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Measurement of Ligand-Induced Activation in Single Viable T Cells Using the lacZ Reporter Gene  

NASA Astrophysics Data System (ADS)

We have used the bacterial ?-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic ?-galactosidase substrate fluorescein di-?-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/A^b complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ^+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ^+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.

Karttunen, Jaana; Shastri, Nilabh



lacZ Transgenic mice to monitor gene expression in embryo and adult  

Microsoft Academic Search

In transgenic experiments, lacZ can be used as a reporter gene for activity of a given promoter. Its main advantage is the ease of visualization in situ, on sections or in whole mount preparations, and the availability of simple protocols. In the following, we describe our procedure for detecting promoter activity in transgenic mice, including choice of lacZ vectors, generation

Andrea Schmidt; Kirsten Tief; Alessandro Foletti; Agnčs Hunziker; Doris Penna; Edith Hummler; Friedrich Beermann



Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.  


The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of beta-galactosidase activity in Tmhs ( tm1Kjn ) heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence. PMID:17876667

Longo-Guess, Chantal M; Gagnon, Leona H; Fritzsch, Bernd; Johnson, Kenneth R



Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation  

PubMed Central

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhstm1Kjn) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of ?-galactosidase activity in Tmhstm1Kjn heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.

Longo-Guess, Chantal M.; Gagnon, Leona H.; Fritzsch, Bernd; Johnson, Kenneth R.



Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.  


IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues. PMID:22371395

Pichery, Mélanie; Mirey, Emilie; Mercier, Pascale; Lefrancais, Emma; Dujardin, Arnaud; Ortega, Nathalie; Girard, Jean-Philippe



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

SciTech Connect

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.

Lennon, E.; Minton, K.W. (Uniformed Services Univ. of the Health Sciences, Bethesda, MD (USA))



A plasmid-based lacZ? gene assay for DNA polymerase fidelity measurement  

PubMed Central

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZ? reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZ?, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated–naphthoylated DEAE–cellulose, resulting in a low background mutation frequency (?1 × 10?4). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system.

Keith, Brian J.; Jozwiakowski, Stanislaw K.; Connolly, Bernard A.



A plasmid-based lacZ? gene assay for DNA polymerase fidelity measurement.  


A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZ? reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZ?, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system. PMID:23098700

Keith, Brian J; Jozwiakowski, Stanislaw K; Connolly, Bernard A



A comprehensive toolbox for the rapid construction of lacZ fusion reporters.  


?-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the ? Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the ?-origin-based suicide vector pNPTS138-R6KT, which can only replicate in ?pir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K(+)-dependent activation of kdp promoter-lacZ fusions in vivo. PMID:23022912

Fried, Luitpold; Lassak, Jürgen; Jung, Kirsten



Fusion of Escherichia coli LacZ to the Cytochrome c Gene of Saccharomyces cerevisiae  

Microsoft Academic Search

Hybrid genes between the Escherichia coli lacZ gene and the iso-1-cytochrome c (CYC1) gene of Saccharomyces cerevisiae were constructed by recombination in vitro. Each of the hybrid genes encodes a chimeric protein with a cytochrome c moiety at the amino terminus and an active beta -galactosidase (beta -D-galactoside galactohydrolase, EC moiety at the carboxy terminus. When these hybrids are

Leonard Guarente; Mark Ptashne



Use of ?-galactosidase (lacZ) gene ?-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis.  


The mutagenic potential of titanium dioxide nanoparticles (TiO(2)-NPs) of an average size 30.6nm was investigated using ?-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-)Escherichia coli DH5? system. Plasmid pUC19 was treated with varying concentrations of TiO(2)-NPs and allowed to transfect the CaCl(2)-induced competent DH5? cells. The data revealed loss in transformation efficiency of TiO(2)-NPs treated plasmids as compared to untreated plasmid DNA in DH5? host cells. Induction of multiple mutations in ?-fragment of lacZ gene caused synthesis of non-functional ?-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO(2)-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO(2)-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO(2)-NPs induced mutations. This study suggested the pUC19/E. coli DH5?lacZ gene ?-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials. PMID:22705419

Ahmad, Javed; Dwivedi, Sourabh; Alarifi, Saud; Al-Khedhairy, Abdulaziz A; Musarrat, Javed



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

SciTech Connect

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

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



Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation.  

PubMed Central

The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability. Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold. Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message. The magnitude of the latter effect was strongly dependent on the sequences at the ribosome-binding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one. The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack. Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites. Images

Petersen, C



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


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 (?qseB) mutant and lsrRK double deletion mutants (?lsrRK). 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

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



Transcribing of Escherichia coli Genes with Mutant T7 RNA Polymerases: Stability of lacZ mRNA Inversely Correlates with Polymerase Speed  

Microsoft Academic Search

When in Escherichia coli the host RNA polymerase is replaced by the 8-fold faster bacteriophage T7 enzyme for transcription of the lacZ gene, the beta-galactosidase yield per transcript drops as a result of transcript destabilization. We have measured the beta-galactosidase yield per transcript from T7 RNA polymerase mutants that exhibit a reduced elongation speed in vitro. Aside from very slow

Olga V. Makarova; Evgeny M. Makarov; Rui Sousa; Marc Dreyfus



Possible Cytotoxic Effect of the Expression of a Connexin 43-LacZ Fusion Gene in Cells of the Vascular Wall  

Microsoft Academic Search

Connexin 43 (Cx43) gap junctions are hypothesized to play a key role in many aspects of vascular function. In an effort to evaluate the importance of connexins in vascular function we took advantage of the fact that a Cx43-LacZ fusion protein has been reported to effectively reduce dye transfer in NIH 3T3 fibroblasts by acting as a dominant negative construct.

Y. Liao; B. R. Duling



The promoter region of the arg3 gene in Saccharomyces cerevisiae: nucleotide sequence and regulation in an arg3-lacZ gene fusion.  

PubMed Central

We have determined the DNA sequence for the 5' end of the arg3 gene of Saccharomyces cerevisiae, including part of the coding region and the 200 nucleotides immediately upstream. A promoter-deletion mutant was found to have lost all of the sequence lying normally in front of the gene except for the 33 nucleotides preceding the AUG codon. The role of the 5' domain in initiation and regulation of arg3 transcription was assessed by a gene fusion experiment. The Escherichia coli lacZ gene, was truncated of the eight amino-terminal codons substituted in vitro, on a 2mu plasmid, for the carboxy-terminal and 3'-flanking regions of arg3, leaving only the first 19 proximal codons and approximately 1600 nucleotides of the region preceding arg3 on the yeast chromosome. The fused gene was expressed in phase and was still submitted to the two mechanisms regulating the wild-type arg3 gene: the general, probably transcriptional control of amino acid biosynthesis and the specific, apparently post-transcriptional control mediated by the products of the argR genes. These results suggest a determining role for the 5' end portion of the arg3 messenger in the specific arginine-mediated control mechanism.

Crabeel, M; Huygen, R; Cunin, R; Glansdorff, N



Structure and properties of a herpesvirus of turkeys recombinant in which US1, US10 and SORF3 genes have been replaced by a lacZ expression cassette  

Microsoft Academic Search

In the process of generating an insertional mutant of herpesvirus of turkeys (HVT) expressing lacZ at the protein kinase (PK) locus, we isolated a recombinant which contained an intact PK gene but the short unique regions US1, US10 and SORF3 had been deleted and replaced by the lacZ cassette. Moreover, the virus contained duplicate copies of gD, gI and gE

V. Zelnik; Pam Tyers; Graham D. Smith; ChunLing Jiang; Norman L. J. Ross



Two versatile shuttle vectors for Thermus thermophilus-Escherichia coli containing multiple cloning sites, lacZ? gene and kanamycin or hygromycin resistance marker.  


Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZ? gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus. PMID:22252135

Fujita, Atsushi; Misumi, Yoshio; Koyama, Yoshinori



A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme.  

PubMed Central

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan [correction of peptidodoglycan] precursors are wrong.

Ulijasz, A T; Grenader, A; Weisblum, B



A mini-promoter lacZ gene fusion for the analysis of fungal transcription control sequences.  


A system for the in vivo analysis of fungal transcription control sequences, based on a mini-promoter, was designed. The mini-promoter, providing all sequences necessary and sufficient for transcription initiation, was derived from the Aspergillus nidulans gpdA promoter region. Transcription initiation was not affected by the introduction of transcription control sequences directly upstream from the mini-promoter. Furthermore, the expression of the mini-promoter was not affected by wide-domain carbon or nitrogen control circuits. Using the mini-promoter vector, a previously identified upstream activating sequence from the A. nidulans gpdA gene was further characterized. PMID:7789794

Punt, P J; Kuyvenhoven, A; van den Hondel, C A



LacZ Transgenic Mice and Immunoelectron Microscopy: An Ultrastructural Method for Dual Localization of ?-Galactosidase and Horseradish Peroxidase  

Microsoft Academic Search

Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, ?-galactosidase, are increasingly becoming available. Similarly, antibody conjugates consisting of specific IgGs coupled to horseradish peroxidase (HRP) are widely used for Western blotting, ELISA, and immunohistochemistry. Here we provide a detailed fixation and histochemical protocol for the simultaneous electron microscopic visualization and discrimination of ?-galactosidase and peroxidase reaction products

Patricia L. St. John; Dale R. Abrahamson



Expression of a reporter gene resembles that of its neighbour: an insertion in the hairy gene of Drosophila  

Microsoft Academic Search

Random insertions of a promotor fused to a reporter gene, such as Lac-Z, reveal regulatory sequences that confer temporal and spatial patterns of gene expression in eukaryotes. These patterns may reflect the activity of a neighbouring gene and thus lead to the isolation of new genes essential for normal development. Here, we demonstrate that this hypothesis is true for an

Laurent Fasano; Nathalie Coré; Stephen Kerridge



Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation.  

PubMed Central

In Escherichia coli transcription of individual genes generally requires concomitant translation, and thus the decay of mRNAs cannot be studied without the complication of translation. Here we have used T7 RNA polymerase to transcribe in vivo lacZ genes carrying ribosome binding sites of variable efficiency. We show that neither cell viability nor growth rate is affected by the T7-driven transcription of these genes, provided that they are present as single chromosomal copy. Furthermore, transcription is now completely uncoupled from translation, allowing large amounts of even completely untranslated mRNAs to be synthesized. Taking advantage of these features, we discuss the influence of the frequency of translation upon the processing and degradation of the lac message. Images

Chevrier-Miller, M; Jacques, N; Raibaud, O; Dreyfus, M



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

PubMed Central

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.

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



A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system  

Microsoft Academic Search

The variability in gene conversion frequency by an RNA-DNA oligonucleotide (RDO) prompted us to develop a system as a means of measuring the conversion frequency rapidly and reproducibly. A shuttle vector was constructed to measure the frequency of targeted gene correction by RDO of the E. coli ?-galactosidase gene containing a single point mutation (G ? A), that resulted in

O Igoucheva; A E Peritz; D Levy; K Yoon



Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus.  

PubMed Central

Transcription of the genes that code for proteins involved in nitrogen fixation in free-living diazotrophs is typically repressed by high internal oxygen concentrations or exogenous fixed nitrogen. The DNA sequence of a regulatory locus required for repression of Rhodobacter capsulatus nitrogen fixation genes was determined. It was shown that this locus, defined by Tn5 insertions and by ethyl methanesulfonate-derived mutations, is homologous to the glnB gene of other organisms. The R. capsulatus glnB gene was upstream of glnA, the gene for glutamine synthetase, in a glnBA operon. beta-Galactosidase expression from an R. capsulatus glnBA-lacZ translational fusion was increased twofold in cells induced by nitrogen limitation relative to that in cells under nitrogen-sufficient conditions. R. capsulatus nifR1, a gene that was previously shown to be homologous to ntrC and that is required for transcription of nitrogen fixation genes, was responsible for approximately 50% of the transcriptional activation of this glnBA fusion in cells induced under nitrogen-limiting conditions. R. capsulatus GLNB, NIFR1, and NIFR2 (a protein homologous to NTRB) were proposed to transduce the nitrogen status in the cell into repression or activation of other R. capsulatus nif genes. Repression of nif genes in response to oxygen was still present in R. capsulatus glnB mutants and must have occurred at a different level of control in the regulatory circuit. Images FIG. 4 FIG. 5

Kranz, R G; Pace, V M; Caldicott, I M



A directional recombination cloning system for restriction- and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters.  


The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements, or enhancers. Here we present a rapid, high-efficiency system for directionally cloning PCR-amplified, PCR-mutated, or synthetic enhancer sequences into the Ganesh family of P element reporter constructs, which contain reporter genes encoding nuclear-localized eGFP, DsRed, or beta-galactosidase. This system, which is scalable for either small projects or high-throughput approaches, makes use of both TOPO and Gateway cloning technologies for directional, efficient cloning, without the need for restriction digestion or ligation reactions. It should be especially useful for those researchers who wish to test large numbers of putative enhancers, those who are undertaking detailed mutational analyses of enhancer sequences, or those who wish to avoid the difficulties sometimes encountered in traditional cloning strategies. PMID:18077106

Swanson, Christina I; Hinrichs, Trish; Johnson, Lisa A; Zhao, Ying; Barolo, Scott



Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements  

PubMed Central

The multicopy ?gt10-lacZ transgene shuttle vector of Muta™Mouse serves as an important tool for genotoxicity studies. Here, we describe a model for ?gt10-lacZ transgene molecular structure, based on characterisation of transgenes recovered from animals of our intramural breeding colony. Unique nucleotide sequences of the 47?513 bp monomer are reported with GenBank® assigned accession numbers. Besides defining ancestral mutations of the ?gt10 used to construct the transgene and the Muta™Mouse precursor (strain 40.6), we validated the sequence integrity of key ? genes needed for the Escherichia coli host-based mutation reporting assay. Using three polymerase chain reaction (PCR)-based chromosome scanning and cloning strategies, we found five distinct in vivo transgene rearrangements, which were common to both sexes, and involved copy fusions generating ?10 defective copies per haplotype. The transgene haplotype was estimated by Southern hybridisation and real-time–polymerase chain reaction, which yielded 29.0 ± 4.0 copies based on spleen DNA of Muta™Mouse, and a reconstructed CD2F1 genome with variable ?gt10-lacZ copies. Similar analysis of commercially prepared spleen DNA from Big Blue® mouse yielded a haplotype of 23.5 ± 3.1 copies. The latter DNA is used in calibrating a commercial in vitro packaging kit for E.coli host-based mutation assays of both transgenic systems. The model for ?gt10-lacZ transgene organisation, and the PCR-based methods for assessing copy number, integrity and rearrangements, potentially extends the use of Muta™Mouse construct for direct, genomic-type assays that detect the effects of clastogens and aneugens, without depending on an E.coli host, for reporting effects.

Shwed, Philip S.; Crosthwait, Jennifer; Douglas, George R.; Seligy, Vern L.



Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically during normal development and determines radiation and drug sensitivity in vivo.  

PubMed Central

To analyze the involvement of p53-dependent transcriptional activation in normal development and in response to DNA damage in vivo, we created transgenic mice with a lacZ reporter gene under the control of a p53-responsive promoter. Five independent strains showed similar patterns of transgene expression. In untreated animals, lacZ expression was limited to the developing nervous system of embryos and newborn mice and was strongly decreased in the adult brain. gamma-irradiation or adriamycin treatment induced lacZ expression in the majority of cells of early embryos and in the spleen, thymus and small intestine in adult mice. Transgene expression was p53 dependent and coincided with the sites of strong p53 accumulation. The lacZ-expressing tissues and early embryos, unlike other adult tissues and late embryos, are characterized by high levels of p53 mRNA expression and respond to DNA damage by massive apoptotic cell death. Analysis of p53-null mice showed that this apoptosis is p53 dependent. These data suggest that p53 activity, monitored by the reporter lacZ transgene, is the determinant of radiation and drug sensitivity in vivo and indicate the importance of tissue and stage specificity of p53 regulation at the level of mRNA expression.

Komarova, E A; Chernov, M V; Franks, R; Wang, K; Armin, G; Zelnick, C R; Chin, D M; Bacus, S S; Stark, G R; Gudkov, A V



pBRINT-T s: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli 1 Published in conjunction with A Wisconsin Gathering Honoring Waclaw Szybalski on the occasion of his 75th year and 20years of Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA. 1  

Microsoft Academic Search

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by

Sylvie Le Borgne; Beatr??z Palmeros; Fernando Valle; Francisco Bolivar; Guillermo Gosset



Fate tracing of neurogenin2-expressing cells in the mouse retina using CreER™: LacZ.  


