehydrogenase. This study is a brief review of the literature on FA and a report of the first molecular evidence for RDH5 gene mutation in a Polish patient with this rare disorder. We present a novel pathogenic RDH5 gene mutation in a 16-year-old female patient with symptoms of night blindness. The patient underwent ophthalmological examinations, including colour vision testing, fundus photography, automated visual field testing, full-field electroretinography (ERG) and spectral optical coherent tomography (SOCT). The patient showed typical FA ERG records, the visual field was constricted and fundus examination revealed numerous characteristic, small, white-yellowish retinal lesions. DNA sequencing of the RDH5 gene coding sequence (exons 2-5) enabled the detection of the homozygous missense substitution c.524A > T (p.Tyr175Phe) in exon 3. This is the first report of RDH5 gene mutation that affects the invariant tyrosine, one of the most conserved amino acid residues in short-chain alcohol dehydrogenases/reductases (SDRs), crucial for these enzymes' activity. The location of this substitution, together with its predicted influence on the protein function, indicate that the p.Tyr175Phe mutation is the cause of FA in our patient. PMID:25820994

  3. Differential gene expression in Neurospora crassa cell types: heterogeneity and multiple copies of rRNA genes. Annual progress report, July 1981-June 1982

    SciTech Connect

    Dutta, S.K.

    1982-01-01

    The significant results obtained were as follows: (I) Multiple copies of isolated rRNA genes from N. crassa were tested for heterogeneity by rRNA: rDNA reassociation kinetics. More than 90% of rDNA copies were identical. The possible heterogeneity of a small fraction of rDNAs could not be attributed to inclusion of any tDNA sequences. (II) Two approaches to study gross differences between rRNA genes from N. crassa cell types-conidia, germinated conidia, and mycelia were undertaken. No difference was seen in either the restriction patterns nor the autoradiographs. Either gross differences between rDNAS of N. crassa cell types were not present or they were not detected by these two approaches. (III) Using similar DNA restriction analysis procedures, differences between closely related heterothallic and homothallic species of Neurospora were detected. (IV) Successful sequencing of 317 bases of the N. crassa slime mutant pMF2 clone which includes the 5.8S rDNA and it's flanking internal spacer regions was achieved. (ERB)

  4. A green fluorescent protein reporter genetic screen that identifies modifiers of Hox gene function in the Drosophila embryo.

    PubMed Central

    Merabet, Samir; Catala, Francoise; Pradel, Jacques; Graba, Yacine

    2002-01-01

    Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. In this study, we describe a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. We found 15 deficiencies that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways, which suggests an extensive use of signaling in the control of Hox gene function. PMID:12242233

  5. Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1991--August 31, 1992

    SciTech Connect

    Barkan, A.

    1992-12-31

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  6. The first report described as an important study: The association of mannose-binding lectin gene 2 polymorphisms in children with Down syndrome

    PubMed Central

    Demirhan, Osman; Taştemir, Deniz; Güneşaçar, Ramazan; Güzel, Ali İrfan; Alptekin, Davut

    2011-01-01

    BACKGROUND: Mannose-binding lectin gene 2 (MBL2) plays a very important role in the first line of host immune response in Down syndrome (DS). The importance of MBL2 gene polymorphisms in children with DS is unclear, and no research has addressed MBL2 gene polymorphisms in patients with DS. This is the first report describing an important association between MBL2 gene polymorphisms and infections in children with DS. MATERIALS AND METHODS: We compared the frequency of single-nucleotide polymorphisms (SNPs) at two codons of the MBL2 gene in a cross sectional cohort of 166 children with DS and 229 controls. Polymorphisms at codons 54 (GGC→GAC) and 57 (GGA→GAA) in exon 1 of the MBL2 gene were typed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique using the restriction enzymes BshN1 (derivated from Bacillus sphaericus) and MboII (derivated from Moraxella bovis), respectively. RESULTS: MBL2 codon 54 GA genotype frequency was found to be lower in patients with DS (22.9%) than those of healthy controls (35.8%), differences were statistically significant (OR = 0.532, 95% CI = 0.339-0.836, P = 0.008). On the other hand, codon 57 polymorphism in the MBL2 gene was detected in none of the DS patients, but only one person in the control group showed codon 57 GA genotype (OR = 1.004, 95% CI = 0.996-1.013, P = 1.000). CONCLUSION: Our data provides an evidence for the first time that a homozygote or heterozygote for the variant, MBL2 alleles, is not associated with infections in patients with DS, and do not influence the incidence of infections. PMID:22090714

  7. Microbial reporter gene assay as a diagnostic and early warning tool for the detection and characterization of toxic pollution in surface waters.

    PubMed

    Hug, Christine; Zhang, Xiaowei; Guan, Miao; Krauss, Martin; Bloch, Robert; Schulze, Tobias; Reinecke, Tim; Hollert, Henner; Brack, Werner

    2015-11-01

    Surface water samples constantly receive a vast mixture of micropollutants mainly originating from wastewater treatment plants (WWTPs). High-throughput live cell arrays provide a promising method for the characterization of the effects of chemicals and the associated molecular mechanisms. In the present study, this test system was evaluated for the first time for the characterization of a set of typical surface water extracts receiving effluent from WWTPs. The extracts containing complex mixtures of micropollutants were analyzed for the expression of 90 stress responsive genes in the Escherichia coli reporter gene assay. The most affected pathways and the genes most sensitive to surface water samples suggested prominent stress-responsive pathways for wastewater-impacted surface water, such as oxidative stress, DNA damage, and drug resistance. Samples strongly affecting particular pathways were identified by statistical analysis of gene expression. Transcription data were correlated with contamination data from chemical screening and percentages of wastewater in the samples. Samples with particular effects and outstanding chemical composition were analyzed. For these samples, hypotheses on the alteration of the transcription of genes involved in drug resistance and DNA repair attributable to the presence of pharmaceuticals were drawn. PMID:26033406

  8. The first report of a Pelecaniformes defensin cluster: characterization of β-defensin genes in the crested ibis based on BAC libraries.