Delineating the final fate of progenitor cells that transiently express a regulatory gene may shed light on how the gene participates in regulating retinal development. We describe the steps in tracing final fates of progenitor cells that once transiently express neurogenin2 (ngn2) during mouse retinal development with the binary, conditional Ngn2-CreER(™)-LacZ reporter system. Ngn2-CreER(™) mice (Zirlinger et al. Proc Natl Acad Sci USA 99:8084-8089, 2002), in which ngn2 promoter drives the expression of Cre-estrogen receptor CreER(™) (Littlewood et al. Nuc Acid Res 23:1686-1690, 1995; Hayashi and McMahon Dev Biol 244:305-318, 2002), are crossed with Rosa26-LoxP-LacZ reporter mice (Soriano Nat Genet 21:70-71, 1999), in which the expression of lacZ requires the removal of "stop" by Cre recombinase (Wagner et al. Transgenic Res 10:545-553, 2001). 4-hydroxytamoxifen (4-OHT), a synthetic ligand with high affinity for ER(™), is administered to double transgenic embryos and/or neonatal mice. Binding of 4-OHT to Cre-ER(™) activates Cre recombinase, which then catalyzes the removal of the "stop" sequence from the LoxP-LacZ transgene, leading to lacZ expression in cells that express ngn2. Retinal tissues are fixed at different time points after 4-OHT treatment and analyzed for LacZ activities by colorimetric reaction. Double-labeling with a cell type-specific marker can be used to define the identity of a LacZ(+) cell. Combining persisted lacZ expression through the life of the cell and the short half-life (0.5-2 h) of 4-OHT (Danielian et al. Curr Biol 8:1323-1326, 1998), this system offers the opportunity to track the final fates of cells that have expressed ngn2 during the brief presence of 4-OHT administered during retinal development. PMID:22688703

Ma, Wenxin; Wang, Shu-Zhen



Enhancer trap integrations in mouse embryonic stem cells give rise to staining patterns in chimaeric embryos with a high frequency and detect endogenous genes  

Microsoft Academic Search

We have generated mouse embryonic stem cell lines that carry lacZ enhancer trap constructs integrated in their genome. Fifty-nine cell lines were analysed for lacZ expression in undifferentiated stem cells and at day 7.5, 8.5 and 12.5 of development in chimaeric embryos obtained after blastocyst injection. In 13 cell lines the lacZ reporter gene was expressed in undifferentiated stem cells

R Korn; M Schoor; H Neuhaus; U Henseling; R Soininen; J Zachgo; A Gossler



Accurate insertional inactivation of lacZ?: construction of pTrueBlue and M13TrueBlue cloning vectors  

Microsoft Academic Search

Color selection plasmid and phage vectors based on insertional inactivation of the lacZ? gene fragment have been in wide use for nearly two decades. The originals (the pUC and M13mp series) and all subsequent derivatives of these vectors contain the same mechanism for insertional inactivation of lacZ? placing the sites for gene interruption between the initiator ATG codon and codon

Steve N. Slilaty; Suzanne Lebel



Comparison of Three Nonviral Transfection Methods for Foreign Gene Expression in Early Chicken Embryos in Ovo  

Microsoft Academic Search

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 embryosin 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

Tatsuo Muramatsu; Yoshimoto Mizutani; Yasushige Ohmori; Jun-ichi Okumura



The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site  

Microsoft Academic Search

Various synthetic DNA sequences were inserted downstream of the fourth codon of the Escherichia coli lacZ gene on plasmids containing a hybrid lacZ-galK operon. Several different sequences, one as short as 10 bp, reduced the functional stability of the lacZ message three- to fourfold, whereas others had little or no effect. Introduction of synthetic sequences into a plasmid containing the

Carsten Petersen



White as a Reporter Gene to Detect Transcriptional Silencers Specifying Position-Specific Gene Expression during Drosophila Melanogaster Eye Development  

PubMed Central

The white(+) gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacW] transposant lines were collected. These represent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to cis-acting suppression of white(+) expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lacZ in P[lacW] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white(+) and lacZ may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacW] insertion site. We speculate that these putative POSE (position-specific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotor-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this ``silencer trap'' may be an efficient way of identifying genes involved in imaginal pattern formation.

Sun, Y. H.; Tsai, C. J.; Green, M. M.; Chao, J. L.; Yu, C. T.; Jaw, T. J.; Yeh, J. Y.; Bolshakov, V. N.



Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes  

Microsoft Academic Search

A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used

A Gossler; A L Joyner; J Rossant; W C Skarnes



Biological Sensor for Sucrose Availability: Relative Sensitivities of Various Reporter Genes  

PubMed Central

A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 ?M sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 ?M. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.

Miller, William G.; Brandl, Maria T.; Quinones, Beatriz; Lindow, Steven E.




PubMed Central

Non-invasive molecular imaging of dynamic processes has benefited tremendously from the use of reporter genes. These genes encode for proteins that emit light, bind radiolabeled probes or, as covered in this review, modulate magnetic resonance (MR) contrast. Reporter genes play a pivotal role in monitoring cell trafficking, gene replacement therapy, protein-protein interactions, neuronal plasticity and embryonic development. Several strategies exist for generating MR contrast: enzyme-catalyzed chemical modification of metal-based contrast agents or (phosphorus) metabolites, using iron-binding and iron-storage proteins to accumulate iron as contrast agent, and using artificial proteins for imaging based on chemical exchange saturation transfer. MR reporter genes have the advantage that the specific signal can be co-registered with soft tissue anatomy and functional tissue information, and have therefore become an active and growing area of scientific interest.

Gilad, Assaf A.; Ziv, Keren; McMahon, Michael T.; van Zijl, Peter C.M.; Neeman, Michal; Bulte, Jeff W.M.



Transcription of individual genes in eukaryotic cells occurs randomly and infrequently  

Microsoft Academic Search

Experimental evidence is presented indicating that the expression of a lacZ reporter gene driven by the HIV-1 long terminal repeat in a series of stably transfected. cloned macrophage cell lines occurs in a very small proportion of cells. The proportion of cells expressing lacZ, rather than the level of expression in each cell, is regulated by external stimuli such as

Ian L Ross; Catherine M Browne; David A Hume



Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy  

PubMed Central

Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse. To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been “knocked in” to the SCL locus, thereby linking ?-galactosidase expression to transcription from the SCL promoter. Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of ?-galactosidase (designated lacZhigh, lacZint, and lacZneg). Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs). These fractions included >99% of the erythroid and >90% of the myeloid CFCs. Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions. These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells. Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were ?-galactosidase-negative.

Elefanty, Andrew G.; Begley, C. Glenn; Metcalf, Donald; Barnett, Louise; Kontgen, Frank; Robb, Lorraine



A stable and sensitive genotoxic testing system based on DNA damage induced gene expression in Saccharomyces cerevisiae  

Microsoft Academic Search

A sensitive and stable genotoxic testing system has been developed based on the induction of a Saccharomyces cerevisiaeRNR3-lacZ reporter gene expression in response to a broad range of DNA-damaging agents and agents that interfere with DNA synthesis. All 11 tested known carcinogenic and genotoxic agents, ranging from DNA alkylating agents, oxidative chemicals and radiations, were able to induce RNR3-lacZ expression

Xuming Jia; Yu Zhu; Wei Xiao



Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice.  


Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(-/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(-/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization. PMID:18252720

Hu, Jan C-C; Hu, Yuanyuan; Smith, Charles E; McKee, Marc D; Wright, J Timothy; Yamakoshi, Yasuo; Papagerakis, Petros; Hunter, Graeme K; Feng, Jerry Q; Yamakoshi, Fumiko; Simmer, James P



Deletion of p66Shc in mice increases the frequency of size-change mutations in the lacZ transgene.  


Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2 O2 . These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism. PMID:23237310

Beltrami, Elena; Ruggiero, Antonella; Busuttil, Rita; Migliaccio, Enrica; Pelicci, Pier Giuseppe; Vijg, Jan; Giorgio, Marco



Progress in gene targeting and gene therapy for retinitis pigmentosa  

SciTech Connect

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.

Farrar, G.J.; Humphries, M.M.; Erven, A. [Trinity College, Dublin (Ireland)] [and others



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

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-?-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.

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



Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.  


Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands. PMID:8095258

Mani, N; Tobin, P; Jayaswal, R K



Construction of Tn5 lac, a Transposon That Fuses lacZ Expression to Exogenous Promoters, and Its Introduction into Myxococcus xanthus  

Microsoft Academic Search

A promoterless trp-lac fusion fragment was inserted near one end of the bacterial transposon Tn5 in the correct orientation to fuse lacZ gene expression to promoters outside Tn5. The resulting transposon, Tn5 lac, retains the kanamycin-resistance gene of Tn5 and transposes in Escherichia coli at 6% the frequency of Tn5 to many different sites in a bacteriophage lambda target. Expression

Lee Kroos; Dale Kaiser



Sequence and chromosomal context effects on variegated expression of keratin 5/lacZ constructs in stratified epithelia of transgenic mice.  

PubMed Central

The expression of transgene loci in mammals often occurs in a heterocellular fashion resulting in variegated patterns of expression. We have examined the effect of chromosomal integration site, copy number, and transcriptionally activating sequences on the variegation of a keratin 5-lacZ (K5Z) construct in the stratified epithelia of transgenic mice. lacZ expression in these mice is always mosaic, and the beta-gal activity per cell is usually higher in the lines with a higher proportion of expressing cells. Similar constructs, in which cDNAs were exchanged by lacZ sequences, showed no variegation. Also, when a strongly active, nonvariegating construct was coinjected with K5Z, most transgenic lines showed an almost homogeneous lacZ expression. The comparison of transgene arrays of different copies inserted at the same locus (obtained by using a lox/Cre system) showed that the reduction of copy number does not lead to an increase in the proportion of cells that express the transgene. Finally, in most of the variegating or nonexpressing lines the transgenes were located both at intermediate positions and at peritelomeric regions in the long chromosome arms. These findings suggest that the probability and efficiency of expression of K5Z genes depend on both long range chromosomal influences and on sequences in the transgene array.

Ramirez, A; Milot, E; Ponsa, I; Marcos-Gutierrez, C; Page, A; Santos, M; Jorcano, J; Vidal, M



Construction and characterization of recombinant fowlpox virus co-expressing F and HN genes of newcastle disease virus and gB gene of infectious larygnotracheitis virus  

Microsoft Academic Search

The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus

Hui-Ling SUN; Yun-Feng WANG; De-Yuan MIAO; Pei-Jun ZHANG; Hai-Dong ZHI; Ling-Long XU; Mei WANG; Guang-Zhi TONG; Ming WANG



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

SciTech Connect

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.

Hoffmann, G.R. [Holy Cross College, Worcester, MA (United States); Janel-Bintz, R.; Fuchs, R.P.P. [CNRS, Strasbourg (France)



Blue\\/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene ( phoZ) expression  

Microsoft Academic Search

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems

D. O Chaffin; C. E Rubens



Magnetic Resonance Reporter Gene Imaging  

PubMed Central

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.

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



Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage.  


Bruton's tyrosine kinase (Btk) is a cytoplasmic protein kinase that is defective in X-linked agammaglobulinaemia in man and in X-linked immunodeficiency in the mouse. There is controversy regarding the stages of B cell development that are dependent on Btk function. To determine the point in B cell differentiation at which defects in Btk become apparent, we generated a mouse model by inactivating the Btk gene through an in-frame insertion of a lacZ reporter by homologous recombination in embryonic stem cells. The phenomenon of X-chromosome inactivation in Btk+/- heterozygous female mice enabled us to evaluate the competition between B cell progenitors expressing wild-type Btk and those expressing the Btk-/lacZ allele in each successive step of development. Although Btk was already expressed in pro-B cells, the first selective disadvantage only became apparent at the transition from small pre-B cells to immature B cells in the bone marrow. A second differentiation arrest was found during the maturation from IgD(low)IgM(high) to IgD(high)IgM(low) stages in the periphery. Our results show that Btk expression is essential at two distinct differentiation steps, both past the pre-B cell stage. PMID:8890160

Hendriks, R W; de Bruijn, M F; Maas, A; Dingjan, G M; Karis, A; Grosveld, F



Recombinant fowlpox virus for in vitro gene delivery to pancreatic islet tissue  

Microsoft Academic Search

The feasibility of using avipox virus as a vector for gene delivery to islet tissue (adult islets and fetal proislets) was examined using a recombinant fowlpox virus (FPV) engineered to express the reporter gene LacZ (FPV-LacZ). The efficiency of in vitro transduction was dose-dependent and influenced by the donor species and maturation status of the islet tissue. Reporter gene expression

Michelle F Solomon; Ian A Ramshaw; Charmaine J Simeonovic



Directed Chromosomal Integration and Expression of the Reporter Gene gusA3 in Lactobacillus acidophilus NCFM ?  

PubMed Central

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 ?-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 ?-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-?-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.

Douglas, Grace L.; Klaenhammer, Todd R.



Dual 19F/1H MR gene reporter molecules for in vivo detection of ?-galactosidase  

PubMed Central

Increased emphasis on personalized medicine and novel therapies require the development of non-invasive strategies for assessing biochemistry in vivo. The detection of enzyme activity and gene expression in vivo is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool since it is non-invasive, and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual 1H/19F nuclear magnetic resonance (NMR) lacZ gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice in vivo. Substrates show a single 19F NMR signal and exposure to ?-galactosidase induces a large 19F NMR chemical shift response. In the presence of ferric ions the liberated aglycone generates intense proton MRI T2 contrast. The dual modality approach allows both the detection of substrate and imaging of product enhancing the confidence in enzyme detection.

Yu, Jian-Xin; Kodibagkar, Vikram D.; Hallac, Rami R.; Liu, Li; Mason, Ralph P.



A new system to place single copies of genes, sites and lacZ fusions on the Escherichia coli chromosome 1 Published in conjunction with A Wisconsin Gathering Honoring Waclaw Szybalski on the occasion of his 75th year and 20 years of the Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA. 1 2 By acceptance of this article, the publisher or recipient acknowledges the right of the US Government and its agents and contractors to retain a non-exclusive, royalty-free license in and to any copyright covering the article. 2  

Microsoft Academic Search

To place a single-copy lacZ fusion on the E. coli chromosome, a method was developed based on in vivo homologous DNA recombination through P1 transduction. The fusions, initially constructed on plasmids, are crossed to ?lacZ fusion vectors which are then lysogenized at the chromosomal ?att site. The features of the new system are: (1) ? lysogens carrying the fusion are

Daiguan Yu; Donald L Court



LacZ ?-galactosidase: structure and function of an enzyme of historical and molecular biological importance.  


This review provides an overview of the structure, function, and catalytic mechanism of lacZ ?-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 ?-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 ?-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

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



Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes. Expression from reporter genes does not always reflect functional mRNA levels.  


Induction of beta-galactosidase from high copy-number plasmids was found to reduce the synthesis of other cellular proteins in Escherichia coli. The reduction depends on the protein in question and on the induction level of the beta-galactosidase. It could be observed transiently within one minute after induction and in some cases also during steady-state induction. Our interpretation is that the concentration of the free ribosomal subunits decreases after induction, leading to an increased competition among the individual ribosome binding sites for ribosomes. The immediate reduction in the synthesis individual proteins after induction of beta-galactosidase was used as an assay to measure in vivo the efficiency of a ribosome binding site. These efficiencies were compared to the calculated affinities between the ribosome binding site of specific mRNA species and the 3' end of 16 S RNA. For several mRNAs with similar Shine-Dalgarno sequences, the sensitivity to competition differed twofold. Our results show, that both transiently during induction of lacZ and also at very high steady-state expression levels, the expression from reporter genes, including the lacZ gene itself, does not reflect the levels of the mRNAs in a simple way. PMID:7685825

Vind, J; Sřrensen, M A; Rasmussen, M D; Pedersen, S



Multi-Scale Molecular Photoacoustic Tomography of Gene Expression  

PubMed Central

Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using lacZ, one of the most widely used reporter genes in biology. We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ?1.0 mm and ?0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 µm and an axial resolution of 15 µm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.

Krumholz, Arie; Guo, Zijian; Erpelding, Todd N.; Zhang, Chi; Zhang, Yu; Xia, Younan; Wang, Lihong V.



Radionuclide reporter gene imaging for cardiac gene therapy  

Microsoft Academic Search

Introduction  In the field of cardiac gene therapy, angiogenic gene therapy has been most extensively investigated. The first clinical trial\\u000a of cardiac angiogenic gene therapy was reported in 1998, and at the peak, more than 20 clinical trial protocols were under\\u000a evaluation. However, most trials have ceased owing to the lack of decisive proof of therapeutic effects and the potential\\u000a risks

Masayuki Inubushi; Nagara Tamaki



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

PubMed Central

Previous studies identified Inka1 as a gene regulated by AP-2? 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-2? 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.

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



Generation of Nkx2.2:lacZ mice using recombination-mediated cassette exchange technology.  


Nkx2.2 encodes a homeodomain transcription factor required for the correct specification and/or differentiation of cells in the pancreas, intestine, and central nervous system (CNS). To follow the fate of cells deleted for Nkx2.2 within these tissues, we generated Nkx2.2:lacZ knockin mice using a recombination-mediated cassette exchange (RMCE) approach. Expression analysis of lacZ and/or ?-galactosidase in Nkx2.2(lacZ/+) heterozygote embryos and adults demonstrates that lacZ faithfully recapitulates endogenous Nkx2.2 expression. Furthermore, the Nkx2.2(lacZ/lacZ) homozygous embryos display phenotypes indistinguishable from the previously characterized Nkx2.2(-/-) strain. LacZ expression analyses in the Nkx2.2(lacZ/lacZ) homozygous embryos indicate that Nkx2.2-expressing progenitor cells within the pancreas are generated in their normal numbers and are not mislocalized within the pancreatic ductal epithelium or developing islets. In the CNS of Nkx2.2(lacZ/lacZ) embryos, LacZ-expressing cells within the ventral P3 progenitor domain display different migration properties depending on the developmental stage and their respective differentiation potential. PMID:22539496

Arnes, Luis; Leclerc, Kevin; Friel, Jessica M; Hipkens, Susan B; Magnuson, Mark A; Sussel, Lori



Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase  

Microsoft Academic Search

We have analyzed the ability of a recombinant replication-defective adenovirus to transfer the thymidine kinase gene of herpes simplex virus (HSV-tk) into hepatocellular carcinoma (HCC) cells to confer sensitivity to ganciclovir. Three HCC cell lines (Hep3B, PLC\\/PRF\\/5, and HepG2) were efficiently infected in vitro by a recombinant adenovirus carrying lacZ reporter gene (AdCMV1acZ). Expression of HSV-tk in HCC cells infected

Cheng Qian; Roberto Bilbao; Oscar Bruńa; Jesus Prieto



PhaF, a Polyhydroxyalkanoate-Granule-Associated Protein of Pseudomonas oleovorans GPo1 Involved in the Regulatory Expression System for pha Genes  

Microsoft Academic Search

The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudo- monas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence




A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions  

SciTech Connect

We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in beta'-galactosidase. Each strain is Lac- and reverts to Lac+ only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac- to Lac+ frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains.

Cupples, C.G.; Miller, J.H.



The mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons.  

PubMed Central

Mutants of the mec-7 beta-tubulin gene of Caenorhabditis elegans lack the large diameter 15-protofilament microtubules normally found only in the set of six touch receptor neurons. Both a mec-7-lacZ reporter gene and affinity-purified anti-mec-7 antibodies were used to show that mec-7 is expressed primarily in the touch neurons. These data are consistent with a possible instructive role for the mec-7 tubulin in determining microtubule protofilament number. The antibodies and the mec-7-lacZ transgene were also used to examine mec-7 expression in mutants affecting the generation, differentiation or maintenance of the touch neurons. Decreased expression was observed in mutants of unc-86 and mec-3, genes that encode transcription factors essential for touch receptor neuron generation and differentiation, respectively. Images

Hamelin, M; Scott, I M; Way, J C; Culotti, J G



Imaging of gene expression in vivo with photoacoustic tomography  

NASA Astrophysics Data System (ADS)

In the post-genomic era, there is an increasing interest in visualizing the expression of functional genes in vivo. With the assistance of the reporter gene technique, various imaging modalities have been adopted for this purpose. In vivo gene expression imaging promises to provide biologists with a powerful tool for deepening our understanding of developmental biology, expanding our knowledge of the genetic basis of disease, and advancing the development of medicine. In this paper, we demonstrate the feasibility of imaging gene expression with photoacoustic imaging, which offers unique absorption contrast with ultrasonic resolution in vivo. We mark tumors in rats with the lacZ reporter gene. The lacZ gene encodes an enzyme ?-galactosidase, which yields a dark blue product when acting on a colorimetric assay called X-gal. Photoacoustic tomography at 650nm clearly visualizes the presence of this blue product. The spectroscopic method can also potentially improve specificity. Considering how many staining methods are used in traditional biology, we believe that photoacoustic techniques will revolutionize the field of molecular imaging. The further development of reporter gene systems with high absorbing products in the NIR region is needed.

Li, Li; Zemp, Roger J.; Lungu, Gina; Stoica, George; Wang, Lihong V.



Regulation of nod gene expression in Bradyrhizobium japonicum  

Microsoft Academic Search

The best inducers of nod:: lacZ translational fusions in Bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein. Upstream of the nodABC genes in B. japonicum is a novel gene, nodY, which is coregulated with nodABC. Measurements of the activity of lacZ fusions to the nodD gene of B. japonicum show that this gene is inducible by soybean seed extract and

Zsofia Banfalvi; Anthony Nieuwkoop; Maria Schell; Linda Besl; Gary Stacey



Seeing the light: luminescent reporter gene assays.  


The luminescent reporter gene assay (LRGA) is arguably the most prominent type of reporter gene assay used in biomolecular and pharmaceutical development laboratories. Part of this popularity is due to the high signal associated with luciferases, the foundation of this technology. This feature makes them ideally suited for high throughput screening applications where potentially millions of chemical compounds can be analyzed in a given assay. Recent technical advancements that enhance signal stability of the luciferases along with development and commercialization of multiple forms of luciferases, their respective substrates, and improvements in expression vectors for reporter gene assay (RGA) applications have broadened their use. While the practical challenges related to the application of luminescent technology in a laboratory setting have been overcome, there remains much to do in laying a systematic approach towards the construction of RGAs, which are essential to the elucidation of the basic biology for genes of interest. This mini-review aims at giving a birds-eye view of the available luciferases, substrates and other luminescent technologies available and provides a general blueprint as well as practical considerations for constructing and interfacing RGAs with chemical biology and functional genomics for the elucidation of fundamental biological questions and for biomedical research. PMID:21564017

Miraglia, Loren J; King, Frederick J; Damoiseaux, Robert



The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals  

Microsoft Academic Search

Summary Genes acrAB encode a multidrug efflux pump in Escherichia coli. We have previously reported that transcription of acrAB is increased under general stress conditions (i.e. 4% ethanol, 0.5 M NaCl, and the stationary phase in Luria-Bertani medium). In this study, lacZ transcriptional fusions and an in vitro gel mobility shift assay have been utilized to study the mechanisms governing

Dzwokai Ma; Marie Alberti; Christy Lynch; Hiroshi Nikaido; John E. Hearst



Mutational spectrum of bleomycin in lacZ mouse kidney: a possible model for mutational spectrum of reactive oxygen species  

Microsoft Academic Search

The mutational spectrum of bleomycin was compared with the spontaneous mutational spectrum in lacZ mouse kidney. Mice were treated with four 20mg\\/kg of doses of bleomycin over a two-week period, leading to a mutant fraction several times greater than that of controls. The major class of bleomycin-induced mutations consisted of small deletions, in particular ?1 deletions at AT base pairs

Joseph B. Guttenplan; Michael Khmelnitsky; Roderick Haesevoets; Wieslawa Kosinska



A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli.  


Temperature-sensitive (TS) mutants of a gene are ones of which the activity or phenotype is very similar to that of wild type only at certain temperature and they provide extremely powerful tool for studying protein function in vivo. Here we report a novel strategy to generate TS phenotype of the interest gene in Escherichia coli based on a temperature-sensitive T7-expression system. A TS T7-RNA polymerase is generated by interrupting it with a TS intein from Saccharomyces cerevisiae vacuolar ATPase subunit (VMA), resulting that the gene flanked by T7-promoter and T7-terminator will be transcribed only at the permissive temperature (18 degrees C), not at the restrictive temperature (37 degrees C). The feasibility to create TS phenotype of this strategy was detected using lacZ as target. Reverse transcriptase polymerase chain reaction (PCR) indicated that at 18 degrees C, transcripts of T7-promoter controlled lacZ were at least 85 times more than those at 37 degrees C. Western blot analysis and enzymatic assay showed that large amounts of active His6-tagged LacZ produced at 18 degrees C but little at 37 degrees C. This strategy appears more promising than other TS creation methods because the target is pre-designed, no modification is introduced, and only simple DNA manipulation is required. PMID:17169451

Liang, Rubing; Liu, Xipeng; Liu, Jianhua; Ren, Qiushi; Liang, Peiji; Lin, Zhixin; Xie, Xiangming



Specific Targeting of Gene Expression to a Subset of Human Trabecular Meshwork Cells Using the Chitinase 3-Like 1 Promoter  

PubMed Central

Purpose To compare the gene expression profile of trabecular meshwork (TM) and Schlemm’s canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway. Methods The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5? promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ. Results ?-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments. Conclusions Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.

Liton, Paloma B.; Liu, Xialin; Stamer, W. Daniel; Challa, Pratap; Epstein, David L.; Gonzalez, Pedro



Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation.  

PubMed Central

The fusogenic activities of enveloped-virus glycoproteins were analyzed by using a quantitative, sensitive, rapid, and highly versatile recombinant vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. One population uniformly expressed vaccinia virus-encoded viral glycoproteins mediating specific binding and fusion activities; the other expressed the corresponding cellular receptor(s). The cytoplasm of one population also contained vaccinia virus-encoded bacteriophage T7 RNA polymerase; the cytoplasm of the other contained a transfected plasmid with the Escherichia coli lacZ gene linked to the T7 promoter. When the two populations were mixed, cell fusion resulted in activation of the LacZ gene in the cytoplasm of the fused cells; beta-galactosidase activity was assessed by colorimetric assay of detergent cell lysates or by in situ staining. We applied this approach to study the human immunodeficiency virus type 1 envelope glycoprotein (Env)-CD4 interaction. Beta-Galactosidase was detected within 1 h after cell mixing and accumulated over the next several hours. Cell fusion dependence was demonstrated by the strict requirement for both CD4 and functional Env expression and by the inhibitory effects of known fusion-blocking monoclonal antibodies and pharmacological agents. Quantitative measurements indicated much higher sensitivity compared with analysis of syncytium formation. The assay was used to probe mechanisms of the cell type specificity for Env-CD4-mediated fusion. In agreement with known restrictions, cell fusion occurred only when CD4 was expressed on a human cell type. Membrane vesicle transfer experiments indicated that CD4 initially produced in either human or nonhuman cells was functional when delivered to human cells, suggesting that the fusion deficiency with nonhuman cells was not associated with irreversible defects in CD4. We also demonstrated that the infectivity specificities of different human immunodeficiency virus type 1 isolates for peripheral blood lymphocytes versus continuous CD4+ cell lines were associated with corresponding fusion selectivities of the respective recombinant Env proteins. The assay enabled analysis of the fusogenic activity of the fusion glycoprotein/hemagglutinin-neuraminidase of the paramyxovirus simian virus 5. This system provides a powerful tool to study fusion mechanisms mediated by enveloped-virus glycoproteins, as well as to screen fusion-blocking antibodies and pharmacological agents. Images

Nussbaum, O; Broder, C C; Berger, E A



Evaluation of the Staphylococcus aureus class C nonspecific acid phosphatase (SapS) as a reporter for gene expression and protein secretion in gram-negative and gram-positive bacteria.  


A phosphatase secreted by Staphylococcus aureus strain 154 has previously been characterized and classified as a new member of the bacterial class C family of nonspecific acid phosphatases. As the acid phosphatase activity can be easily detected with a cost-effective plate screen assay, quantitatively measured by a simple enzyme assay, and detected by zymography, its potential use as a reporter system was investigated. The S. aureus acid phosphatase (sapS) gene has been cloned and expressed from its own regulatory sequences in Escherichia coli, Bacillus subtilis, and Bacillus halodurans. Transcriptional and translational fusions of the sapS gene with selected heterologous promoters and signal sequences were constructed and expressed in all three of the host strains. From the range of promoters evaluated, the strongest promoter for heterologous protein production in each of the host strains was identified, i.e., the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis, and the B. halodurans sigma(D) promoter in B. halodurans. This is the first report on the development of a class C acid phosphatase gene as a reporter gene with the advantage of being able to function in both gram-positive and gram-negative host strains. PMID:17905879

du Plessis, Erika; Theron, Jacques; Berger, Eldie; Louw, Maureen



Use of Genetic Recombination as a Reporter of Gene Expression  

Microsoft Academic Search

An understanding of the patterns of gene expression in response to specific environmental signals can yield insight into a variety of complex biological systems such as microbial-host interactions, developmental cycles, cellular differentiation, ontogeny, etc. To extend the utility of the reporter gene fusion approach to such studies, we have constructed a gene expression reporter cassette that permits the generation of

Andrew Camilli; David T. Beattie; John J. Mekalanos



Human reporter genes: potential use in clinical studies.  


The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies; (c) and assessments of endogenous molecular events using different inducible reporter gene imaging systems. PMID:17921031

Serganova, Inna; Ponomarev, Vladimir; Blasberg, Ronald



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


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

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



Production of LacZ Inducible T Cell Hybridoma Specific for Human and Mouse gp10025–33 Peptides  

PubMed Central

Identification and quantification of immunogenic peptides and tumor-derived epitopes presented on MHC-I molecules are essential for basic studies and vaccines generation. Although lymphocytes derived from transgenic mice can serve as sensitive detectors of processes of antigen presentation and recognition, they are not always available. The use of cell lines might be extremely useful. In this study, we generated a lacZ inducible CD8+ hybridoma (BUSA14) capable of recognizing both human and mouse gp10025–33 melanoma antigens presented on dendritic and tumor cell lines. This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNF?. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface.

Tzehoval, Esther; Eisenbach, Lea



Imaging hNET Reporter Gene Expression with  

Microsoft Academic Search

The norepinephrine transporter (NET) has recently been sug- gested as a useful reporter gene. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked hNET-green fluorescent protein (GFP) hybrid reporter gene for both nuclear and optical imaging. Methods: A retroviral vector pQCXhNET-IRES-GFP was constructed and used to generate several reporter cell lines and xenografts. Transduced cells were

Maxim A. Moroz; Inna Serganova; Pat Zanzonico; Ludmila Ageyeva; Tatiana Beresten; Ekaterina Dyomina; Eva Burnazi; Ronald D. Finn; Michael Doubrovin; Ronald G. Blasberg


Efficient gene delivery to pig airway epithelia and submucosal glands using helper-dependent adenoviral vectors.  


Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR). Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e127; doi:10.1038/mtna.2013.55; published online 8 October 2013. PMID:24104599

Cao, Huibi; Machuca, Tiago N; Yeung, Jonathan C; Wu, Jing; Du, Kai; Duan, Cathleen; Hashimoto, Kohei; Linacre, Virginia; Coates, Allan L; Leung, Kitty; Wang, Jian; Yeger, Herman; Cutz, Ernest; Liu, Mingyao; Keshavjee, Shaf; Hu, Jim



Use of genetic recombination as a reporter of gene expression.  


An understanding of the patterns of gene expression in response to specific environmental signals can yield insight into a variety of complex biological systems such as microbial-host interactions, developmental cycles, cellular differentiation, ontogeny, etc. To extend the utility of the reporter gene fusion approach to such studies, we have constructed a gene expression reporter cassette that permits the generation of transcriptional fusions to tnpR encoding resolvase, a site-specific recombinase of the transposable element gamma delta. Induction of the transcriptional fusions results in production of resolvase, which in turn, catalyzes excision of a linked tetracycline-resistance reporter gene flanked by direct repeats of res, the DNA sequences at which resolvase functions. The loss of tetracycline resistance in descendant bacteria serves as a permanent and heritable marker of prior gene expression. This gene fusion approach will allow us to assay the induction of gene expression in as few as one cell. Additionally, gene expression can be assayed at a later time and/or different place from the inducing environment facilitating the study of gene expression in complex environments such as animal tissues. PMID:8146167

Camilli, A; Beattie, D T; Mekalanos, J J



Cloning-free regulated monitoring of reporter and gene expression  

PubMed Central

Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

al-Haj, Latifa; Al-Ahmadi, Wijdan; Al-Saif, Maher; Demirkaya, Omer; Khabar, Khalid SA



tRNA genes protect a reporter gene from epigenetic silencing in mouse cells  

PubMed Central

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.

Ebersole, Thomas; Kim, Jung-Hyun; Samoshkin, Alexander; Kouprina, Natalay; Pavlicek, Adam; White, Robert J



In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart.  


High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models. PMID:12960971

Iwatate, M; Gu, Y; Dieterle, T; Iwanaga, Y; Peterson, K L; Hoshijima, M; Chien, K R; Ross, J



Effect of SNPs in protein kinase Cz gene on gene expression in the reporter gene detection system  

Microsoft Academic Search

AIM: To investigated the effects of the SNPs (rs411021, rs436045, rs427811, rs385039 and rs809912) on gene expression and further identify the susceptibility genes of type 2 diabetes. METHODS: Ten allele fragments (49 bp each) were synthesized according to the 5 SNPs mentioned above. These fragments were cloned into luciferase reporter gene vector and then transfected into HepG2 cells. The activity

Zhuo Liu; Hong-Xia Sun; Yong-Wei Zhang; Yun-Feng Li; Jin Zuo; Yan Meng; Fu-De Fang



Multifactorial induction of an orphan PKS-NRPS gene cluster in Aspergillus terreus.  


Mining the genome of the pathogenic fungus Aspergillus terreus revealed the presence of an orphan polyketide-nonribosomal-peptide synthetase (PKS-NRPS) gene cluster. Induced expression of the transcriptional activator gene adjacent to the PKS-NRPS gene was not sufficient for the activation of the silent pathway. Monitoring gene expression, metabolic profiling, and using a lacZ reporter strain allowed for the systematic investigation of physiological conditions that eventually led to the discovery of isoflavipucine and dihydroisoflavipucine. Phytotoxin formation is only activated in the presence of certain amino acids, stimulated at alkaline pH, but strictly repressed in the presence of glucose. Global carbon catabolite repression by CreA cannot be abolished by positive-acting factors such as PacC and overrides the pathway activator. Gene inactivation and stable isotope labeling experiments unveiled the molecular basis for flavipucine/fruit rot toxin biosynthesis. PMID:21236704

Gressler, Markus; Zaehle, Christoph; Scherlach, Kirstin; Hertweck, Christian; Brock, Matthias



pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans.  


Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene. pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance. Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption. PMID:11982330

D'Haeze, Wim; Verplancke, Christa; Mironov, Vladimir; Holsters, Marcelle



Angiographically guided utero-placental gene transfer in rabbits with adenoviruses, plasmid\\/liposomes and plasmid\\/polyethyleneimine complexes  

Microsoft Academic Search

We examined the feasibility of gene transfer to rabbit placenta using adenoviruses, plasmid\\/liposomes and plasmid\\/polyethyleneimine (PEI) complexes. Pregnant New Zealand White rabbits (n = 17) were anesthetized and local gene transfer was done via a catheter inserted in uterine arteries under direct angiographic control. Either nuclear targeted LacZ adenoviruses (1.0 × 1010 p.f.u.), nuclear targeted LacZ plasmid (500 ?g)\\/liposome (DOTMA:DOPE

A Heikkilä; M O Hiltunen; M P Turunen; L Keski-Nisula; A-M Turunen; H Räsänen; T T Rissanen; V-M Kosma; H Manninen; S Heinonen; S Ylä-Herttuala



Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene  

Microsoft Academic Search

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and

Markus Fuhrmann; Amparo Hausherr; Lars Ferbitz; Thomas Schödl; Markus Heitzer; Peter Hegemann



Marker and reporter genes: illuminating tools for environmental microbiologists  

Microsoft Academic Search

Fluorescent and luminescent marker and reporter genes provide easily detectable phenotypes to microbial cells and are therefore valuable tools for the study of microorganisms in the environment. Although these tools are becoming widely adopted, there are still issues that remain to be solved, such as the dependence of the reporter output on the physiological status of the cell. Eventually it

Janet K Jansson



Noninvasive Monitoring of Target Gene Expression by Imaging Reporter Gene Expression in Living Animals Using Improved Bicistronic Vectors  

Microsoft Academic Search

Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this

Yanling Wang; Meera Iyer; Alexander J. Annala; Steve Chappell; Vincent Mauro; Sanjiv S. Gambhir


Chlorpyrifos-Induced hsp70 Expression and Effect on Reproductive Performance in Transgenic Drosophila melanogaster ( hsp70 - lacZ ) Bg 9  

Microsoft Academic Search

Expression of hsp70 in the third-instar larval tissues of transgenic Drosophila melanogaster (hsp70-lacZ) following dietary exposure to organophosphate insecticide chlorpyrifos for various time intervals was investigated. Effect\\u000a of the chemical on different developmental stages of the fly was also evaluated by looking at survivorship, hatchability,\\u000a emergence, fecundity, fertility, and reproductive performance. Our results showed that the toxicant evokes profound cytotoxic

A. Nazir; I. Mukhopadhyay; D. K. Saxena; D. Kar Chowdhuri



Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction.  


We reported previously that predelivery of heme oxygenase-1 (HO-1) gene to the heart by adeno-associated virus-2 (AAV-2) markedly reduces ischemia and reperfusion (I/R)-induced myocardial injury. However, the effect of preemptive HO-1 gene delivery on long-term survival and prevention of postinfarction heart failure has not been determined. We assessed the effect of HO-1 gene delivery on long-term survival, myocardial function, and left ventricular (LV) remodeling 1 yr after myocardial infarction (MI) using echocardiographic imaging, pressure-volume (PV) analysis, and histomorphometric approaches. Two groups of Lewis rats were injected with 2 x 10(11) particles of AAV-LacZ (control) or AAV-human HO-1 (hHO-1) in the anterior-posterior apical region of the LV wall. Six weeks after gene transfer, animals were subjected to 30 min of ischemia by ligation of the left anterior descending artery followed by reperfusion. Echocardiographic measurements and PV analysis of LV function were obtained at 2 wk and 12 mo after I/R. One year after acute MI, mortality was markedly reduced in the HO-1-treated animals compared with the LacZ-treated animals. PV analysis demonstrated significantly enhanced LV developed pressure, elevated maximal dP/dt, and lower end-diastolic volume in the HO-1 animals compared with the LacZ animals. Echocardiography showed a larger apical anterior-to-posterior wall ratio in HO-1 animals compared with LacZ animals. Morphometric analysis revealed extensive myocardial scarring and fibrosis in the infarcted LV area of LacZ animals, which was reduced by 62% in HO-1 animals. These results suggest that preemptive HO-1 gene delivery may be useful as a therapeutic strategy to reduce post-MI LV remodeling and heart failure. PMID:17322421

Liu, Xiaoli; Simpson, Jeremy A; Brunt, Keith R; Ward, Christopher A; Hall, Sean R R; Kinobe, Robert T; Barrette, Valerie; Tse, M Yat; Pang, Stephen C; Pachori, Alok S; Dzau, Victor J; Ogunyankin, Kofo O; Melo, Luis G



Transformation of Nonselectable Reporter Genes in Marine Diatoms.  