    PubMed

    Lan, Hong; Chen, Hui; Chen, Li-Cheng; Wang, Bei-Bing; Sun, Li; Ma, Mei-Ying; Fang, Sheng-Guo; Wan, Qiu-Hong

    2014-01-01

    Defensins play a key role in the innate immunity of various organisms. Detailed genomic studies of the defensin cluster have only been reported in a limited number of birds. Herein, we present the first characterization of defensins in a Pelecaniformes species, the crested ibis (Nipponia nippon), which is one of the most endangered birds in the world. We constructed bacterial artificial chromosome libraries, including a 4D-PCR library and a reverse-4D library, which provide at least 40 equivalents of this rare bird's genome. A cluster including 14 β-defensin loci within 129 kb was assigned to chromosome 3 by FISH, and one gene duplication of AvBD1 was found. The ibis defensin genes are characterized by multiform gene organization ranging from two to four exons through extensive exon fusion. Splicing signal variations and alternative splice variants were also found. Comparative analysis of four bird species identified one common and multiple species-specific duplications, which might be associated with high GC content. Evolutionary analysis revealed birth-and-death mode and purifying selection for avian defensin evolution, resulting in different defensin gene numbers among bird species and functional conservation within orthologous genes, respectively. Additionally, we propose various directions for further research on genetic conservation in the crested ibis. PMID:25372018

  9. Isolated duodenal myeloid sarcoma associated with the CBFβ/MYH11 fusion gene followed by acute myeloid leukemia progression: A case report and literature review

    PubMed Central

    HUANG, BO; YOU, PENG; ZHU, PING; DU, ZUNGUO; WU, BEIQIAN; XU, XIAOPING; CHEN, ZI

    2014-01-01

    Myeloid sarcoma (MS) is a rare disease that presents as an extramedullary tumorous mass of immature myeloid precursors. The majority of MS are identified in acute myeloid leukemia (AML) patients and rarely present as a primary isolated MS without AML. In addition, inversion of chromosome 16 [inv(16)] and the CBFβ/MYH11 fusion gene are rarely associated with MS. The current study reports a female patient with an isolated duodenal MS, who developed AML-M4 associated with the CBFβ/MYH11 fusion gene and 48,XX,inv(16),+13,+22. A review of previously reported cases of isolated MS with the CBFβ/MYH11 fusion gene was also performed. Isolated MS with the CBFβ/MYH11 fusion gene was often observed in abdominal lesions, with the intestinal tract being the predominantly involved site. In addition, patients with isolated MS with the CBFβ/MYH11 fusion exhibited a high risk of developing systemic AML. The diagnosis of isolated MS may be particularly challenging and, therefore, determining the optimal standard treatment for isolated MS is required. PMID:25120702

  10. Nitrogen catabolite repression-sensitive transcription as a readout of Tor pathway regulation: the genetic background, reporter gene and GATA factor assayed determine the outcomes.

    PubMed

    Georis, Isabelle; Feller, André; Tate, Jennifer J; Cooper, Terrance G; Dubois, Evelyne

    2009-03-01

    Nitrogen catabolite repression (NCR)-sensitive genes, whose expression is highly repressed when provided with excess nitrogen and derepressed when nitrogen is limited or cells are treated with rapamycin, are routinely used as reporters in mechanistic studies of the Tor signal transduction pathway in Saccharomyces cerevisiae. Two GATA factors, Gln3 and Gat1, are responsible for NCR-sensitive transcription, but recent evidence demonstrates that Tor pathway regulation of NCR-sensitive transcription bifurcates at the level of GATA factor localization. Gln3 requires Sit4 phosphatase for nuclear localization and NCR-sensitive transcription while Gat1 does not. In this article, we demonstrate that the extent to which Sit4 plays a role in NCR-sensitive transcription depends upon whether or not (i) Gzf3, a GATA repressor homologous to Dal80, is active in the genetic background assayed; (ii) Gat1 is able to activate transcription of the assayed gene in the absence of Gln3 in that genetic background; and (iii) the gene chosen as a reporter is able to be transcribed by Gln3 or Gat1 in the absence of the other GATA factor. Together, the data indicate that in the absence of these three pieces of information, overall NCR-sensitive gene transcription data are unreliable as Tor pathway readouts. PMID:19104072

  11. [Construction and identification of recombinant firefly luciferase report vector containing human acyl coenzyme a: cholesterol acyltransferase 1 gene P7 promoter].

    PubMed

    Ge, Jing; Cheng, Bei; He, Ping; Wen, Hui; Lu, Han; Chen, Xin; Zeng, Yongli

    2008-12-01

    The DNA segment of the human acyl coenzyme A: cholesterol acyltransferasel (ACAT1) gene P7 promoter was amplified by PCR from human monocytic leukemia cell line (THP-1) and cloned to TA vector, then the positive clone was confirmed by restriction enzymes and sequencing. The targeted segment was subcloned to Firefly luciferase report vector pGL3-Enhancer. The recombinant plasmid pGL3E-P7 was transfected transiently into THP-1, then the expression of luciferase could be detected in THP-1 by pGL3E-P7 transfection. We successfully constructed luciferase reporter vector containing P7 promoter of the human ACAT1 gene, and established a new means to study the transcriptional regulation mechanisms of ACAT1 during atherosclerosis. PMID:19166214

  12. Patched homolog 1 gene mutation (p.G1093R) induces nevoid basal cell carcinoma syndrome and non-syndromic keratocystic odontogenic tumors: A case report