: We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms. PMID:10383998

Falciatore; Casotti; Leblanc; Abrescia; Bowler



Reporter gene assays for algal-derived toxins.  


We have modified the cell-based directed cytotoxicity assay for sodium channel and calcium channel active phycotoxins using a c-fos-luciferase reporter gene construct. In this report we describe the conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method. N2A mouse neuroblastoma cells, which express voltage-dependent sodium channels, were stably transfected with the reporter gene c-fos-luc, which contains the firefly luciferase gene under the transcriptional regulation of the human c-fos response element. The characteristics of the N2A reporter gene assay were determined by dose response with brevetoxin and ciguatoxin. Brevetoxin-1 and ciguatoxin-1 induced c-fos-luc with an EC50 of 4.6 and 3.0 ng ml(-1), respectively. Saxitoxin caused a concentration-dependent inhibition of brevetoxin-1 induction of c-fos-luc with an EC50 of 3.5 ng ml(-1). GH4C1 rat pituitary cells, which lack voltage-dependent sodium channels but express voltage-dependent calcium channels, were also stably transfected with the c-fos-luc. GH4C1 cells expressing c-fos-luciferase were responsive to maitotoxin (1 ng ml(-1)) and a putative toxin produced by Pfiesteria piscicida. Although reporter gene assays are not designed to replace existing detection methods used to measure toxin activity in seafood, they do provide a valuable means to screen algal cultures for toxin activity, to conduct assay-guided fractionation and to characterize pharmacologic properties of algal toxins. PMID:11122538

Fairey, E R; Ramsdell, J S



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

SciTech Connect

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.

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



Organization and regulation of the mannopine cyclase-associated opine catabolism genes in Agrobacterium tumefaciens 15955.  

PubMed Central

We have isolated and characterized Tn3HoHo1- and Tn5-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR). Characterization of the transposon-induced lacZ fusion mutants by beta-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines. Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus. The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements. Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid. Analysis of beta-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source. The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen. This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability. Images

Hong, S B; Dessaux, Y; Chilton, W S; Farrand, S K



Engineering BAC reporter gene constructs for mouse transgenesis.  


A culmination of large-scale ideas and efforts has truly allowed for the use of large genomic DNA clones housed in Bacterial Artificial Chromosome (BAC) vectors for biological research. Fundamental advances that have allowed this to happen include (1) the completion of genome sequencing projects and the establishment of highly annotated web-accessible databases allowing for the rapid identity and purchase of BAC clones containing genes of interest. (2) The generation of methodologies to modify BACs genetically, allowing for the rapid creation of gene targeting constructs or transgenic reporter gene constructs using homologous recombination in bacteria.Recent efforts on our part have capitalized on these advances by using BACs and bacterial recombination methods to generate fluorescent protein reporter transgenic mice to study skeletal biology. The rationale for using BAC genomic DNA clones to engineer reporter gene constructs is based on their much larger size, thus increasing the likelihood that most, if not all, of a gene's respective cis regulator elements are present, giving a truer representation of the endogenous gene's expression. In a relatively short amount of time, we have become extremely proficient at generating BAC reporters. Contrary to the widely perceived notion that working with BACs is complex and difficult, we decided to write this chapter to encourage laboratories that are currently using traditional molecular cloning methods to engineer transgenic DNA constructs to strongly consider learning BAC methodologies. As an example, we walk through the steps we took to generate the transgenic reporter mouse line, Tenascin C (TNC)-mCherry. PMID:21080280

Fu, Yu; Maye, Peter



Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish ( Danio rerio ) Tissues  

Microsoft Academic Search

:   We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-?m microcarriers and 1 ?g of pCMVL plasmid DNA at a delivery pressure of 200 psi.\\u000a Time course studies revealed luciferase activity peaking at

Jacob Torgersen; Philippe Collas; Peter Aleström



Pleiotrophin gene therapy for peripheral ischemia: evaluation of full-length and truncated gene variants.  


Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model. PMID:23630585

Fang, Qizhi; Mok, Pamela Y; Thomas, Anila E; Haddad, Daniel J; Saini, Shereen A; Clifford, Brian T; Kapasi, Neel K; Danforth, Olivia M; Usui, Minako; Ye, Weisheng; Luu, Emmy; Sharma, Rikki; Bartel, Maya J; Pathmanabhan, Jeremy A; Ang, Andrew A S; Sievers, Richard E; Lee, Randall J; Springer, Matthew L



Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool  

Microsoft Academic Search

In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N2-fixation (nif) genes in response to molecular O2 and ammonium.We investigated the influence of membrane-bound oxidoreduc- tases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH˘- ˘lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in

Roman Grabbe; Ruth A. Schmitz



Bioluminescence Reporter Gene-Based Detection of MicroRNAs.  


MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that inhibit the expression of cognate genes in multicellular organisms. These small RNAs have been demonstrated to play crucial roles in a variety of biological processes including cell differentiation, proliferation, and survival. Knowledge of specific expression patterns of miRNAs is critical for functional studies. Here, we describe a bioluminescence reporter gene-based method to measure miRNA activity in cultured cells and mice using a Gaussia luciferase reporter gene controlled by miRNA binding sites in its 3'untranslated region. This method can be used to noninvasively monitor the expression patterns of functionally active miRNAs involved in different biological processes or diseases in mice. PMID:24166370

Ko, Hae Young; Lee, Young Sik; Kim, Soonhag



Positron emission tomography reporter gene imaging in the myocardium: for monitoring of angiogenic gene therapy in ischemic heart disease.  


Cardiac angiogenic gene therapy has emerged as a novel treatment approach for patients with intractable ischemic heart disease, aiming at facilitating neovascularization to augment blood flow in the ischemic myocardium by introducing genes encoding for angiogenic factors. While several clinical trials for cardiac angiogenic gene therapy are currently in progress, there remains a discrepancy between impressive preclinical results and their limited clinical findings. On the other hand, positron emission tomography (PET) reporter gene imaging has been developed to monitor expression of transgenes in vivo. PET reporter genes encode for proteins that retain complementary positron-emitting tracers (PET reporter probes), and theoretically any therapeutic gene can be linked and coexpressed with an appropriate PET reporter gene. Consequently, PET reporter gene imaging with a PET reporter probe affords external determination of the location, magnitude, and duration of expression of therapeutic genes noninvasively. Since PET imaging can be performed in various species ranging from mice to humans, in vivo cardiac PET reporter gene imaging could play a critical role in identifying the "missing link" as a powerful translational research tool. In this article, we discuss the role of PET reporter gene imaging in basic and clinical research on cardiac angiogenic gene therapy. PMID:16305630

Inubushi, Masayuki; Tamaki, Nagara


Genetics in methylotrophic bacteria: Appendix. Final report  

SciTech Connect

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.

Lidstrom, M.E.



Use of reporter genes to measure xenobiotic-mediated activation of CYP gene transcription.  


Transcriptional activation of CYP gene expression by xenobiotics may have fundamental effects on body physiology. It may result in the altered pharmacokinetics of other chemicals in the body, both xenobiotic and endogenous substrates, potentially altering their effects. This may often result in no observable clinical effect, but in a significant number of cases these interactions lead to altered physiology or failure of a therapeutic drug. It is therefore important to be able to screen novel chemical entities for their ability to activate CYP gene expression. In addition, through mechanistic studies of how such transcriptional activation occurs, the ability to predict and avoid such potential interactions is improved. Reporter gene assays provide a simple, high-throughput methodology for examining the transcriptional activation of CYP gene expression by xenobiotics. They are suitable for use in screening as well as mechanistic studies and are of use in both the drug discovery/development and research arenas. PMID:16719405

Plant, Nick



Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system.  


Differentiation of a specific organ or tissue requires sequential activation of regulatory genes. However, little is known about how serial gene expression is temporally regulated. Here, we present evidence that differential expression of single-minded (sim) target genes can be attributed, in part, to the number of Sim and Tango (Tgo) heterodimer binding sites within their enhancer regions. The Sim, termed a master regulator, directs ventral midline differentiation of Drosophila central nervous system (CNS). According to data on the onset timing of ventral midline gene expression, sim target genes are classified into at least 2 groups (early and late). The sim and rhomboid (rho) genes are activated during early midline differentiation whereas orthodenticle (otd), CG10249, and slit (sli) genes undergo activation during later stages of midline differentiation. Germline transformation and in situ hybridization with transgenic embryos demonstrate that enhancers activating sim and rho expression contain 4 Sim-Tgo binding sites whereas only 1 Sim-Tgo binding site is found in an enhancer of sli. A mutagenized version of the rho enhancer lacking either 1, 2, or 3 Sim-Tgo binding sites mediated progressively more delayed expression of a lacZ reporter gene in the ventral midline. In contrast, a modified sli enhancer displayed progressively earlier onset of lacZ expression when 1, 2, or 3 more Sim-Tgo binding sites were added. Taken together, these results suggest that the number of Sim-Tgo-binding sites is decisive in determining the timing of gene expression in the developing ventral midline. We also discuss a combinatorial model accounting for the sequential expression of sim target genes. PMID:23701883

Hong, Joung-Woo; Park, Kye Won; Levine, Michael S



The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium  

PubMed Central

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3? and pUGiGM3? contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3? and pUMiGM3? contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3? untranslated region. In pUGGM3? and pUMGM3?, the promoters were fused directly with egfp, whereas in pUGiGM3? and pUMiGM3?, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5? end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5? of the egfp gene (pUGiGM3? and pUMiGM3?) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5? intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5? intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3? paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.

Ma, Biao; Mayfield, Mary B.; Gold, Michael H.



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...


Involvement of Snf7p and Rim101p in the transcriptional regulation of TIR1 and other anaerobically upregulated genes in Saccharomyces cerevisiae.  


Despite the scientific and applied interest in the anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is upregulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically upregulated TIR1 gene was fused to lacZ revealed a loss of anaerobic upregulation in an snf7Delta mutant. Anaerobic upregulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p. Analysis of lacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic upregulation of TIR1 involved Rim101p. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, could not totally elucidate the TIR1 regulation, suggesting the involvement of a more complex regulation network. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p. Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited an Snf7p/Rim101p-dependent anaerobic upregulation, among which, besides TIR1, are four other anaerobic genes SML1, MUC1, AAC3 and YBR300C. These results provide new evidence on the implication of the Rim101p cascade in the transcriptional regulation of anaerobic metabolism in S. cerevisiae. PMID:20402793

Snoek, Ishtar S I; Tai, Siew L; Pronk, Jack T; Yde Steensma, H; Daran, Jean-Marc



Research report Rat B50 gene transcription and translation  

Microsoft Academic Search

Previously we reported that the rat B-50\\/GAP-43 gene contains two promoters (P1 and P2). This study describes the contribution of these two promoters to the mRNA population in several paradigms leading to an altered B-50 mRNA expression. In 8-day-old rat brain we found that P1 transcripts (1676 + 50 nt) account for 5% and P2 transcripts (1462 + 46 nt)

Bart J. L. Eggen; Dieta Brandsma; Willem Hendrik Gispen; Loes H. Schrama


Duplicated Proteasome Subunit Genes in Drosophila Melanogaster Encoding Testes-Specific Isoforms  

PubMed Central

Using the previously cloned proteasome ?-type subunit gene Pros28.1, we screened a Drosophila melanogaster genomic library using reduced stringency conditions to identify closely related genes. Two new genes, Pros28.1A (map position 92F) and Pros28.1B (map position 60D7), showing high sequence similarity to Pros28.1, were identified and characterized. Pros28.1A encodes a protein with 74% amino acid identity to PROS28.1, while the Pros28.1B gene product is 58% identical. The Pros28.1B gene has two introns, located in exactly analogous positions as the two introns in Pros28.1, while the Pros28.1A gene lacks introns. Northern blot analysis reveals that the two new genes are expressed only in males, during the pupal and adult stages. Tissue-specific patterns of expression were examined using transgenic flies carrying lacz-fusion reporter genes. This analysis revealed that both genes are expressed in germiline cells during spermatogenesis, although their expression patterns differed. Pros28.1A expression is first detected at the primary spermatocyte stage and persists into the spermatid elongation phase of spermiogenesis, while Pros28.1B expression is prominent only during spermatid elongation. These genes represent the most striking example of cell-type-specific proteasome gene expression reported to date in any system and support the notion that there is structural and functional heterogeneity among proteasomes in metazoans.

Yuan, X.; Miller, M.; Belote, J. M.



Highly efficient regulation of gene expression by tetracycline in a replication-defective herpes simplex viral vector.  


Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases. PMID:16574491

Yao, Feng; Theopold, Christoph; Hoeller, Daniela; Bleiziffer, Oliver; Lu, Zheming



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

PubMed Central

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 SM22? promoter, containing ?-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 SM22? (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.

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



Antagonism of platelet-derived growth factor by perivascular gene transfer attenuates adventitial cell migration after vascular injury: new tricks for old dogs?  

Microsoft Academic Search

Migration of adventitial fibroblasts con- tributes to vascular remodeling after angioplasty. This study has used perivascular gene transfer of a truncated platelet-derived growth factor PDGF receptor (PDG- FXR) to investigate whether antagonism of PDGF sig- naling alters adventitial cell migration after balloon injury in rat carotid arteries. Adenoviruses coordinating expression of -galactosidase (LacZ) and PDGFXR or LacZ and green fluorescent

Chandike M. Mallawaarachchi; Peter L. Weissberg; Richard C. M. Siow



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


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.

Gambhir; Sanjiv (Portola Valley, CA), Pritha; Ray (Mountain View, CA)



Enhancement of Reporter Gene Detection Sensitivity by Insertion of Specific Mini-Peptide-Coding Sequences  

PubMed Central

Two important aspects for gene therapy are to increase the level of gene expression and track the gene delivery site and expression, and a sensitive reporter gene may be one of the options for preclinical studies and possibly for human clinical trials. We report the novel concept of increasing the activity of the gene products. With the insertion of the mini-peptide-coding sequence CWDDWLC into the plasmid DNA of a SEAP reporter gene, we observed vast increases in the enzyme activity in vitro in all murine and human cell lines used. Also, in vivo injection of this CWDDWLC-SEAP encoding gene resulted in the same increases in reporter gene activity, but these increases did not correspond to alterations in the level of the gene products in the serum. Minor sequence changes in this mini-peptide negate the activity increase of the reporter gene. We report the novel concept of increasing the activity of gene products as another method to improve the reporting sensitivity of reporter genes. This improved reporter gene could complement any improved vector for maximizing the reporter sensitivity. Also, this strategy has the potential to be used to discover peptides that improve the activity of therapeutic genes.

Cutrera, Jeffry; Dibra, Denada; Xia, Xueqing; Li, Shulin



rAAV Vectors as Safe and Efficient Tools for the Stable Delivery of Genes to Primary Human Chondrosarcoma Cells In Vitro and In Situ  

PubMed Central

Treatment of chondrosarcoma remains a major challenge in orthopaedic oncology. Gene transfer strategies based on recombinant adenoassociated viral (rAAV) vectors may provide powerful tools to develop new, efficient therapeutic options against these tumors. In the present study, we tested the hypothesis that rAAV is adapted for a stable and safe delivery of foreign sequences in human chondrosarcoma tissue by transducing primary human chondrosarcoma cells in vitro and in situ with different reporter genes (E. coli lacZ, firefly luc, Discosoma sp. RFP). The effects of rAAV administration upon cell survival and metabolic activities were also evaluated to monitor possibly detrimental effects of the gene transfer method. Remarkably, we provide evidence that efficient and prolonged expression of transgene sequences via rAAV can be safely achieved in all the systems investigated, demonstrating the potential of the approach of direct application of therapeutic gene vectors as a means to treat chondrosarcoma.

Madry, Henning; Venkatesan, Jagadeesh K.; Schmitt, Gertrud; Schetting, Sarah; Ekici, Myriam; Kohn, Dieter; Cucchiarini, Magali



Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells  

Microsoft Academic Search

Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured

Mark R. Soboleski; Jason Oaks; William P. Halford



Ganglioglioma associated with alterations of NBN gene. A case report.  


We report a case of a 13-year-old girl with a tumour of the right fronto-parietal region of the brain. The tumour consisted of two components: a well-differentiated astroglial component with Rosenthal fibres and a neoplastic neuronal component. The final histopathology established diagnosis of ganglioglioma WHO grade I. The patient was selected from a group of children with central nervous system (CNS) tumours screened for the most common molecular variants in the NBN gene (exons 5 and 6). Molecular analysis revealed the presence of c.511A>G (p.Ile171Val) substitution on one allele. This is the first patient with ganglioglioma and confirmed mutation in the NBN gene. PMID:19813148

Grajkowska, Wies?awa; Piekutowska-Abramczuk, Dorota; Ciara, Elzbieta; Dembowska-Baginska, Bozena; Perek, Danuta; Roszkowski, Marcin; Daszkiewicz, Pawel; Matyja, Ewa; Pronicki, Maciej; Chrzanowska, Krystyna H



Generation of Shox2-Cre allele for tissue specific manipulation of genes in the developing heart, palate, and limb.  


Shox2 is expressed in several developing organs in a tissue specific manner in both mice and humans, including the heart, palate, limb, and nervous system. To better understand the spatial and temporal expression patterns of Shox2 and to systematically dissect the genetic cascade regulated by Shox2, we created Shox2-LacZ and Shox2-Cre knock-in mouse lines. We show that the Shox2-LacZ allele expresses beta-galactosidase reporter gene in a fashion that recapitulates the endogenous Shox2 expression pattern in developing organs, including the sinoatrial node (SAN), the anterior portion of the palate, and the proximal region of the limb bud. Conditional deletion of Shox2 in mice carrying the Shox2-Cre allele yielded SAN phenotypes that resemble conventional Shox2 knockout mice. Our results indicate that the Shox2-Cre allele offer a useful tool for tissue specific manipulation of genes in a number of developing organs, particularly in the developing SAN. PMID:23620086

Sun, Cheng; Zhang, Tao; Liu, Chao; Gu, Shuping; Chen, YiPing



Photoacoustic molecular imaging of ferritin as a reporter gene  

NASA Astrophysics Data System (ADS)

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.

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



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

SciTech Connect

Progress is reported in gene transfer experiments using the soybean seed storage protein gene. The sequencing of gene Gmg ..cap alpha..' 17.1 has been completed. Several deletion mutants of this gene are being prepared for experiments to transfer the gene into the Ti-plasmid of Agrobacterium tumefaciens. The purpose is to determine which, if any, of the upstream sequences are those which regulate the developmental expression of the gene. (ACR)

Beachy, R.N.



Novel Fe3+-Based 1H MRI ?-Galactosidase Reporter Molecules**  

PubMed Central

There is increasing interest in the development of reporter agents to reveal enzyme activity in vivo using small animal imaging. We have previously demonstrated the feasibility of detecting lacZ gene activity using the commercially available 3,4-cyclohexenoesculetin-?-D-galactopyranoside (S-Gal™) as a 1H MRI reporter. Specifically, ?-galactosidase (?-gal) releases the aglycone, which forms an MR contrast-inducing paramagnetic precipitate in the presence of Fe3+. Contrast was primarily T2-weighted signal loss, but T1 effects were also observed. Since T1-contrast generally provides signal enhancement as opposed to loss, it appeared attractive to explore whether analogues could be generated with enhanced characteristics. We now report the design and successful synthesis of novel analogues together with characterization of 1H MRI contrast based on both T1 and T2 response to ?-gal activity in vitro for the lead agent.

Yu, Jian-Xin; Gulaka, Praveen K.; Liu, Li; Kodibagkar, Vikram D.; Mason, Ralph P.



Expression of a mouse metallothionein-Escherichia coli. beta. -galactosidase fusion gene (MT-. beta. gal) in early mouse embryos  

SciTech Connect

The authors have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli {beta}-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl {beta}-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. They observed staining indicative of exogenous {beta}-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO{sub 4}. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.

Stevens, M.E.; Meneses, J.J.; Pedersen, R.A. (Univ. of California, San Francisco (United States))



Comparison of bidirectional and bicistronic inducible systems for coexpression of connexin genes and fluorescent reporters.  


Gene expression studies often require inducible coexpression of both a gene of interest and a reporter gene. Fusion of fluorescent reporters can, however, modify protein structure and function. We have generated inducible expression systems for two connexin genes: Cx30 and Cx43. It has been reported recently that reporter fusion to connexins can modify their function. Therefore, we compared two methods of independent reporter coexpression and examined colocalization with induced connexin expression. Identical levels of connexin expression were observed for both the bidirectional and bicistronic expression systems. In contrast, however, reporter gene expression by the bidirectional promoter provided brighter average fluorescent pixel intensity than expression of a reporter gene in a bicistronic transcript. Moreover, as a result of this difference in reporter expression, bidirectional expression systems provided equal or better colocalization between the connexins and reporter gene fluorescence. The results of our study indicate that bidirectional reporter expression provides a robust indicator of transfection and gene expression and, therefore, may favor the use of bidirectional over bicistronic reporters in the design of expression systems where the gene of interest, such as a connexin gene, contains translational motifs or long intronic regions. PMID:22929700

Wan, Carthur K; Shaikh, Shamim B; Green, Colin R; Nicholson, Louise F B



PET/CT imaging of human somatostatin receptor 2 (hsstr2) as reporter gene for gene therapy  

NASA Astrophysics Data System (ADS)

Localized information on region-selective gene expression in small animals is widely obtained by use of reporter genes inducing light emission. Using these reporter genes for imaging deep inside the human body fluorescent probes are hindered by attenuation, scattering and possible fluorescence quenching. This can be overcome by use of radio-peptide receptors as reporter genes. Therefore, the feasibility of the somatostatin receptor 2 expression vector system for expression imaging was checked against a control vector containing luciferase gene. For in vivo transduction of vector DNA into the rat forelimb muscles the in vivo electroporation technique was chosen because of its high regio-selectivity. The gene expression was imaged by high-sensitive CCD camera (luciferase activity) and by PET/CT using a Ga-68-DOTATOC as radio peptide probe. The relative sstr2 expression was enhanced by gene transduction at maximum to a factor of 15. The PET/CT images could be fully quantified. The above demonstrated feasibility of radio-peptide PET/CT reporter gene imaging may serve in the future as a tool for full quantitative understanding of regional gene expression, especially in large animals and humans.

Hofmann, M.; Gazdhar, A.; Weitzel, T.; Schmid, R.; Krause, T.



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

SciTech Connect

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.

Mishra, N.C.



Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli.  


A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases. PMID:2182377

Sahasrabudhe, N A; Ranjekar, P K



Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression  

SciTech Connect

The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.



733. The Human XIST Gene Promoter Prevents Silencing of an Integrated Reporter Gene  

Microsoft Academic Search

Epigenetic silencing and position dependent expression are long-standing problems which continue to limit the development of gene replacement therapy. As a strategy to overcome this problem we have tested the ability of the human XIST (X inactivation-specific transcript) gene promoter to overcome epigenetic silencing. The XIST gene is one of a relatively small cohort of genes which are expressed from

Michael R. Greene; Christopher H. Lowrey



MyoD– lacZ transgenes are early markers in the neural retina, but MyoD function appears to be inhibited in the developing retinal cells  

Microsoft Academic Search

Recent findings suggest that eye and skeletal muscle development in vertebrates share the same regulatory network. In that network, Pax3 gene is apparently activated through Dach\\/Eya\\/Six feedback loop to mediate MyoD-driven myogenesis. The purpose of this study was to investigate previously reported MyoD–lacZ expression in the developing mouse neural retina and to gain insight into the potential role of MyoD

Boris Kablar



Detection of the reporter and selection genes in transformed hop (Humulus lupulus L.)  

Microsoft Academic Search

Agrobacterium-mediated transformation of hop nodal explants with meristems was used for the introduction of a gus reporter gene and nptII plant selection gene into Slovenian hop cv. Aurora. Emerging hop regenerants were previously tested for the gus gene expression by histochemical analysis of ?-glucoronidase (GUS) activity. Approximately six months after the transformation procedure, PCR molecular analysis of shoots originating from




Odontoblast-specific expression of cre recombinase successfully deletes gene segments flanked by loxP sites in mouse teeth.  


Embryonic or neonatal lethality of mice with targeted disruption of critical genes preclude them from further characterization of specific roles of these genes during postnatal development and aging. In order to study the molecular roles of such genes in teeth, we generated transgenic mouse lines expressing bacteriophage Cre recombinase under the control of the mouse dentin sialophosphoprotein (dspp) gene promoter. The expression of Cre recombinase protein was mainly detected in the nucleus of the odontoblasts. The efficiency of Cre activity was analyzed by crossing the Dspp-Cre mice with ROSA26 reporter (R26R) mice. The offspring with both genotypes have shown specific deletion of intervening sequences flanked by loxP sites upstream of the reporter gene, thereby facilitating the expression of the beta-galactosidase (beta-gal) gene in the teeth. The activity of beta-gal was initially observed in the odontoblasts of 1-day-old mice and increased with tooth development. Almost all of the odontoblasts have shown lacZ activity by 3 weeks of age. We could not detect Cre recombinase activity in any other cells, including ameloblasts. These studies indicate that the Dspp-Cre transgenic mice will be valuable to generate odontoblast-specific gene knockout mice so as to gain insight into the molecular roles of critical genes in the odontoblasts during dentinogenesis. PMID:12533791

Sreenath, T L; Cho, A; Thyagarajan, T; Kulkarni, A B



The objectivity of reporters: interference between physically unlinked promoters affects reporter gene expression in transient transfection experiments.  


Despite inherent limitations, the ease and rapidity of their use make transiently expressed reporter gene assays the most frequently used techniques for analyzing promoters and transcriptional regulators. The results of transient reporter gene assays are generally accepted to reflect transcriptional processes correctly, though these assays study regulatory sequences outside of the chromosomal environment and draw conclusions on transcription based on enzyme activity determination. For transient reporter gene assays, often more than one promoter is introduced into one cell. In addition to the one driving the primary reporter gene expression, a further one might serve to ensure the production of an internal control second reporter or/and a trans-acting factor. We demonstrate here by various examples that interference between physically unlinked promoters can profoundly affect reporter expression. Results of reporter gene assays performed by combinations of the cytomegalovirus promoter and various other promoter constructs (human immunodeficiency virus [HIV], Human T-cell Leukemia Virus Type I (HTLV-I), NF-?B-responsive, and p53-responsive) and trans-activator factors (HIV-Tat and p53) in different host cell lines (U2OS, HeLa, and L929) prove that interference between active transcription units can modify transcription responses dramatically. Since the interference depends on the promoters used, on the amount of transfected DNA, on the host cells, and on other factors, extra caution is required in interpreting results of transient reporter gene assays. PMID:22994211

Huliák, Ildikó; Sike, Adám; Zencir, Sevil; Boros, Imre M



Optical imaging of Renilla luciferase reporter gene expression in living mice  

Microsoft Academic Search

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme\\/protein (FL). In the current study, we validate

S. Bhaumik; S. S. Gambhir



MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation.  


Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high-resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell-based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies. PMID:23225197

Vandsburger, Moriel H; Radoul, Marina; Cohen, Batya; Neeman, Michal



The Ice Nucleation Gene fromPseudomonas syringaeas a Sensitive Gene Reporter for Promoter Analysis inZymomonas mobilis  

Microsoft Academic Search

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 promoterlessversionoftheinaZgenewasplacedunderthecontrolofthreedifferentpromoters:Ppdc(pyruvate decarboxylase), a homologous strong promoter from Z.




Comprehensive Set of Integrative Plasmid Vectors for Copper-Inducible Gene Expression in Myxococcus xanthus  

PubMed Central

Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 ?M during growth and 60 ?M during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

Gomez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Munoz, Aurelio; Garcia-Bravo, Elena; Garcia-Hernandez, Raquel; Martinez-Cayuela, Marina; Perez, Juana; S?gaard-Andersen, Lotte



A screen for genes that function in leg disc regeneration in Drosophila melanogaster  

PubMed Central

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported here aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. We screened a collection of 97 recessive lethal P-lacZ enhancer trap lines for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, we identified six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration.

McClure, Kimberly D.; Schubiger, Gerold



Yeast excision-repair gene is inducible by DNA damaging agents  

SciTech Connect

Plasmids containing various RAD-lacZ gene fusions were integrated into the chromosome of haploid yeast cells. These integrant strains were tested for expression of Escherichia coli ..beta..-galactosidase after treatment with agents that damage DNA or interfere with normal DNA replication. The authors did not observe induction of single-copy RAD1-lacZ or RAD3-lacZ fusion genes under the experimental conditions used. However, exposure of cells containing an integrated RAD2-lacZ fusion gene to UV-radiation, ..gamma..-radiation, 4-nitroquinoline 1-oxide, or nalidixic acid resulted in 4- to 6-fold enhanced expression of ..beta..-galactosidase. Induction of the RAD2 gene after treatment of untransformed cells with 4-nitroquinoline 1-oxide was confirmed by direct examination of RAD2 mRNA. Lower levels of induction (approx. = 50%) were observed after treatment of cells with other chemicals. Induction of the RAD2-lacZ fusion gene was also observed in cells transformed with single-copy and multicopy autonomously replicating plasmids.

Robinson, G.W.; Nicolet, C.M.; Kalainov, D.; Friedberg, E.C.



Deletion of the Saccharomyces cerevisiae TRR1 gene encoding thioredoxin reductase inhibits p53-dependent reporter gene expression.  


The prevalence of p53 gene mutations in many human tumors implies that p53 protein plays an important role in preventing cancers. Central among the activities ascribed to p53 is its ability to stimulate transcription of other genes that inhibit cells from entering S phase with damaged DNA. Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription. Although p53 strongly stimulated reporter gene expression in wild type yeast, it only weakly stimulated reporter gene expression in Deltatrr1 yeast that lacked the gene encoding thioredoxin reductase. Furthermore, ectoptic expression of TRR1 in Deltatrr1 yeast restored p53-dependent reporter gene activity to high levels. Immunoblot assays established that the Deltatrr1 mutation affected the activity and not the level of p53 protein. The results suggest that p53 can form disulfides and that these disulfides must be reduced in order for the protein to function as a transcription factor. PMID:9488661

Pearson, G D; Merrill, G F



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


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 ?-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 ?M using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for ?-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

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



Sex differences in the regulation of tyrosine hydroxylase gene transcription by estrogen in the locus coeruleus of TH9-LacZ transgenic mice  

Microsoft Academic Search

Although estrogen is recognized increasingly as having an important role in modulating extrahypothalamic brain function, the mechanisms through which this occur are not well established. The norepinephrine (NE) neurons of the locus coeruleus provide an important neuromodulatory influence upon multiple neural networks throughout the brain and estrogen has been implicated in their regulation. Using a tyrosine hydroxylase (TH) promoter-LacZ transgenic

Niren R Thanky; Jin H Son; Allan E Herbison



Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting  

PubMed Central

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julian; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J



A trial of somatic gene targeting in vivo with an adenovirus vector  

Microsoft Academic Search

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected

Asami Ino; Yasuhiro Naito; Hiroyuki Mizuguchi; Naofumi Handa; Takao Hayakawa; Ichizo Kobayashi



New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment.  


Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents. PMID:20737269

Svobodová, Katerina; Cajthaml, Tomás



The Study of Drug-Receptor Interaction Using Reporter Gene Systems in Mammalian Cells  

Microsoft Academic Search

\\u000a “Reporter gene” is the term used to describe a plasmid containing either an inducible or constitutive promoter element that\\u000a controls the expression of a readily measurable enzyme or other protein. The reporter protein typically has a unique activity\\u000a or structure to enable it to be distinguished from other proteins present. The choice of reporter gene, which is often an\\u000a enzyme,

D. M. Ignar; S. Rees


Signal Transduction Pathways that Regulate CAB Gene Expression, (Final Report).  

National Technical Information Service (NTIS)

The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals)....

J. Chory



Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae.  

PubMed Central

The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.

Arvanitis, N; Vargas, C; Tegos, G; Perysinakis, A; Nieto, J J; Ventosa, A; Drainas, C



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

Microsoft Academic Search

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




Signal transduction pathways that regulate CAB gene expression. Progress report  

SciTech Connect

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.

Chory, J.



An RNA Hairpin Sequesters the Ribosome Binding Site of the Homing Endonuclease mobE Gene?  

PubMed Central

Previous transcript mapping of the bacteriophage Aeh1 nrd operon revealed a predicted RNA hairpin upstream of the homing endonuclease mobE gene. We enzymatically mapped the hairpin, showing that the mobE ribosome binding site is sequestered. Cloning of the hairpin upstream of lacZ resulted in reduced ?-galactosidase activity, consistent with translational regulation.

Gibb, Ewan A.; Edgell, David R.



Mapping and regulation of the cel genes in Erwinia chrysanthemi  

Microsoft Academic Search

Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the fusions, (iii) recombinational exchange

Jean-Luc Aymeric; Annick Guiseppi; Marie-Claire Pascal; Marc Chippaux



Regulated spatial expression of fusion gene constructs with the 5? upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos  

Microsoft Academic Search

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5' flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for ß-galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another

Akira Hikosaka; Noriyuki Satoh; Kazuhiro W. Makabe



Truncated-gene reporter system for studying the regulation of manganese peroxidase expression  

Microsoft Academic Search

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of Phanerochaete chrysosporium is regulated by Mn, heat shock (HS), and H2O2 at the level of gene transcription. We have constructed a homologous gene reporter system to further examine the regulation\\u000a of two mnp genes, mnp1 and mnp2, encoding individual MnP isozymes. Internal deletions of 234 and 359 bp were made

Jessica M. Gettemy; Dan Li; Margaret Alic; Michael H. Gold



Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants  

Microsoft Academic Search

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems\\u000a for a ?-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter\\u000a in transgenic tobacco plants. On treatment

Susumu Kodama; Kumiko Okada; Hideyuki Inui; Hideo Ohkawa



Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile  

PubMed Central

Background Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. Methods Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). Results Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. Conclusions We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer.



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


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

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



Noninvasive Optical Imaging of Firefly Luciferase Reporter Gene Expression in Skeletal Muscles of Living Mice  

Microsoft Academic Search

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demon-strate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD

Joseph C. Wu; Gobalakrishnan Sundaresan; Meera Iyer; Sanjiv S. Gambhir



Gene therapy for C-26 colon cancer using heparin-polyethyleneimine nanoparticle-mediated survivin T34A  

PubMed Central

Background Gene therapy provides a novel method for the prevention and treatment of cancer, but the clinical application of gene therapy is restricted, mainly because of the absence of an efficient and safe gene delivery system. Recently, we developed a novel nonviral gene carrier, ie, heparin-polyethyleneimine (HPEI) nanoparticles for this purpose. Methods and results HPEI nanoparticles were used to deliver plasmid-expressing mouse survivin-T34A (ms-T34A) to treat C-26 carcinoma in vitro and in vivo. According to the in vitro studies, HPEI nanoparticles could efficiently transfect the pGFP report gene into C-26 cells, with a transfection efficiency of 30.5% ± 2%. Moreover, HPEI nanoparticle-mediated ms-T34A could efficiently inhibit the proliferation of C-26 cells by induction of apoptosis in vitro. Based on the in vivo studies, HPEI nanoparticles could transfect the Lac-Z report gene into C-26 cells in vivo. Intratumoral injection of HPEI nanoparticle-mediated ms-T34A significantly inhibited growth of subcutaneous C-26 carcinoma in vivo by induction of apoptosis and inhibition of angiogenesis. Conclusion This research suggests that HPEI nanoparticle-mediated ms-T34A may have a promising role in C-26 colon carcinoma therapy.

Zhang, Ling; Gao, Xiang; Men, Ke; Wang, BiLan; Zhang, Shuang; Qiu, Jinfeng; Huang, Meijuan; Gou, MaLing; Huang, Ning; Qian, ZhiYong; Zhao, Xia; Wei, YuQuan



A Tn3 derivative that can be used to make short in-frame insertions within genes.  