    PubMed Central

    PONTI, GIOVANNI; POLLIO, ANNAMARIA; PASTORINO, LORENZA; PELLACANI, GIOVANNI; MAGNONI, CRISTINA; NASTI, SABINA; FORTUNA, GIULIO; TOMASI, ALDO; SCARRÀ, GIOVANNA BIANCHI; SEIDENARI, STEFANIA

    2012-01-01

    Mutations in the Patched homolog 1 (PTCH1) gene lead to an autosomal dominant disorder known as nevoid basal cell carcinoma syndrome (NBCCS) or Gorlin syndrome (GS). Several PTCH1 mutations have been observed in NBCCS associated with keratocystic odontogenic tumors (KCOTs), including non-syndromic KCOTs. The missense mutation c.3277G>C (p.G1093R) in exon 19 of the PTCH1 gene has only been reported in non-syndromic KCOTs. The present study reports for the first time a familial case (father and daughter) of NBCCS and KCOTs, carrying the same c.3277G>C (p.G1093R) germline mutation. This observation suggests that this missense mutation is involved in the pathogenesis of NBCCS as well as in a subset of non-syndromic KCOTs. The identification of a missense mutation may lead to an earlier diagnosis of NBCCS. PMID:22844361

  13. (The isolation and characterization of beta-glucosidase gene and beta-glucosidase of Trichoderma viride): Progress report

    SciTech Connect

    Stafford, D.W.

    1983-01-01

    Our project was to isolate and characterize the enzyme ..beta..-glucosidase and to clone and characterize the ..beta..-glucosidase gene; our goal is to clone and characterize each of the cellulase genes from Trichoderma. The induction of the Trichoderma reesei cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. Although the induction of the cellulase complex has previously been well documented, the induction of ..beta..-glucosidase had been questioned. 49 refs., 6 figs., 2 tabs.

  14. Organization and regulation of the genes for nitrogen fixation in Rhodopseudomonas capsulata: Progress report, June 5, 1987-June 4, 1988

    SciTech Connect

    Haselkorn, R.

    1988-02-01

    We have cloned a number of fragments of DNA containing genes necessary for nitrogen fixation from the photosynthetic bacterium Rhodobacter capsulatus. The nif genes are locally clustered but the clusters are on non-neighboring DNA restriction fragments. We sought to determine the physical linkage of these fragments, to determine their relationship with the corresponding nif genes of Klebsiella, and to determine the nucleotide sequence of some of the fragments. So far we have identified six or seven regulatory genes among these, using a nifH::lac fusion. Four of the regulatory genes are required for expression of nifH. Two of these, nifR1 and nifR2, have sequences homologous to ntrC and ntrB of enteric bacteria. A third, nifR4, has sequence homology, in the C-terminal region, to the ntrA genes of Rhizobium and Klebsiella. Constitutive expression of nifR4 in R. capsulatus, from a plasmid clone, complemented a nifR4 chromosomal mutant but not a nifR1 mutant. Moreover, both oxygen and ammonia regulation of nitrogenase were maintained under these conditions. These results are consistent with a model requiring both nifR1 and nifR4 for nitrogenase gene expression; they rule out our earlier suggestion that nifR1 is needed only to turn on nifR4. Current efforts are focused on the purification of RNA polymerase and the products of nifR1, nifR2, and nifR4 to study nif gene transcription in vitro, particularly with the goal of determining the role of DNA supercoiling in transcription.

  15. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1988--April 14, 1989

    SciTech Connect

    Okita, T.W.

    1989-12-31

    During this period researchers have been successful in determining the structure of the rice pyrophosphorylase gene. Potato tuber ADPglucose pyrophosphorylse purification and structure studies were carried out as well as recombinant DNA studies. Evidence suggests that the tuber form is made up of subunits with similar molecular weights and immunological relatedness. In contrast, the spinach leaf enzyme and presumably the maize endosperm species is composed of two dissimilar sununits encoded by different genes.

  16. Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli.

    PubMed Central

    Kawamukai, M; Matsuda, H; Fujii, W; Utsumi, R; Komano, T

    1989-01-01

    The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin. PMID:2546924

  17. First report of a clinical, multidrug-resistant Enterobacteriaceae isolate coharboring fosfomycin resistance gene fosA3 and carbapenemase gene blaKPC-2 on the same transposon, Tn1721.

    PubMed

    Li, Gang; Zhang, Ying; Bi, Dexi; Shen, Pinghua; Ai, Fuqi; Liu, Hong; Tian, Yueru; Ma, Yiming; Wang, Bei; Rajakumar, Kumar; Ou, Hong-Yu; Jiang, Xiaofei

    2015-01-01

    In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla(KPC-2) and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla(KPC-2) was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored bla(KPC-2). Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla(KPC-2) colocated in the same Tn1721-Tn3-like composite transposon on a novel IncP group plasmid. PMID:25367902

  18. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

    NASA Technical Reports Server (NTRS)

    Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  19. SSTR2-based reporters for assessing gene transfer into non-small cell lung cancer: evaluation using an intrathoracic mouse model.