PubMed Central

A Tn3 derivative was constructed to make small in-frame insertions within genes. The transposon contains the URA3 gene, the tetA gene, a truncated lacZ, and phage P1 loxP recombination sites at either end. Insertions that have fused lacZ to an open reading frame are lac+ because they express the truncated lacZ. In the presence of the phage P1 cyclization recombinase cre, the transposon can delete the URA3, tetA, and lacZ genes between the two loxP sites. The remaining short imperfect palindrome contains the ends of Tn3 and a loxP site and does not contain a translational termination codon in the correct reading frame. We have analyzed several insertions within the yeast HO gene. Several insertions inactivate HO and prohibit initiation of mating-type switching. In contrast, an epitope inserted in the central portion encodes a functional HO endonuclease. Images

Hoekstra, M F; Burbee, D; Singer, J; Mull, E; Chiao, E; Heffron, F



Experience report: issues in comparing gene function annotation in text  

Microsoft Academic Search

Annotating function of genes accurately is one of the most important tasks in molecular biology and medical sciences. The new sequencing technology, called the next generation sequencing technology, made sequencing the whole genomes possible with a fraction of cost of sequencing by using the traditional sequencing technology. As a result, the amount of sequence data has been growing very rapidly,

Youngik Yang; Sun Kim




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, ...


Successive silencing of tandem reporter genes in potato (Solanum tuberosum) over 5 years of vegetative propagation  

PubMed Central

Background and Aims Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. Methods To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. Key Results A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. Conclusions We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

Nocarova, Eva; Opatrny, Zdenek; Fischer, Lukas



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

SciTech Connect

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.

Lapeyre, J.N.; Marini, F.; Gratzner, H.G. (M. D. Anderson Cancer Center, Houston, TX (United States) AMC ImmunoDiagnostics, Houston, TX (United States))



Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report  

SciTech Connect

Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have been to use a different transgene as the target of gene silencing. We tested two other transgenes. Both encoded proteins unrelated to the first but they were all expressed from the same type of promoter and they all had a similar tendency to become post-transcriptionally silenced. After screening over 80 F2 segregants from each cross between our egs1 mutant and Arabidopsis of the same ecotype homozygous for the new transgene, we were disappointed to find that the egs1 mutation did not enhance post-transcription silencing of the two new genes. In 80 plants we expected to have between 4 and 6 plants that were homozygous for the transgene and for the mutant egs1 allele. If egs1 mutations could enhance gene silencing of the new transgene, these plants would not express it. However all the double homozygotes still expressed the transgene. Therefore, we could not change the target transgene for mapping. This was the state of the cloning at the time for renewal of the grant in 1999. Because the selection of new meaningful recombinant plants had become extremely inefficient using the original rolB transgene, we abandoned the attempt at map based cloning and did not apply for further funding.

Grant, S. R.



Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes.  

PubMed Central

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.

Parcells, M S; Anderson, A S; Morgan, T W



A Novel Mutation in ABCC8 Gene in a Newborn with Congenital Hyperinsulinism -A Case Report.  


Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycemia in infancy. The genetic basis of CHI includes a variety of defects in key genes regulating insulin secretion. Mutations in at least seven genes are found in 50% of cases. The most common forms of medically unresponsive CHI, which requires a near-total pancreatectomy are associated with autosomal recessive mutations in the ABCC8 and KCNJ11 genes encoding the two subunits of the pancreatic ?-cell ATP-sensitive potassium channel. We report a neonate with CHI and have a novel homozygous splicing mutation in the ABCC8 gene. PMID:23607867

Ustün, Nuran Uzunalic; Dilli, Dilek; Kundak, Ahmet Afsin; Okumus, Nurullah; Erdo?an, Derya; Apayd?n, Sema



Consensus: a framework for evaluation of uncertain gene variants in laboratory test reporting.  


Accurate interpretation of gene testing is a key component in customizing patient therapy. Where confirming evidence for a gene variant is lacking, computational prediction may be employed. A standardized framework, however, does not yet exist for quantitative evaluation of disease association for uncertain or novel gene variants in an objective manner. Here, complementary predictors for missense gene variants were incorporated into a weighted Consensus framework that includes calculated reference intervals from known disease outcomes. Data visualization for clinical reporting is also discussed. PMID:22640420

Crockett, David K; Ridge, Perry G; Wilson, Andrew R; Lyon, Elaine; Williams, Marc S; Narus, Scott P; Facelli, Julio C; Mitchell, Joyce A



Virus Attenuation after Deletion of the Cytomegalovirus Fc Receptor Gene Is Not due to Antibody Control  

Microsoft Academic Search

The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological signif- icance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ




Functional analysis of the sporulation-specific SPR6 gene of Saccharomyces cerevisiae  

Microsoft Academic Search

The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation. The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size. This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused

L. A. Kallal; M. Bhattacharyya; S. N. Grove; R. F. Iannacone; T. A. Pugh; D. A. Primerano; M. J. Clancy



Plasmids expressing the herpes simplex virus thymidine kinase gene in mammalian and bacterial cells  

Microsoft Academic Search

Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk- cells as well as E. coli tk- cells to the Tk+ phenotype and are

Michael Strauss; Udo Kiessling; Ruth Kähler



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

SciTech Connect

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.

Haber, J. E.



Making reporter gene constructs to analyze cis-regulatory elements.  


Cis-regulatory sequences control when, where, and how much genes are transcribed. A better understanding on these elements is a fundamental keystone to better understand development, cell differentiation, and morphogenesis. Several methods based on in silico analysis or ChIP-seq experiments have been developed to detect cis-acting sequences. Here, we describe a protocol to isolate such sequences from genomic DNA and to clone them into expression vectors for functional assays using the Gateway cloning technology. PMID:22065451

Bessa, José; Gómez-Skarmeta, José Luis



Effect of Ku80 Deficiency on Mutation Frequencies and Spectra at a LacZ Reporter Locus in Mouse Tissues and Cells  

Microsoft Academic Search

Non-homologous end joining (NHEJ) is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs) and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based

Rita A. Busuttil; Denise P. Muńoz; Ana Maria Garcia; Francis Rodier; Woo Ho Kim; Yousin Suh; Paul Hasty; Judith Campisi; Jan Vijg; David J. Chen



Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography  

PubMed Central

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression (r2 > 0.80). Over a similar range of HSV1-tk expression in vivo, the percent injected dose retained per gram of liver was 0–23% for ganciclovir and 0–3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.

Gambhir, Sanjiv S.; Barrio, Jorge R.; Phelps, Michael E.; Iyer, Meera; Namavari, Mohammad; Satyamurthy, Nagichettiar; Wu, Lily; Green, Leeta A.; Bauer, Eileen; MacLaren, Duncan C.; Nguyen, Khoi; Berk, Arnold J.; Cherry, Simon R.; Herschman, Harvey R.



442. LV Expressing MR Reporter Genes Allows In Vivo Monitoring of Stem Cell Gene Therapy  

Microsoft Academic Search

Somatic stem cells (SSC) have raised interest because of their therapeutic potential in both cell-based and gene therapy applications. Towards this goal, tracking the fate of either delivered cells or of genetically modified endogenous cells is of utmost importance. Diverse imaging approaches are available for cell tracking and among these MRI shows a greater resolution and allows direct anatomic correlation

Mario Amendola; Letterio S. Politi; Marcello Cadioli; Rossella Galli; Elena Binda; Andrea Falini; Sonia Levi; Giuseppe Scotti; Alessandra Biffi; Luigi Naldini



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

SciTech Connect

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.

Kuchka, M.R.



Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.  


Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver. PMID:7753194

Nuez, B; Michalovich, D; Bygrave, A; Ploemacher, R; Grosveld, F



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


Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals. PMID:22914496

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



In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes.  


A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When a luciferase reporter gene was employed, gene expression was found in all tissues examined including lung, heart, spleen, liver and kidney with the highest expression in the lung. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 micrograms per mouse at which concentration approximately 20 ng luciferase protein per milligram extracted tissue protein could be detected in the lung. Increasing the DNA to 100 micrograms per mouse resulted in toxicity and death of the animal. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. High expression was also found in the spleen 6 h after injection but dropped very rapidly thereafter. The in vivo gene expression by LPD was dependent upon the route of administration since intraportal injection of LPD led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection. Using lacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both the lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be nontoxic and only weakly immunogenic in humans, this novel vector may be useful for clinical gene therapy. PMID:9349425

Li, S; Huang, L



Characterization of the Arxula adeninivorans AHOG1 gene and the encoded mitogen-activated protein kinase.  


Arxula adeninivorans is an osmo-resistant yeast species that can tolerate high levels of osmolytes like NaCl, PEG400 and ethylene glycol. As in other yeast species, this tolerance is elicited by components of the high osmolarity glycerol (HOG) response pathway. In the present study, we isolated and characterized as a key component of this pathway the A. adeninivorans AHOG1 gene encoding the mitogen-activated protein (MAP) kinase Ahog1p, an enzyme of 45.9 kDa. The gene includes a coding sequence of 1,203 bp disrupted by a 57-bp intron. The identity of the gene was confirmed by complementation of a hog1 mutation in a Saccharomyces cerevisiae mutant strain and the high degree of homology of the derived amino acid sequence with that of MAP kinases from other yeasts and fungi. Under stress-free conditions, the inactive Ahoglp is present in low levels. When exposed to osmotic stress, Ahoglp is rendered active by phosphorylation. In addition, AHOG1 expression is increased. Assessment of the AHOG1 promoter activity with a lacZ reporter gene confirmed its inducibility by osmolytes, a characteristic not observed in homologous HOG1 genes of other yeast species. This specific property could account for the fast adaptation and high osmo-resistance encountered in this species. PMID:15526205

Böer, Erik; Wartmann, Thomas; Dlubatz, Karen; Gellissen, Gerd; Kunze, Gotthard



DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.  

PubMed Central

The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA. Images

Hediger, M A; Johnson, D F; Nierlich, D P; Zabin, I



Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi  

Microsoft Academic Search

The recent development of variants of the green fluorescent protein (GFP) with altered codon composition facilitated the efficient expression of this reporter protein in a number of fungal species. In this report, we describe the construction and application of a series of plasmids, which support the expression of an enhanced gfp (egfp) gene in filamentous fungi and assist the study

Stefanie Pöggeler; Sandra Masloff; Birgit Hoff; Severine Mayrhofer; Ulrich Kück



Screening and identification of substances that regulate nephrin gene expression using engineered reporter podocytes  

Microsoft Academic Search

Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was

K Yamauchi; Y Takano; A Kasai; K Hayakawa; N Hiramatsu; N Enomoto; J Yao; M Kitamura



Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene  

PubMed Central

Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2. Although fluorescence was observed, there were discrepancies between in vivo imaging and ex vivo imaging as well as between nuclear imaging and fluorescent imaging. Conclusion These studies showed that the SSTR2-EGFP fusion construct can be used for in vivo nuclear and optical imaging of gene transfer.

Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.



Anterior epidermis-specific expression of the cuticle gene EDG84A is controlled by many cis-regulatory elements in Drosophila melanogaster.  


During insect metamorphosis, a pulse of ecdysteroids induces many different morphological changes depending on different parts of the body. In Drosophila, although a number of transcription factors are expressed in a stage-specific manner in response to an ecdysteroid pulse, little is known on the regulatory mechanism for space-specific gene expression during metamorphosis. The EDG84A gene encoding pupal cuticle protein is one of the targets of ecdysteroid-inducible transcription factor betaFTZ-F1 and is expressed only in anterior epidermis of the body during mid- to late prepupal period, whereas betaFTZ-F1 is expressed in almost all tissues. To address the regulatory mechanism of the tissue-specific expression of the EDG84A gene, we established transgenic fly lines which carry various upstream regions of the gene fused to the LacZ gene and examined the expression pattern of the reporter gene. Results of the transgenic fly reporter assays showed that the space-specific expression is controlled by at least four positive and two negative elements within a 263-bp region near the transcription start site, and at least three of them showed space-specific effects to the anterior body trunk. These results suggest that both high expression level and differential expression are achieved through many cis-regulatory elements. PMID:16025347

Kayashima, Yasunari; Hirose, Susumu; Ueda, Hitoshi



Gene therapy for intraperitoneally disseminated pancreatic cancers by Escherichia coli uracil phosphoribosiltransferase (UPRT) gene mediated by restricted replication-competent adenoviral vectors.  


Although patients with unresectable pancreatic tumors have been treated with 5-fluorouracil (5FU)-based combination chemotherapy, the drug resistance of cancer cells presents a crucial therapeutic problem. It was reported that UPRT overcomes 5FU resistance. UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits thymidylate synthetase (TS). We first demonstrated that injecting an E1-deficient adenoviral vector (Adv) expressing UPRT (AxCAUPRT) followed by 5-FU treatment resulted in a volume reduction of xenotransplanted human tumors. In examining the therapeutic effect of AxCAUPRT/5-FU against peritoneal dissemination, we found that non-selective gene transduction of AxCAUPRT caused severe adverse effects arising from the increase of F-dUMP in normal intestine. Because the therapeutic gene delivered by a restricted replication-competent Adv lacking 55 kDa E1B protein (AxE1AdB) is speculated to be expressed selectively in tumors, mice with established tumors were injected with AxE1AdB and E1-deleted Adv expressing the lacZ reporter gene (AxCAlacZ). The expression of the reporter gene (lacZ) was selectively enhanced in disseminated tumors. The therapeutic advantage of restricted replication competent Adv that expresses UPRT (AxE1AdB-UPRT) was evaluated in an intraperitoneal disseminated tumor model. To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the Adv, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. Our results showed that the AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer. PMID:12353234

Oonuma, Masaru; Sunamura, Makoto; Motoi, Fuyuhiko; Fukuyama, Shouji; Shimamura, Hiromune; Yamauchi, Jun-Ichiro; Shibuya, Kazuhiko; Egawa, Shin-Ichi; Hamada, Hirofumi; Takeda, Kazunori; Matsuno, Seiki



The naphthalene catabolic (nag) genes of Polaromonas naphthalenivorans CJ2: Evolutionary implications for two gene clusters and novel regulatory control  

SciTech Connect

Polaromonas naphthalenivorans CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site, is able to grow on mineral salts agar media with naphthalene as the sole carbon source. Beginning from a 484-bp nagAc-like region, we used a genome walking strategy to sequence genes encoding the entire naphthalene degradation pathway and additional flanking regions. We found that the naphthalene catabolic genes in P. naphthalenivorans CJ2 were divided into one large and one small gene cluster, separated by an unknown distance. The large gene cluster is bounded by a LysR-type regulator (nagR). The small cluster is bounded by a MarR-type regulator (nagR2). The catabolic genes of P. naphthalenivorans CJ2 were homologous to many of those of Ralstonia U2, which uses the gentisate pathway to convert naphthalene to central metabolites. However, three open reading frames (nagY, nagM, and nagN), present in Ralstonia U2, were absent. Also, P. naphthalenivorans carries two copies of gentisate dioxygenase (nagI) with 77.4% DNA sequence identity to one another and 82% amino acid identity to their homologue in Ralstonia sp. strain U2. Investigation of the operons using reverse transcription PCR showed that each cluster was controlled independently by its respective promoter. Insertional inactivation and lacZ reporter assays showed that nagR2 is a negative regulator and that expression of the small cluster is not induced by naphthalene, salicylate, or gentisate. Association of two putative Azoarcus-related transposases with the large cluster and one Azoarcus-related putative salicylate 5-hydroxylase gene (ORF2) in the small cluster suggests that mobile genetic elements were likely involved in creating the novel arrangement of catabolic and regulatory genes in P. naphthalenivorans.

Jeon, C.O.; Park, M.; Ro, H.S.; Park, W.; Madsen, E.L. [Cornell University, Ithaca, NY (United States). Dept. of Microbiology



Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.  


A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature. PMID:10609651

Schughart, K; Bischoff, R; Rasmussen, U B; Hadji, D A; Perraud, F; Accart, N; Boussif, O; Silvestre, N; Cordier, Y; Pavirani, A; Kolbe, H V



Reporter genes and highly regulated promoters as tools for transformation experiments in Volvox carteri.  

PubMed Central

The multicellular alga Volvox is an attractive model for the study of developmental processes. With the recent report of successful transformation, regulated promoters as well as reporter genes working in this organism are now required. The Volvox genes encoding arylsulfatase and the extracellular glycoprotein ISG are strictly regulated. The former is transcribed only under conditions of sulfur starvation, whereas the latter operates under extreme developmental control--i.e., it is transcribed for only a few minutes in Volvox embryos at the stage of embryonic inversion. The gene encoding the sexual pheromone of Volvox carteri was placed under the control of the arylsulfatase promoter. In response to sulfur deprivation, V. carteri transformed by this construct synthesized and secreted biologically active pheromone. In addition, the gene encoding Volvox arylsulfatase was placed under the control of the ISG promoter. Transformed algae synthesized arylsulfatase mRNA only during embryonic inversion. These experiments demonstrate the usefulness of both the arylsulfatase and the sexual pheromone reporter genes. In addition, the highly regulated arylsulfatase promoter allows the construction of inducible expression vectors for cloned genes. Images

Hallmann, A; Sumper, M



NANOG Reporter Cell Lines Generated by Gene Targeting in Human Embryonic Stem Cells  

PubMed Central

Background Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency, but the precise role and regulation of NANOG are not well defined. Methodology/Principal Findings To facilitate the study of NANOG expression and regulation in viable hESC cultures, we generated fluorescent NANOG reporter cell lines by gene targeting in hESCs. In these reporter lines, the fluorescent reporter gene was co-expressed with endogenous NANOG and responded to experimental induction or repression of the NANOG promoter with appropriate changes in expression levels. Furthermore, NANOG reporter lines facilitated the separation of hESC populations based on NANOG expression levels and their subsequent characterization. Gene expression arrays on isolated hESC subpopulations revealed genes with differential expression in NANOGhigh and NANOGlow hESCs, providing candidates for NANOG downstream targets hESCs. Conclusion/Significance The newly derived NANOG reporter hESC lines present novel tools to visualize NANOG expression in viable hESCs. In future applications, these reporter lines can be used to elucidate the function and regulation of NANOG in pluripotent hESCs.

Fischer, Yvonne; Ganic, Elvira; Ameri, Jacqueline; Xian, Xiaojie; Johannesson, Martina; Semb, Henrik



Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays.  


Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical-analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical-analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical-analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes. PMID:19286037

Houtman, Corine J; Sterk, Saskia S; van de Heijning, Monique P M; Brouwer, Abraham; Stephany, Rainer W; van der Burg, Bart; Sonneveld, Edwin



Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.  


Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 +/- 0.1 versus 1.9 +/- 0.1) x (10(6) p/s/cm(2)/sr)] but similar level of fluorine-18-labeled 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 +/- 0.15 versus 1.37 +/- 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttk-expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. PMID:17409415

Ray, Pritha; Tsien, Roger; Gambhir, Sanjiv Sam



CAT5, a new gene necessary for derepression of gluconeogenic enzymes in Saccharomyces cerevisiae.  


PCK1 encoding phosphoenolpyruvate carboxykinase is transcriptionally regulated by two upstream activating elements. By screening for mutants that failed to derepress a UAS2PCK1-CYC1-lacZ reporter gene we isolated the new recessive derepression mutation cat5. The CAT5 gene encodes a protein of 272 amino acids showing a 42% identity to the ZC395.2 gene product of Caenorhabditis elegans whose function is unknown. Deletion of CAT5 caused a complete loss of glucose derepression affecting gluconeogenic key enzymes. Respiration, but not mitochondrial cytochrome c oxidase activity, was also affected. CAT5 expression is 5- to 6-fold repressed by glucose, and CAT5 transcriptional activation was dependent on CAT1 (SNF1), CAT8 and CAT5 itself. The CAT5 gene is necessary for UAS1PCK1 and UAS2PCK1 protein binding since a carbon source-specific interaction was no longer detectable in cat5 mutants. Glucose derepression of gluconeogenesis depends on the active Cat1 (Snf1) protein kinase and the Cat8 zinc cluster activator. Mig1p-independent overexpression of CAT8 did not stimulate activation of gluconeogenic promoters in cat1 and in cat5 mutants. Since Cat8p multicopy expression suppresses the ethanol growth deficiency in cat1 (snf1) mutants, these results indicate that activation of Cat8p by the Cat1p (Snf1p) kinase and the Cat5p protein might be necessary for release from glucose repression. PMID:8557031

Proft, M; Kötter, P; Hedges, D; Bojunga, N; Entian, K D



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

NASA Astrophysics Data System (ADS)

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.

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



Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y.  


A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene. PMID:12756537

Rahman, R N Z A; Chin, J H; Salleh, A B; Basri, M



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

PubMed Central

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.

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



Brain-specific gene expression by immortalized microglial cell-mediated gene transfer in the mammalian brain  

Microsoft Academic Search

The intra-arterial injection of immortalized microglia transfected with the lacZ gene, resulted in the expression of ?-galactosidase in the rat brain at 48 h and the activity of ?-galactosidase was detected for up to 3 weeks post-injection. More than 30-fold higher activity of ?-galactosidase was detected in the brain than in the liver, lung or spleen at 48 h post-injection.

Makoto Sawada; Fumihiro Imai; Hiromi Suzuki; Motoharu Hayakawa; Tetsuo Kanno; Toshiharu Nagatsu



Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris).  


Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species. PMID:14610892

Basu, Chhandak; Kausch, Albert P; Luo, Hong; Chandlee, Joel M



Genetic Evidence for Transcriptional Activation by the Yeast Ime1 Gene Product  

PubMed Central

IME1 is required in yeast for meiosis and for expression of IME2 and other early meiotic genes. IME1 is a 360-amino acid polypeptide with central and C-terminal tyrosine-rich regions. We report here that a fusion protein composed of the lexA DNA-binding domain and IME1 activates transcription in vivo of a reporter gene containing upstream lexA binding sites. Activation by the fusion protein shares several features with natural IME1 activity: both are dependent on the RIM11 gene product; both are impaired by the same ime1 missense mutations; both are restored by intragenic suppressors. The central tyrosine-rich region is sufficient to activate transcription when fused to lexA. Deletion of this putative activation domain results in a defective IME1 derivative. Function of the deletion derivative is restored by fusion to the acidic Herpesvirus VP16 activation domain. The C-terminal tyrosine-rich region is dispensable for transcriptional activation; rather it renders activation dependent upon starvation and RIM11. Immunofluorescence studies indicate that an IME1-lacZ fusion protein is concentrated in the nucleus. These observations are consistent with a model in which IME1 normally stimulates IME2 expression by providing a transcriptional activation domain at the IME2 5' regulatory region.

Smith, H. E.; Driscoll, S. E.; Sia, RAL.; Yuan, H. E.; Mitchell, A. P.



High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers.  


The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors. PMID:23216086

Guasch-Vidal, B; Estévez, J; Dardanelli, M S; Soria-Díaz, M E; de Córdoba, F Fernández; Balog, C I A; Manyani, H; Gil-Serrano, A; Thomas-Oates, J; Hensbergen, P J; Deelder, A M; Megías, M; van Brussel, A A N



Construction and characterization of a reporter gene cell line for assessment of human glucocorticoid receptor activation.  


Glucocorticoids are widely used drugs in human pharmacotherapy. There is an increasing demand for tools allowing detection of the ligands for glucocorticoid receptor (GR), with regard to pre-clinical drug testing and environmental applications. We constructed human luciferase reporter gene cell line AZ-GR derived from HeLa human cervix carcinoma cells, which were stably transfected with reporter plasmid containing three copies of glucorticoid response element (GRE) upstream of luciferase reporter gene. We isolated five dexamethasone-responsive clones, and we further characterized two most responsive ones (AZ-GR). Dose-response analyses were performed with 22 different natural and synthetic steroids and the values of EC(50) were calculated. AZ-GR cells displayed high specificity and sensitivity to glucocorticoids, very low responsiveness to mineralocorticoids, but no responsiveness to estrogens, gestagens or androgens. Time-course analyses revealed that AZ-GR cells allow detection of GR activators soon after 14 h of the treatment (6-10-fold induction by 100 nM dexamethasone). Functionality of AZ-GR cells was not affected with cryopreservation. Generated reporter gene cell lines fully maintained responsiveness to glucocorticoids for 32 days in the culture and over 16 passages without significant alterations. The sensitivity of the assay allows high throughput format using 96-well plates. Collectively, we present here glucocorticoid-responsive stable reporter gene cell line that allows high throughput, rapid, sensitive and selective detection of GR activators, with possible use in pre-clinical research and environmental applications. PMID:23089292

Novotna, Aneta; Pavek, Petr; Dvorak, Zdenek



Improved efficiency and stability of multiple cloned gene insertions at the ? sequences of Saccharomyces cerevisiae  

Microsoft Academic Search

Two ?-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first\\u000a vector was a traditional construct containing

F. W. F. Lee; N. A. Da Silva



A Genetically Modified Adenoviral Vector Exhibits Enhanced Gene Transfer of Human Smooth Muscle Cells  

Microsoft Academic Search

Adenoviral vector-based gene therapy is a promising approach for the treatment of restenosis postangioplasty. However, a high concentration of adenoviral vector can cause cellular activation, damage, and an enhanced immune response. One approach to solving this problem is to increase gene transfer efficiency by directing adenoviral vector entry via an alternate receptor system. We have constructed an adenoviral vector, Av9LacZ,

Enming J. Su; Susan C. Stevenson; Michele Rollence; Jennifer Marshall-Neff; Gene Liau



Bacterial ?-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection  

Microsoft Academic Search

Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial ?-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven

Alexander V. Zelenin; Victor A. Kolesnikov; Olga A. Tarasenko; Ramin A. Shafei; Inessa A. Zelenina; Vyacheslav V. Mikhailov; Maria L. Semenova; Dmitry V. Kovalenko; Olga V. Artemyeva; Tatyana E. Ivaschenko; Oleg V. Evgrafov; George Dickson; Vladislav S. Baranovand



Photodynamic therapy-mediated oxidative stress as a molecular switch for the temporal expression of genes ligated to the human heat shock promoter.  


Oxidative stress associated with photodynamic therapy (PDT) is a transcriptional inducer of genes encoding stress proteins, including those belonging to the heat shock protein (hsp) family. The efficiency of PDT to function as a molecular switch by initiating expression of heterologous genes ligated to the human hsp promoter was examined in the present study. Selective and temporal reporter gene expression was documented after PDT in mouse radiation-induced fibrosarcoma cells stably transfected with recombinant vectors containing an hsp promoter ligated to either the lac-z or CAT reporter genes and in transfected radiation-induced fibrosarcoma tumors grown in C3H mice. Hyperthermia treatments were included as a positive control for all experiments. Expression vectors containing either human p53 or tumor necrosis factor (TNF)-alpha cDNA under the control of an hsp promoter were also constructed and evaluated. A p53 null and TNF-alpha-resistant human ovarian carcinoma (SKOV-3) cell line was stably transfected with either the p53 or TNF-alpha constructs. Inducible expression and function of p53 as well as inducible expression, secretion, and biological activity of TNF-alpha were documented after PDT or hyperthermia in transfected SKOV cells. These results demonstrate that PDT-mediated oxidative stress can function as a molecular switch for the selective and temporal expression of heterologous genes in tumor cells containing expression vectors under the control of an hsp promoter. PMID:10749134

Luna, M C; Ferrario, A; Wong, S; Fisher, A M; Gomer, C J



Gene transcription and electromagnetic fields. Final progress report  

SciTech Connect

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.

Henderson, A.S.



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

PubMed Central

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.

Zhou, Z.; Elledge, S. J.



The cytochrome c gene proximal enhancer drives activity-dependent reporter gene expression in hippocampal neurons  

PubMed Central

The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP+ neurons, while the frequency of mEPSCs was not significantly different. Increasing neural activity by applying a GABAA receptor antagonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity.

Delgado, Jary Y.; Owens, Geoffrey C.



Mutations in a Novel Gene, NHS, Cause the Pleiotropic Effects of Nance-Horan Syndrome, Including Severe Congenital Cataract, Dental Anomalies, and Mental Retardation  

PubMed Central

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features, and, in some cases, mental retardation. NHS has been mapped to a 1.3-Mb interval on Xp22.13. We have confirmed the same localization in the original, extended Australian family with NHS and have identified protein-truncating mutations in a novel gene, which we have called “NHS,” in five families. The NHS gene encompasses ?650 kb of genomic DNA, coding for a 1,630–amino acid putative nuclear protein. NHS orthologs were found in other vertebrates, but no sequence similarity to known genes was identified. The murine developmental expression profile of the NHS gene was studied using in situ hybridization and a mouse line containing a lacZ reporter-gene insertion in the Nhs locus. We found a complex pattern of temporally and spatially regulated expression, which, together with the pleiotropic features of NHS, suggests that this gene has key functions in the regulation of eye, tooth, brain, and craniofacial development.

Burdon, Kathryn P.; McKay, James D.; Sale, Michele M.; Russell-Eggitt, Isabelle M.; Mackey, David A.; Wirth, M. Gabriela; Elder, James E.; Nicoll, Alan; Clarke, Michael P.; FitzGerald, Liesel M.; Stankovich, James M.; Shaw, Marie A.; Sharma, Shiwani; Gajovic, Srecko; Gruss, Peter; Ross, Shelley; Thomas, Paul; Voss, Anne K.; Thomas, Tim; Gecz, Jozef; Craig, Jamie E.



A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression  

SciTech Connect

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of this analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes.

Schultz, J.A.; Gremke, L.; Storti, R.V. (University of Illinois of Medicine, Chicago (United States)); Tansey, T. (Georgetown University, Washington, D.C., VA (United States))



High rate of mutation reporter gene inactivation during human T cell proliferation  

Microsoft Academic Search

Caspase activation and degradation of deoxyribonucleic acid (DNA) damage response factors occur during in vitro T-cell proliferation,\\u000a and an increased frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-negative variants have been reported in\\u000a conditions associated with in vivo T-cell proliferation. We have applied two human somatic cell mutation reporter assays,\\u000a for the HPRT and phosphatidylinositol glycan class A (PIG-A) genes, to human T

Aida Gabdoulkhakova; Gunnel Henriksson; Nadezhda Avkhacheva; Alexander Sofin; Anders Bredberg



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

PubMed Central

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.

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



Aldehyded Dextran and ?-Poly(L-lysine) Hydrogel as Nonviral Gene Carrier  

PubMed Central

Background. The expression term of the gene transfected in cells needs to belong enough inorder to make a gene therapy clinically effective. The controlled release of the transfected gene can be utilized. The new biodegradable hydrogel material created by 20?w/w% aldehyded dextran and 10?w/w% ?-poly(L-lysine) (ald-dex/PLL) was developed. We examined whether it could be as a nonviral carrier of the gene transfer. Methods. A plasmid (Lac-Z) was mixed with ald-dex/PLL. An in vitro study was performed to assess the expression of Lac-Z with X-gal stain after gene transfer into the cultured 293 cells and bone marrow cells. As a control group, PLL was used as a cationic polymer. Results. We confirmed that the transfection efficiency of the ald-dex/PLL had a higher transfection efficiency than PLL in 293?cells (plasmid of 2??g: ald-dex/PLL 1.1%, PLL 0.23%, plasmid of 16??g: ald-dex/PLL 1.23%, PLL 0.48%). In bone marrow cells, we confirmed the expression of Lac-Z by changing the quantity of aldehyded dextran. In the groups using ald-dextran of the quantity of 1/4 and 1/12 of PLL, their transfection efficiency was 0.43% and 0.41%, respectively. Conclusions. This study suggested a potential of using ald-dex/PLL as a non-carrier for gene transfer.

Togo, Yumiko; Takahashi, Katsu; Saito, Kazuyuki; Kiso, Honoka; Tsukamoto, Hiroko; Hyon, Suong-Hyu



Aldehyded Dextran and ? -Poly(L-lysine) Hydrogel as Nonviral Gene Carrier.  


Background. The expression term of the gene transfected in cells needs to belong enough inorder to make a gene therapy clinically effective. The controlled release of the transfected gene can be utilized. The new biodegradable hydrogel material created by 20?w/w% aldehyded dextran and 10?w/w% ? -poly(L-lysine) (ald-dex/PLL) was developed. We examined whether it could be as a nonviral carrier of the gene transfer. Methods. A plasmid (Lac-Z) was mixed with ald-dex/PLL. An in vitro study was performed to assess the expression of Lac-Z with X-gal stain after gene transfer into the cultured 293 cells and bone marrow cells. As a control group, PLL was used as a cationic polymer. Results. We confirmed that the transfection efficiency of the ald-dex/PLL had a higher transfection efficiency than PLL in 293?cells (plasmid of 2? ? g: ald-dex/PLL 1.1%, PLL 0.23%, plasmid of 16? ? g: ald-dex/PLL 1.23%, PLL 0.48%). In bone marrow cells, we confirmed the expression of Lac-Z by changing the quantity of aldehyded dextran. In the groups using ald-dextran of the quantity of 1/4 and 1/12 of PLL, their transfection efficiency was 0.43% and 0.41%, respectively. Conclusions. This study suggested a potential of using ald-dex/PLL as a non-carrier for gene transfer. PMID:24027586

Togo, Yumiko; Takahashi, Katsu; Saito, Kazuyuki; Kiso, Honoka; Huang, Boyen; Tsukamoto, Hiroko; Hyon, Suong-Hyu; Bessho, Kazuhisa



Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication  

PubMed Central

Summary The use of influenza A virus-inducible reporter gene segments in detecting influenza A virus replication was investigated. The RNA polymerase I promoter/terminator cassette was used to express RNA transcripts encoding green fluorescence protein or firefly luciferase flanked by the untranslated regions of the influenza A/WSN/33 NP segment. Reporter gene activity was detected after reconstitution of the influenza A virus polymerase complex from cDNA or after virus infection, and was influenza A virus-specific. Reporter gene activity could be detected as early as 6 hours post infection and was virus dose-dependent. Inhibitory effects of antibodies or amantadine could be detected a nd quantified rapidly, providing a means of not only identifying influenza A virus -specific replication, but of determining the antigenic subtype as well as antiviral drug susceptibility. Induction of virus-specific reporter genes provides a rapid, sensitive method for detecting virus replication, quantifying virus titers and assessing antiviral sensitivity as well as antigenic subtype.

Lutz, Andrew; Dyall, Julie; Olivo, Paul D.; Pekosz, Andrew



[Gene sequencing by scanning molecular exciton microscopy]. Progress report, October 1, 1990--September 30, 1991  

SciTech Connect

This report details progress made in setting up a laboratory for optical microscopy of genes. The apparatus including a fluorescence microscope, a scanning optical microscope, various spectrometers, and supporting computers is described. Results in developing photon and exciton tips, and in preparing samples are presented. (GHH)

Not Available



Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays  

Microsoft Academic Search

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX® bioassay), measuring

Corine J. Houtman; Saskia S. Sterk; Monique P. M. van de Heijning; Abraham Brouwer; Rainer W. Stephany; Bart van der Burg; Edwin Sonneveld



A mutant of herpes simplex virus type 1 in which the UL13 protein kinase gene is disrupted.  


Gene UL13 of herpes simplex virus type 1 (HSV-1) has previously been proposed to encode a protein kinase. An HSV-1 mutant with UL13 inactivated by insertion of the Escherichia coli lacZ gene was constructed. This UL13-lacZ mutant was found to grow to near wild-type (wt) titres in tissue culture. Comparison of silver-stained SDS-PAGE profiles of wt and UL13-lacZ virions demonstrated that the UL13 protein is a readily detectable component of wt virions, located in the tegument and probably equivalent to the previously described species VP18.8. Studies of in vitro phosphorylation with nuclear extracts of virus-infected cells and with detergent-treated virions showed that the UL13 protein is involved in phosphorylation of the tegument protein VP22. Extracts of cells engineered to express UL13, and infected with UL13-lacZ virus, were also capable of VP22 phosphorylation. PMID:8383174

Coulter, L J; Moss, H W; Lang, J; McGeoch, D J



Ush1c gene expression levels in the ear and eye suggest different roles for Ush1c in neurosensory organs in a new Ush1c knockout mouse.  