    PubMed

    Singh, S P; Han, L; Murali, R; Solis, L; Roth, J; Ji, L; Wistuba, I; Kundra, V

    2011-01-01

    The most common cause of cancer-related deaths in North America is lung cancer, 85% of which is non-small cell lung cancer (NSCLC). Gene therapy is a promising approach, but has been hindered by lack of methods for localizing and quantifying gene expression in vivo. Human somatostatin receptor subtype-2 (SSTR2)-based reporters can be used to follow gene expression in vivo using ligands with greater affinity for this subtype. NSCLCs can express SSTR subtypes, which may interfere with SSTR2-based reporters. We assessed whether a SSTR2-based reporter can serve as a reporter of gene transfer into NSCLCs. SSTR subtype expression was assessed in NSCLC cell lines A549, H460, and H1299 using RT-PCR. After infection with an adenovirus containing hemagglutinin-A-tagged-SSTR2 (Ad-HA-SSTR2) or control insert, expression was assessed by immunologic techniques and binding to clinically-approved (111)In-octreotide. In vivo, after magnetic resonance (MR) imaging, intrathoracic H460 tumors were injected with Ad-HA-SSTR2 or control virus (n?=?6 mice/group) under ultrasound guidance. Intravenous injection of (111)In-octreotide 2 days later was followed by planar and single-photon emission computed tomography (SPECT) imaging. Biodistribution into tumors was assessed in vivo using anatomic MR and functional gamma-camera images and ex vivo using excised organs/tumors. In human lung tumor samples (n?=?70), SSTR2 expression was assessed using immunohistochemistry. All three NSCLC cell lines expressed different SSTR subtypes, but none expressed SSTR2. Upon Ad-HA-SSTR2 infection, HA-SSTR2 expression was seen in all three cell lines using antibodies targeting the HA domain or (111)In-octreotide targeting the receptor domain (p?reporters can serve as reporters of gene transfer into NSCLCs. PMID:20653396

  20. Schizophrenia and the androgen receptor gene: Report of a sibship showing co-segregation with Reifenstein Syndrome but no evidence for linkage in 23 multiply affected families

    SciTech Connect

    Arranz, M.; Sharma, T.; Sham, P.; Kerwin, R.

    1995-10-09

    Crow et al. have reported excess sharing of alleles by male sibling pairs with schizophrenia, at a triplet repeat marker within the androgen receptor gene, indicating that mutations at or near this gene may be a risk factor for males. In this report, we describe a pair of male siblings concordant for both schizophrenia and Reifenstein syndrome, which is caused by a mutation in this gene. This provides support for the hypothesis that the androgen receptor may contribute to liability to develop schizophrenia. Because of this, we have examined a collection of 23 pedigrees multiply affected by schizophrenia for linkage to the androgen receptor. We have found no evidence for linkage by both the LOD score and affected sibling-pair methods, under a range of genetic models with a broad and narrow definition of phenotype, and when families with male-to-male transmission are excluded. However, because of the small number of informative male-male pairs in our sample, we cannot confirm or refute the excess allele sharing for males reported by Crow. 35 refs., 1 fig., 2 tabs.

  1. A New Fluorescence-Based Reporter Gene Vector as a Tool for Analyzing and Fishing Cells with Activated Wnt Signaling Pathway

    PubMed Central

    Reischmann, Patricia; Müller, Oliver

    2013-01-01

    The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β-galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way. PMID:24066239

  2. A new fluorescence-based reporter gene vector as a tool for analyzing and fishing cells with activated wnt signaling pathway.

    PubMed

    Apfel, Johanna; Reischmann, Patricia; Müller, Oliver

    2013-01-01

    The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β -galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way. PMID:24066239

  3. Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

    PubMed

    Zhang, Huimin; Zheng, Beiwen; Gao, Rongsui; Feng, Youjun

    2015-09-01

    The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus. PMID:26050090

  4. Availability of iron to Pseudomonas fluorescens in rhizosphere and bulk soil evaluated with an ice nucleation reporter gene.

    PubMed Central

    Loper, J E; Henkels, M D

    1997-01-01

    The biological availability of iron in the rhizosphere was assessed by evaluating ice nucleation activity (INA) expressed in situ by Pseudomonas fluorescens Pf-5 containing a transcriptional fusion (pvd-inaZ) of an iron-regulated promoter to an ice nucleation reporter gene (inaZ). Pf-5 containing pvd-inaZ expresses INA that is inversely related to the iron availability of a growth medium (J. E. Loper and S. E. Lindow, Appl. Environ. Microbiol. 60:1934-1941, 1994). INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ was at a maximum within 12 to 24 h following inoculation of the bacterium onto bean roots and typically decreased gradually during the following 4 days. Iron availability in the soil, which was altered by the addition of chelators, influenced INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ. In soil adjusted to a pH of 7.0 or 8.0 by adding Ca(OH)2, rhizosphere populations of Pf-5 containing pvd-inaZ expressed greater INA, indicating lower iron availability, than they did in the nonamended soil at a pH of 5.4. Similarly, rhizosphere populations of Pf-5 containing pvd-inaZ expressed less INA in an agricultural soil of pH 5.4 than in other agricultural soils ranging in pH from 6.4 to 7.7. These results conform to the predictions of chemical models stating that pH is a major factor influencing iron availability in soil solutions. The results of this study indicate that P. fluorescens Pf-5 encountered an iron-limited environment immediately after it was inoculated onto bean roots planted in agricultural field soils. One to two days after the bacterium was inoculated onto root surfaces, however, iron became more available to rhizosphere populations of Pf-5. We speculate that iron acquisition systems of plants and other rhizosphere organisms may provide available sources of iron to established rhizosphere populations of P. fluorescens. PMID:8979343

  5. Associations of ADH and ALDH2 gene variation with self report alcohol reactions, consumption and dependence: an integrated analysis

    PubMed Central

    Macgregor, Stuart; Lind, Penelope A.; Bucholz, Kathleen K.; Hansell, Narelle K.; Madden, Pamela A.F.; Richter, Melinda M.; Montgomery, Grant W.; Martin, Nicholas G.; Heath, Andrew C.; Whitfield, John B.

    2009-01-01

    Alcohol dependence (AD) is a complex disorder with environmental and genetic origins. The role of two genetic variants in ALDH2 and ADH1B in AD risk has been extensively investigated. This study tested for associations between nine polymorphisms in ALDH2 and 41 in the seven ADH genes, and alcohol-related flushing, alcohol use and dependence symptom scores in 4597 Australian twins. The vast majority (4296) had consumed alcohol in the previous year, with 547 meeting DSM-IIIR criteria for AD. There were study-wide significant associations (P < 2.3 × 10−4) between ADH1B-Arg48His (rs1229984) and flushing and consumption, but only nominally significant associations (P < 0.01) with dependence. Individuals carrying the rs1229984 G-allele (48Arg) reported a lower prevalence of flushing after alcohol (P = 8.2 × 10−7), consumed alcohol on more occasions (P = 2.7 × 10−6), had a higher maximum number of alcoholic drinks in a single day (P = 2.7 × 10−6) and a higher overall alcohol consumption (P = 8.9 × 10−8) in the previous year than those with the less common A-allele (48His). After controlling for rs1229984, an independent association was observed between rs1042026 (ADH1B) and alcohol intake (P = 4.7 × 10−5) and suggestive associations (P < 0.001) between alcohol consumption phenotypes and rs1693482 (ADH1C), rs1230165 (ADH5) and rs3762894 (ADH4). ALDH2 variation was not associated with flushing or alcohol consumption, but was weakly associated with AD measures. These results bridge the gap between DNA sequence variation and alcohol-related behavior, confirming that the ADH1B-Arg48His polymorphism affects both alcohol-related flushing in Europeans and alcohol intake. The absence of study-wide significant effects on AD results from the low P-value required when testing multiple single nucleotide polymorphisms and phenotypes. PMID:18996923

  6. Associations of ADH and ALDH2 gene variation with self report alcohol reactions, consumption and dependence: an integrated analysis.

    PubMed

    Macgregor, Stuart; Lind, Penelope A; Bucholz, Kathleen K; Hansell, Narelle K; Madden, Pamela A F; Richter, Melinda M; Montgomery, Grant W; Martin, Nicholas G; Heath, Andrew C; Whitfield, John B

    2009-02-01

    Alcohol dependence (AD) is a complex disorder with environmental and genetic origins. The role of two genetic variants in ALDH2 and ADH1B in AD risk has been extensively investigated. This study tested for associations between nine polymorphisms in ALDH2 and 41 in the seven ADH genes, and alcohol-related flushing, alcohol use and dependence symptom scores in 4597 Australian twins. The vast majority (4296) had consumed alcohol in the previous year, with 547 meeting DSM-IIIR criteria for AD. There were study-wide significant associations (P<2.3 x 10(-4)) between ADH1B-Arg48His (rs1229984) and flushing and consumption, but only nominally significant associations (P<0.01) with dependence. Individuals carrying the rs1229984 G-allele (48Arg) reported a lower prevalence of flushing after alcohol (P=8.2 x 10(-7)), consumed alcohol on more occasions (P=2.7 x 10(-6)), had a higher maximum number of alcoholic drinks in a single day (P=2.7 x 10(-6)) and a higher overall alcohol consumption (P=8.9 x 10(-8)) in the previous year than those with the less common A-allele (48His). After controlling for rs1229984, an independent association was observed between rs1042026 (ADH1B) and alcohol intake (P=4.7 x 10(-5)) and suggestive associations (P<0.001) between alcohol consumption phenotypes and rs1693482 (ADH1C), rs1230165 (ADH5) and rs3762894 (ADH4). ALDH2 variation was not associated with flushing or alcohol consumption, but was weakly associated with AD measures. These results bridge the gap between DNA sequence variation and alcohol-related behavior, confirming that the ADH1B-Arg48His polymorphism affects both alcohol-related flushing in Europeans and alcohol intake. The absence of study-wide significant effects on AD results from the low P-value required when testing multiple single nucleotide polymorphisms and phenotypes. PMID:18996923

  7. The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis.

    PubMed Central

    Hoffmaster, A R; Koehler, T M

    1997-01-01

    The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atx4-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes. First, total protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electrophoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing CO2-enhanced atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin coregulated) were Tox+, indicating that these genes may not be involved in anthrax toxin gene activation. Our data indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins. PMID:9234759

  8. Mutations in the Spt4, Spt5, and Spt6 Genes Alter Transcription of a Subset of Histone Genes in Saccharomyces Cerevisiae

    PubMed Central

    Compagnone-Post, P. A.; Osley, M. A.

    1996-01-01

    The SPT4, SPT5, and SPT6 gene products define a class of transcriptional repressors in Saccharomyces cerevisiae that are thought to function through their effects on chromatin assembly or stability. Mutations in these genes confer a similar range of phenotypes to mutations in HIR genes, which encode transcriptional repressors that regulate expression of many of the core histone genes. Here we show that mutations in the three SPT genes also affect transcription of the histone genes that reside at the HTA1-HTB1 locus. HTA1-lacZ transcription was reduced in each spt mutant background, an effect that required a negative site in the HTA1 promoter. The transcriptional effect could be reversed by the overproduction of histones H2A and H2B in an spt4 mutant and histones H3 and H4 in all three spt mutants. Suppression of the spt4 transcriptional defect was dependent on the overproduction of both histones H2A and H2B, and required the presence of N-terminal amino acids in both histones. The results are consistent with the idea that the effects of the spt mutations on nucleosome assembly and/or stability activate repressors of HTA1 transcription. PMID:8844144

  9. Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    Dervyn, E; Poncet, S; Klier, A; Rapoport, G

    1995-01-01

    The CryIVD protein is involved in the overall toxicity of the Bacillus thuringiensis subsp. israelensis parasporal inclusions and is one of the four major components of the crystals. Determination of the DNA sequence indicated that the cryIVD gene is the second gene of an operon which includes three genes. The first one encodes a 19-kDa polypeptide and has sequence homology with the orf1 gene of the Bacillus thuringiensis cryIIA and cryIIC operons. The second and third genes have already been identified and encode the CryIVD crystal protein and the P20 polypeptide, respectively. The promoter region was located by deletion analysis, and the 5' end of the mRNA was determined by primer extension mapping. Transcription of the cryIVD gene in B. thuringiensis subsp. israelensis strains is induced 9 h after the beginning of sporulation. Sequence analysis indicated two potential promoters, a strong one and a weak one, recognized respectively by the RNA polymerase associated with the sigma 35 or the sigma 28 factor of B. thuringiensis (sigma E and sigma K of Bacillus subtilis, respectively). Transcriptional lacZ fusion integrated in single copy into the chromosome of various B. subtilis sporulation mutants confirmed the sigma E dependence of cryIVD gene transcription. PMID:7730255

  10. Systematic analysis of RNAi reports identifies dismal commonality at gene-level & reveals an unprecedented enrichment in pooled shRNA screens

    PubMed Central

    Bhinder, Bhavneet; Djaballah, Hakim

    2013-01-01

    RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes. PMID:23848309

  11. Differential gene expression in neurospora crassa cell types: amplification of rRNA genes. Progress report, July 1979-30 June 1980

    SciTech Connect

    Dutta, S.K.

    1980-01-01

    The significant results obtained during 1979 to 1980 of the current research program are as follows: (1) the differential rRNA gene amplification in germinated conidia of N.crassa was confirmed. N.crassa rDNAs showed differences in degrees of homology with isolated DNAs from other Neurospora species which could be due to heterogeneity in internal spacers. Studies with N.crassa rDNA clones were initiated to study their heterogeneities. The organization of the Institutional Biohazard Committee (IBC) for Recombinant DNA research was completed and necessary certifications for the laboratory and the workers were obtained in accordance with the P/sub 2/EK/sub 1/ containment regulation of N.I.H. Known 17S and 26S N.crassa rDNA probes are being used to detect differences, if any, in restriction cleavage sites in rDNAs of different cell types and developmental mutants of N.crassa. DNAs from these N.crassa cells are restricted with EcoR/sub 1/ and Hind III and cleaved fragments separated by gel electrophoresis are transferred into nitrocellulose papers. Experiments are underway now to see if there are any changes in cleavage sites by annealing with /sup 32/P or /sup 3/H-17S or 26S rDNA probes followed by autoradiography.

  12. (Nuclear genes from nicotiana encoding the small subunit of ribulose-1,5-bisphosphate carboxylase). Progress report

    SciTech Connect

    Cashmore, A.R.

    1985-01-01

    Two pea nuclear genes encoding ribulose-1,5-bisphosphate carboxylase (rbcS) were isolated and completely sequenced. These sequence studies include approximately 1 kb of 5' noncoding region and several hundred nucleotides of 3' noncoding sequences. The two genes are tightly linked being separated by 10 kb of DNA and they are oriented with their 3' ends towards one another. The two genes (ss3.6 and ss8.0) correspond to two of five EcoRI fragments of pea DNA that hybridize to a rbcS hybridization probe. The two genes ss3.6 and ss8.0 are quite divergent at their 5' and their 3' ends and in the first of the two intervening sequences. In direct contrast the second of the two intervening sequences is total conserved between the two genes. This conservation of sequence identity could result directly from evolutionary forces selecting against any sequence change. Such selection would presumably reflect a very sequence-dependent function for these introns. A role in splicing is one possibility and a transcriptional regulatory element is another possibility. 9 refs.

  13. Construction of a cytosolic firefly luciferase reporter cassette for use in PCR-mediated gene deletion and fusion in Saccharomyces cerevisiae.

    PubMed

    Ainsworth, W B; Rome, C M; Hjortsø, M A; Benton, M G

    2012-12-01

    Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(-SKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern. PMID:23172625

  14. Proteus mirabilis urease: operon fusion and linker insertion analysis of ure gene organization, regulation, and function.

    PubMed

    Island, M D; Mobley, H L

    1995-10-01

    Urease is an inducible virulence factor of uropathogenic Proteus mirabilis. Although eight contiguous genes necessary for urease activity have been cloned and sequenced, the transcriptional organization and regulation of specific genes within the Proteus gene cluster has not been investigated in detail. The first gene, ureR, is located 400 bp upstream and is oriented in the direction opposite the other seven genes, ureDABCEFG. The structural subunits of urease are encoded by ureABC. Previously, UreR was shown to contain a putative helix-turn-helix DNA-binding motif 30 residues upstream of a consensus sequence which is a signature for the AraC family of positive regulators; this polypeptide is homologous to other DNA-binding regulatory proteins. Nested deletions of ureR linked to either ureD-lacZ or ureA-lacZ operon fusions demonstrated that an intact ureR is required for urea-induced synthesis of LacZ from either ureA or ureD and identified a urea-regulated promoter in the ureR-ureD intergenic region. However, lacZ operon fusions to fragments encompassing putative promoter regions upstream of ureA and ureF demonstrated that no urea-regulated promoters occur upstream of these open reading frames; regions upstream of ureR, ureE, and ureG were not tested. These data suggest that UreR acts as a positive regulator in the presence of urea, activating transcription of urease structural and accessory genes via sequences upstream of ureD. To address the role of the nonstructural regulatory and accessory genes, we constructed deletion, cassette, and linker insertion mutations throughout the ure gene cluster and determined the effect of these mutations on production and regulation of urease activity in Escherichia coli. Mutations were obtained, with locations determine by DNA sequencing, in all genes except ureA and ureE. In each case, the mutation resulted in a urease-negative phenotype. PMID:7559355

  15. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase genes. Progress report, [April 15, 1990--April 14, 1991

    SciTech Connect

    Okita, T.W.

    1990-12-31

    The long term goal of this project is to assess the feasibility of increasing the conversion of photosynthate a key regulatory enzyme in starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is primarily regulated by the gene activation, expression, and allosteric regulation of ADPglucose pyrophosphorylase, as well as starch synthase, and branching enzyme. During the last year we have elucidated the structure of both subunits which compose this tetrameric enzyme and determined the temporal and spatial expression of the genes encoding each subunit as well as their correlation to starch biosynthesis. Genomic clones to both subunits have also been isolated and the gene structure of the small subunit determined. Transgenic potato plants have been produced containing deletions of the small subunit promoter. Currently, cis acting elements and their involvement in spatial and temporal expression are under investigation.

  16. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1987--April 14, 1988

    SciTech Connect

    Okita, T.W.

    1988-12-31

    Many agronomically important crops are viewed as significant resources of renewable energy. Overall crop productivity could be increased if the efficiency of photoassimilate conversion into dry matter such as starch were improved in storage tissues. Starch production is controlled by the catalytic activity of ADPglucose pyrophosphorylase in the first step of starch biosynthesis. This research focuses on the genetic structure and molecular mechanisms by which it is controlled during plant development and how it is affected by environmental and hormonal conditions. The current goal is to isolate the genes for this enzyme present in both cereal endosperm and potato tuber tissues, and to elucidate its structure and the controlling sequences responsible for gene expression. The long term goal is the improvement of starch production in storage organs by manipulating this gene so that it encodes an enzyme refractive to inorganic phosphate inhibition.

  17. Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1992--April 15, 1993

    SciTech Connect

    Barkan, A.

    1993-04-20

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that control the timing and cell-type specificity of chloroplast gene expression. Studies are being conducted with nuclear mutants of maize that are defective in the biogenesis or translation of chloroplast mRNAs. Currently studies are focused on two nuclear mutants with specific and unique lesions in chloroplast RNA processing (crp mutants). Crp1 mutants (formerly called hcf136) fail to accumulate the cytochrome f/b6 complex. The protein loss is due to a defect in the metabolism of transcripts encoding the petB and petD gene products, two subunits of the missing complex. Mutant seedlings lack the monocistronic petB and petD MRNAS, which both arise in nominal plants by endonucleolytic cleavage of the polycistronic primary transcript of the psbB gene cluster. Precursor mRNAs accumulate normally in crp1, indicating that its defect is due either to a failure to cleave the precursors, or a failure to stabilize the fully processed mRNAs. We are interested in both the biochemistry of this site-specific RNA processing and in the role of the processing in generating translatable mRNAs. To address the latter, we are quantifying the rates of synthesis of the petB and petD gene products with the goal of determining whether the missing transcripts are more efficiently translated than their precursors. To address the biochemistry of the defect in RNA metabolism, the crp1 gene is being cloned via the transposon tag. crp2 (formerly called hcf142) lacks the predominant mRNA encoding petA, but appears to be otherwise unimpaired in chloroplast RNA metabolism. The precise role of crp2 in synthesizing or stabilizing the petA mRNA is being investigated through biochemical studies.

  18. Differential gene expression in Neurospora crassa cell types: heterogeneity and amplification of rRNA genes. Progress report, July 1980-June 30, 1981

    SciTech Connect

    Dutta, S.K.

    1981-01-01

    The significant results obtained during 1980-1981 year of the current research program are as follows: I. Studies on heterogeneity of multiple copies of rDNAs from N. crassa cell types are being continued, such as: (1) Autoradiographs of Southern transfers of EcoR/sub 1/ restricted fragments of nuclear DNA from conidia, germinated conidia (sprouts) and mycelia of N. crassa were compared after hybridization with /sup 32/P-rDNA probe. The nuclear DNA of two hours sprout and of 16 hours mycelia gave similar hybridization patterns with EcoR/sub 1/ digest, but no such hybridization pattern was evident in conidial DNA digest; (2) Procedure for concentration of rDNAs from Neurospora species and cell types was standardized; restriction analysis of purified rDNAs is being done; (3) 35S total rDNA clone, 17S rDNA clone and 26S rDNA subclone are being used to see gross differences in the precursor rRNAs of different cell types; (4) Comparison of DNA:DNA homologies of rRNA genes with different Neurospora species. II. Post-mitochondrial DNAs of N. crassa are found to be rDNA-like and were further characterized by electron microscopic studies and are found to be approximately twice the size of SV-40 DNAs. These N. crassa post-mitochondrial DNAs hybridized with /sup 32/P-labeled N. crassa nuclear DNAs. III. Previous studies on differential RNase sensitive DNA polymerase activity in N. Crassa cell types and on evolution of sexual morphogenesis in the genus Neurospora are completed and published. RNase sensitive DNA polymerase activity is found to be in the post-mitochondrial fraction. Heterothallism in the genus Neurospora is evolved from homothallism.

  19. Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression.

    PubMed Central

    Sawers, G; Böck, A

    1989-01-01

    The sequence of the 5' regulatory region of the gene encoding pyruvate formate-lyase is presented together with a detailed analysis of the transcriptional signals required for its expression. The sequence data revealed that a gene coding for an open reading frame (orf) of unknown function is situated just upstream of the pfl gene. Analysis of RNA transcripts by Northern blot hybridization demonstrated that the genes for orf and pfl were cotranscribed as an operon but that the pfl gene was also transcribed alone. S1 nuclease protection analysis, primer extension, and construction of lacZ fusions with sequential deletions in the pfl 5' regulatory sequence revealed that transcription initiated from at least six promoters which spanned 1.2 kilobases of DNA. Three of these lay within the orf structural gene and were responsible for the high expression of pfl. All transcripts originating from these promoters terminated in the 3' untranslated region of the pfl gene at a strong rho-independent transcription terminator. All of the promoters were coordinately regulated by anaerobiosis, pyruvate, nitrate, and the fnr gene product, and the sequences thought to be responsible for this regulation lay 0.8 to 1.3 kilobases upstream of the translational initiation codon of the pfl gene. There were two sequences within this region which showed strong homology with that proposed to be required for recognition by the Fnr protein. Images PMID:2651404

  20. Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.

    PubMed

    Grassi, G; Maccaroni, P; Meyer, R; Kaiser, H; D'Ambrosio, E; Pascale, E; Grassi, M; Kuhn, A; Di Nardo, P; Kandolf, R; Kpper, J-H

    2003-10-01

    The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes. As a better understanding of these biological processes may contribute to the development of novel cancer treatments and to study the complex mechanisms regulating gene silencing, we established a cellular system suitable to dissect the mechanisms regulating DNA methylation and histone acetylation. For this purpose, we stably transfected the neuroblastoma cell line U87 with a cytomegalovirus promoter-driven reporter gene construct whose expression was analyzed following treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine or histone deacetylation inhibitor trichostatin A. Both substances reactivated the silenced cytomegalovirus promoter, but with different reaction kinetics. Furthermore, whereas the kinetics of reactivation by trichostatin A did not substantially change over the time range considered (5 days), reactivation induced by 5'-aza-2'-deoxycytidine showed profound differences between day 1 and longer time points. We showed that this effect is related to the down-regulation of DNA replication by 5'-aza-2'-deoxycytidine. Finally, we have shown that the simultaneous administration of trichostatin A and 5'-aza-2'-deoxycytidine results in reactivation of the CMV promoter according to a cooperative, not synergistic or additive, mechanism. In conclusion, our cellular system should represent a powerful tool to investigate the complex mechanisms regulating gene silencing and to identify new anticancer drugs. PMID:12869421

  1. (Analysis of cyanobacterial photosystem 2 genes by cloning and mutagenesis): Progress report, May 1986 to December 1986

    SciTech Connect

    Not Available

    1986-01-01

    We have isolated proteins that appear to be regulated by changes in environmental conditions, either iron or light. Iron-regulated proteins include: a 36 kDa protein that is involved in iron-acquisition, and a 34 kDa intrinsic thylakoid chlorophyll-binding protein associated with PS II. Utilizing a lambda gtll library we have cloned and sequenced the 36 kDa protein and are performing site-directed mutagenesis to determine the function of the protein. Insertion of a transposon into this gene and transformation of the mutated gene into a wild type host resulted in a strain which is unable to grow under low-iron conditions. We have obtained a 42 kDa carotenoid-binding protein that is induced upon growth in high light conditions, and cloned the gene for this protein. We have also isolated a carotenoid-binding protein that is localized exclusively in the plasmalemma. We have also cloned the 33 kDa extrinsic maganese-binding protein that is involved in oxygen-evolution. The gene has been cloned and sequenced and contains a 28 amino acid signal sequence. 4 figs.

  2. Brief Report: The Dopamine-3-Receptor Gene ("DRD3") Is Associated with Specific Repetitive Behavior in Autism Spectrum Disorder (ASD)

    ERIC Educational Resources Information Center

    Staal, Wouter G.; de Krom, Mariken; de Jonge, Maretha V.

    2012-01-01

    Recently the "DRD3" gene has been associated with ASD in two independent samples. Follow up analysis of the risk allele of the SNP rs167771 in 91 subjects revealed a significant association with a specific type of repetitive behavior: the factor "insistence on sameness" (IS) derived from the Autism Diagnostic Interview. This risk allele was

  3. CELL DIVISION GENE CLUSTER IN SPIROPLASMA KUNKELII: FUNCTIONAL CHARACTERIZATION OF FTSZ AND THE FIRST REPORT OF FTSA IN MOLLICUTES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ftsZ gene, found in representatives of all bacterial groups, encodes an essential protein engaged in prokaryotic cell division. We cloned and characterized the ftsZ homologue (ftsZsk) from a pathogenic strain of the wall-less bacterium Spiroplasma kunkelii that causes corn stunt disease. The 1...

  4. Brief Report: The Dopamine-3-Receptor Gene ("DRD3") Is Associated with Specific Repetitive Behavior in Autism Spectrum Disorder (ASD)

    ERIC Educational Resources Information Center

    Staal, Wouter G.; de Krom, Mariken; de Jonge, Maretha V.

    2012-01-01

    Recently the "DRD3" gene has been associated with ASD in two independent samples. Follow up analysis of the risk allele of the SNP rs167771 in 91 subjects revealed a significant association with a specific type of repetitive behavior: the factor "insistence on sameness" (IS) derived from the Autism Diagnostic Interview. This risk allele was…

  5. Immunoglobulin gene translocations in chronic lymphocytic leukemia: A report of 35 patients and review of the literature

    PubMed Central

    DE BRAEKELEER, MARC; TOUS, CORINE; GUÉGANIC, NADIA; LE BRIS, MARIE-JOSÉE; BASINKO, AUDREY; MOREL, FRÉDÉRIC; DOUET-GUILBERT, NATHALIE

    2016-01-01

    Chronic lymphocytic leukemia (CLL) represents the most common hematological malignancy in Western countries, with a highly heterogeneous clinical course and prognosis. Translocations involving the immunoglobulin (IG) genes are regularly identified. From 2000 to 2014, we identified an IG gene translocation in 18 of the 396 patients investigated at diagnosis (4.6%) and in 17 of the 275 analyzed during follow-up (6.2%). A total of 4 patients in whom the IG translocation was identified at follow-up did not carry the translocation at diagnosis. The IG heavy locus (IGH) was involved in 27 translocations (77.1%), the IG κ locus (IGK) in 1 (2.9%) and the IG λ locus (IGL) in 7 (20.0%). The chromosome band partners of the IG translocations were 18q21 in 16 cases (45.7%), 11q13 and 19q13 in 4 cases each (11.4% each), 8q24 in 3 cases (8.6%), 7q21 in 2 cases (5.7%), whereas 6 other bands were involved once (2.9% each). At present, 35 partner chromosomal bands have been described, but the partner gene has solely been identified in 10 translocations. CLL associated with IG gene translocations is characterized by atypical cell morphology, including plasmacytoid characteristics, and the propensity of being enriched in prolymphocytes. The IG heavy chain variable region (IGHV) mutational status varies between translocations, those with unmutated IGHV presumably involving cells at an earlier stage of B-cell lineage. All the partne