Usher syndrome (USH) is the most common form of deaf-blindness in humans. Molecular characterization revealed that the USH gene products form a macromolecular protein network in hair cells of the inner ear and in photoreceptor cells of the retina via binding to PDZ domains in the scaffold protein harmonin encoded by the Ush1c gene in mice and humans. Although several mouse mutants for the Ush1c gene have been described, we generated a targeted null mutation Ush1c mouse model in which the first four exons of the Ush1c gene were replaced with a reporter gene. Here, we assessed the expression pattern of the reporter gene under control of Ush1c regulatory elements and characterized the phenotype of mice defective for Ush1c. These Ush1 knockout mice are deaf but do not recapitulate vision defects before 10 months of age. Our data show LacZ expression in multiple layers of the retina but in neither outer nor inner segments of the photoreceptor layers in mice bearing the knockout construct at 1-5 months of age. The fact that Ush1c expression is much higher in the ear than in the eye suggests a different role for Ush1c in ear function than in the eye and may explain why Ush1c mutant mice do not recapitulate vision defects. PMID:20211154

Tian, Cong; Liu, Xue Z; Han, Fengchan; Yu, Heping; Longo-Guess, Chantal; Yang, Bin; Lu, Changjun; Yan, Denise; Zheng, Qing Y



A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans  

Microsoft Academic Search

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene

Baris Tursun; Luisa Cochella; Inés Carrera; Oliver Hobert; Anne C. Hart



Mutational Analysis of pre-mRNA Splicing in Saccharomyces cerarisiae Using a Sensitive New Reporter Gene, CUPl  

Microsoft Academic Search

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron- containing fragment from the Saccharomyces cerevisiae ACTl gene has been fused to CUPl, the yeast metallothionein homolog. CUPl is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the

Cammie F. Lesser; Christine Guthrie



Characterization of the cis-regulatory region of the Drosophila homeotic gene Sex combs reduced  

SciTech Connect

The Drosophilia 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. This analysis provides an entry point for the study of how Scr transcription is regulated at the molecular level. 60 refs., 7 figs., 1 tab.

Gindhart, J.G. Jr.; King, N.A.; Kaufman, T.C. [Indiana Univ., Bloomington, IN (United States)



Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure  

PubMed Central

BACKGROUND/AIMS—Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.?METHODS—An E1/E3 deleted recombinant adenovirus (denoted AdCMV?Gal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. ?-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.?RESULTS—Intravenous or intraperitoneal injection of AdCMV?Gal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMV?Gal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMV?Gal caused a higher ?-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with standard AdCMV?Gal vectors.?CONCLUSIONS—Local administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation.???Keywords: adenovirus; gene transfer; colitis; colon

Wirtz, S; Galle, P; Neurath, M



Mapping our genes: Federal genome projects: How vast. How fast. : Volume 1, Contractor reports  

SciTech Connect

This report contains contractor contributions solicited by the US Office of Technology Assessment in support of its recommendations for federal organization of the human genome project. The individual reports contained herein are entitled: Bibliometric analysis of work on human gene mapping; Medical implications of extensive physical and sequence characterization of the human genome; Mapping the human genome: Some implications; Mapping and sequencing the human genome: Considerations from the history of particle accelerators; Mapping the human genome: Historical background; Long-term implications of mapping and sequencing the human genome: Ethical and philosophical implications. Each report is also separately abstracted and indexed for the Energy Data Base. (DT)

Reisher, S.R.; Friedmann, T.; Glover, J.; Heilbron, J.L.; Judson, H.F.



Vagaries of Fluorochrome Reporter Gene Expression in Foxp3+ Regulatory T Cells  

PubMed Central

CD4+CD25+ regulatory T (Treg) cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4+CD8+ double-positive (DP) and CD4+CD8? single-positive stages of thymic development, as well as in postthymic CD4+ T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3+ Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC)-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3+ DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4+ T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3+ Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3+ Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene.

Tsai, Pei-Yun; Sparwasser, Tim; Kretschmer, Karsten



Rapid, Specific Detection of Alphaviruses from Tissue Cultures Using a Replicon-Defective Reporter Gene Assay  

PubMed Central

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFP?nsp4 and pVaXJ-GLuc?nsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.

Wang, Huanqin; Li, Jiandong; Zhang, Quanfu; He, Ying; Li, Jia; Fu, Juanjuan; Li, Dexin; Liang, Guodong



An upstream activating sequence from the Aspergillus nidulans gpdA gene.  


Introduction of a previously identified promoter element of the Aspergillus nidulans gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase), the so-called gpd box, into the upstream region of the highly regulated A. nidulans amdS gene (encoding acetamidase), significantly increased (up to 30-fold) the expression of the lacZ reporter gene fused to these expression signals. This increase was dependent on the orientation of the gpd box and on the site of introduction into the amdS upstream region. The presence of additional gpdA sequences which flank the gpd box reduced or even extinguished positive effects of the gpd box. omega-Amino acid and carbon catabolite regulation of the amdS promoter were retained after introduction of the gpd box, indicating that the gpd box does not abolish interactions of the regulatory proteins, AmdR and CreA, with the amdS transcription control sequences. Based on the results, it is suggested that the gpd box comprises at least two separate activities: one being orientation dependent, but relatively independent of position of the gpd box in the upstream region, and the other is only functional near other sites of transcriptional control. Most likely, both activities are not involved in regulation of the amdS promoter. PMID:1398125

Punt, P J; Kramer, C; Kuyvenhoven, A; Pouwels, P H; van den Hondel, C A



Rhizobium meliloti Genes Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions.  


Rhizobium meliloti trc genes controlling the catabolism of trigonelline, a plant secondary metabolite often abundant in legumes, are closely linked to nif-nod genes on the symbiotic megaplasmid pSym [Boivin, C., Malpica, C., Rosenberg, C., Denarie, J., Goldman, A., Fleury, V., Maille, M., Message, B., and Tepfer, D. (1989). In Molecular Signals in the Microbe-Plant Symbiotic and Pathogenic Systems. (Berlin: Springer-Verlag), pp. 401-407]. To investigate the role of trigonelline catabolism in the Rhizobium-legume interaction, we studied the regulation of trc gene expression in free-living and in endosymbiotic bacteria using Escherichia coli lacZ as a reporter gene. Experiments performed with free-living bacteria indicated that trc genes were organized in at least four transcription units and that the substrate trigonelline was a specific inducer for three of them. Noninducing trigonelline-related compounds such as betaines appeared to antagonize the inducing effect of trigonelline. None of the general or symbiotic regulatory genes ntrA, dctB/D, or nodD seemed to be involved in trigonelline catabolism. trc fusions exhibiting a low basal and a high induced [beta]-galactosidase activity when present on pSym were used to monitor trc gene expression in alfalfa tissue under symbiotic conditions. Results showed that trc genes are induced during all the symbiotic steps, i.e., in the rhizosphere, infection threads, and bacteroids of alfalfa, suggesting that trigonelline is a nutrient source throughout the Rhizobium-legume association. PMID:12354952

Boivin, C.; Camut, S.; Malpica, C. A.; Truchet, G.; Rosenberg, C.



Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087.  


Erwinia herbicola strain Eh1087 produces the broad-spectrum phenazine antibiotic D-alanylgriseoluteic acid (AGA). In this report, a cluster of 16 ehp (Erwinia herbicola phenazine) plasmid genes required for the production of AGA by Eh1087 is described. The extent of the gene cluster was revealed by the isolation of 82 different Eh1087 AGA- mutants, all found to possess single mini-Tn5lacZ2 insertions within a 14 kbp DNA region. Additional transposon insertions that did not affect antibiotic production by Eh1087 were created to define the boundaries of the gene cluster. The size and location of genes between these boundaries were derived from a combination of DNA sequence analyses, minicell protein analyses and the correlation between mutation position and the production of coloured AGA intermediates by many ehp mutants. Precursor-feeding and complementation experiments resulted in 15 ehp genes being assigned to one of four functional groups according to their role in the synthesis of AGA. Group 1 is required for the synthesis of the phenazine nucleus in the form of antibiotic precursor one (AP1, phenazine-1,6-dicarboxylic acid). Group 2 is responsible for conversion of AP1 to AP2, which is subsequently modified to AP3 (griseoluteic acid) and exported by the group 3 gene products. Group 4 catalyses the addition of D-alanine to AP3 to create AGA, independently of groups 1, 2 and 3. A gene that is divergently transcribed from the 15 AGA synthesis ehp genes confers resistance to AGA. PMID:12139622

Giddens, Stephen R; Feng, Yunjiang; Mahanty, H Khris




NSDL National Science Digital Library

Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.

Excellence, Access



Chimeric fluorescent reporter as a tool for generation of transgenic Eimeria (Apicomplexa, Coccidia) strains with stage specific reporter gene expression.  


Progress in transfection of Eimeria sporozoites leads to transformed oocysts, however the output of mutants after passages in the host animals is low. Further enrichment of transgenic oocysts was dependent on fluorescent activated cell sorting and could not be achieved by drug selection. In this study, we fused the Toxoplasma gondii DHFR-TSm2m3 pyrimethamine resistance gene with the yellow fluorescent protein (YFP) encoding sequence to provide continuous pyrimethamine resistance and fluorescence in the Eimeria parasite from a single transcript. The permanent YFP signal of transgenic parasites allows differentiating transgenic parasites from wild type parasites throughout the entire life cycle. The output of transformed oocysts increased up to more than 30% after initial transfection and completion of the life cycle in the host animal. Within three passages under pyrimethamine treatment, a strain with 100% transformed sporulated oocysts of the parasite could be isolated. This new method provides the potential to produce and monitor transgenic Eimeria strains without additional fluorescence activated cell sorting (FACS). The chimeric fluorescent reporter can be utilized as a continuous internal control for plasmids containing stage specific promoter. By this means we utilized an Eimeria tenella gamogony gene specific regulatory sequence to confer macrogamont specific tandem dimer tomato (tdtomato) reporter gene expression in Eimeria nieschulzi. PMID:22449589

Hanig, Sacha; Entzeroth, Rolf; Kurth, Michael



Itm2a Is a Pax3 Target Gene, Expressed at Sites of Skeletal Muscle Formation In Vivo  

PubMed Central

The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2–4 with loxP sites and inserting an IRESnLacZ reporter in the 3? UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed.

Lagha, Mounia; Mayeuf-Louchart, Alicia; Chang, Ted; Montarras, Didier; Rocancourt, Didier; Zalc, Antoine; Kormish, Jay; Zaret, Kenneth S.; Buckingham, Margaret E.; Relaix, Frederic



Itm2a is a Pax3 target gene, expressed at sites of skeletal muscle formation in vivo.  


The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2-4 with loxP sites and inserting an IRESnLacZ reporter in the 3' UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed. PMID:23650549

Lagha, Mounia; Mayeuf-Louchart, Alicia; Chang, Ted; Montarras, Didier; Rocancourt, Didier; Zalc, Antoine; Kormish, Jay; Zaret, Kenneth S; Buckingham, Margaret E; Relaix, Frederic



[Preparation and gene expression of transferrin modified gene loaded procationic liposomes].  


A novel transferrin modified non-viral gene delivery system Tf-PLPD was developed and the related characteristics was investigated. Blank procationic liposomes were prepared by film dispersion-filteration method. PLPD was prepared as follows by first mixing the plasmid DNA and protamine together, then the resulted polyplexes were incubated for 10 min at room temperature, followed by addition of preformed blank procationic liposomes. Transferrin was adsorbed at the surface of PLPD via electrostatic interactions to form Tf-PLPD. Central composite design (CCD) was employed to optimize the formulation. The HepG2 cells were transfected using lacZ as reporter gene and characteristics such as the morphology, the mean particle size, the zeta potential and the transfection efficiency in HepG2 cells were further investigated by different methods. The resulting PLPD had a regular spherical surface with an average size of (228. 9 +/- 8. 0) nm (polydispersity index, PDI = 0. 122 +/- 0. 02, n = 3) , a zeta potential of ( - 25. 08 +/-2. 50) mV (n = 3) and a transfection efficiency of (12. 18 +/- 3. 80) mU x mg(-1) (protein). The Tf-PLPD had an average size of (240 +/- 12) nm (polydispersity index, PDI = 0. 150 +/- 0. 03, n = 3), a zeta potential of ( - 24. 10 +/- 2. 50) mV ( n = 3) and a transfection efficiency of (24. 26 +/- 2. 60) mU x mg(-1) (protein) , 20 times greater than that of the naked plasmid DNA. The presence of serum didn' t affect the tansfection activity of PLPD or Tf-PLPD. Compared to one kind of cationic liposomes (liposome-protamine-DNA, LPD), the PLPD and Tf-PLPD had much less cytotoxicity to three hepatic cell lines (including HepG2, SMMC7721 and Chang' s normal hepatocyte). The results indicated that the Tf-PLPD is a perspective non-viral vector for gene delivery systems. PMID:17518055

Zhong, Zhi-rong; Liu, Ji; Deng, Yong; Zhang, Zhi-rong; Song, Qing-guo; He, Qin



A novel mutation in the SH3BP2 gene causes cherubism: case report  

PubMed Central

Background Cherubism is a rare hereditary multi-cystic disease of the jaws, characterized by its typical appearance in early childhood, and stabilization and remission after puberty. It is genetically transmitted in an autosomal dominant fashion and the gene coding for SH3-binding protein 2 (SH3BP2) may be involved. Case presentation We investigated a family consisting of 21 members with 3 female affected individuals with cherubism from Northern China. Of these 21 family members, 17 were recruited for the genetic analysis. We conducted the direct sequence analysis of the SH3BP2 gene among these 17 family members. A disease-causing mutation was identified in exon 9 of the gene. It was an A1517G base change, which leads to a D419G amino acid substitution. Conclusion To our knowledge, the A1517G mutation has not been reported previously in cherubism. This finding is novel.

Li, Cui-Ying; Yu, Shi-Feng



Systemic delivery of AAV8 in utero results in gene expression in diaphragm and limb muscle: Treatment implications for muscle disorders  

PubMed Central

One of the major challenges in the treatment of primary muscle disorders, which often affect many muscle groups, is achieving efficient, widespread transgene expression in muscle. In utero gene transfer can potentially address this problem by accomplishing gene delivery when the tissue mass is small and the immune system is immature. Previous studies with systemic in utero adeno-associated viral (AAV) vector serotype 1 gene delivery to embryonic day 16 (E-16) pups resulted in high levels of transduction in diaphragm and intercostal muscles, but no detectable transgene expression in limb muscles. Recently newer AAV serotypes such as AAV8 have demonstrated widespread and high transgene expression in skeletal muscles and diaphragm by systemic delivery in adult and neonatal mice. We tested AAV8 vector gene delivery by intraperitoneal administration in E-16 mice in utero. Using an AAV8 vector carrying a lacZ reporter gene, we observed high level transduction of diaphragm and intercostal muscles and more moderate transduction of multiple limb muscles and heart. Our current studies demonstrate the potential of AAV8 to achieve widespread muscle transduction in utero and suggest its therapeutic potential for primary muscle disorders.

Koppanati, Bhanu Munil; Li, Juan; Xiao, Xiao; Clemens, Paula R.



Detection of Indicator Bacteria and Pathogens in Water by Polym Erase Chain Reaction (PCR) and Gene Probe Methods  

Microsoft Academic Search

Sensitive methods for detecting coliform bacteria, and pathogenic bacteria (Salmonella and Shiqella) in environmental waters that do not require culturing of bacteria were developed by using the polymerase chain reaction\\u000a (PCR) and gene probes. Cells were collected by filtration and DNA was released by freeze-thaw cycling. PCR amplification of\\u000a region of lacZ gene was used as a target for detection

R. Atlas; A. Bej; M. Mahbubani; R. Steffan; M. Perlin; J. DiCesare; L. Haff


L-cysteine biosynthesis in Bacillus subtilis: identification, sequencing, and functional characterization of the gene coding for phosphoadenylylsulfate sulfotransferase.  

PubMed Central

Random Tn917 mutagenesis of Bacillus subtilis followed by selection of lipoic acid auxotrophs led to the isolation of the cysH gene. The gene was sequenced and found to encode a phosphoadenylylsulfate sulfotransferase with a molecular mass of 27 kDa. Expression of lacZ fused to the cysH promoter was repressed by cysteine and sulfide and induced by sulfur limitation, indicating that cysH is controlled at the level of transcription.

Mansilla, M C; de Mendoza, D



Transcriptional Regulation and Evolution of Lactose Genes in the Galactose-Lactose Operon of Lactococcus lactis NCDO2054  

Microsoft Academic Search

The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes. Initially the b-galac- tosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment. Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and




Analysis of protein tyrosine kinase inhibitors in recombinant yeast lacking the ERG6 gene.  


Studies of small-molecule-protein interactions in yeast can be hindered by the limited permeability of yeast to small molecules. This diminished permeability is thought to be related to the unique sterol composition of fungal membranes, which are enriched in the steroid ergosterol. We report the construction of the novel Saccharomyces cerevisiae yeast strain DCY250, which is compatible with yeast two-hybrid-based systems and bears a targeted disruption of the ERG6 gene to ablate ergosterol biosynthesis and enhance permeability to small molecules. The small-molecule inhibitors of protein tyrosine kinases (PTKs) PP1, PP2, herbimycin A, and staurosporine were investigated with yeast tribrid systems that detect the activity of the PTKs v-Abl and v-Src. These tribrid systems function by expression of the PTK, a B42 activation domain fused to the phosphotyrosine-binding Grb2 SH2 domain, a DNA-bound LexA-GFP-(AAYANAA)(4) universal PTK substrate, and a lacZ reporter gene. Yeast genetic systems that lack functional ERG6 were found to be as much as 20-fold more sensitive to small-molecule inhibitors of PTKs than systems with ERG6, and these deficient systems may provide a useful platform for the discovery and analysis of small-molecule-protein interactions. PMID:12512083

Clark, Daniel D; Peterson, Blake R



A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.  


Activation of hepatic stellate cells (HSCs) and other resident mesenchymal cells into myofibroblasts expressing alpha smooth muscle actin (alphaSMA) and collagen I is a key event in liver fibrogenesis. However, the temporal expression profiles of alphaSMA and collagen I genes in these cells is unknown. To address this question, we studied alphaSMA and collagen alpha1(I) transcriptional patterns in primary cultures of HSCs, and additionally, in an in vivo model of secondary biliary fibrosis using transgenic mice that express the Discomsoma sp. red fluorescent protein (RFP) and the enhanced green fluorescent protein (EGFP) reporter genes under direction of the mouse alphaSMA and collagen alpha1(I) promoter/enhancers, respectively. The alphaSMA-RFP mice were crossed with collagen-EGFP mice to generate double transgenic mice. Reporter gene expression in cultured HSCs demonstrated that both transgenes were induced at day 3 with continued expression through day 14. Interestingly, alphaSMA and collagen alpha1(I) transgenes were not coexpressed in all cells. Flow cytometry analysis showed three different patterns of gene expression: alphaSMA-RFP positive cells, collagen-EGFP positive cells, and cells expressing both transgenes. AlphaSMA-only and alphaSMA/collagen expressing cells showed higher expression levels of synaptophysin, reelin, MMP13, TIMP1, and ICAM-1 compared to collagen-only expressing cells, as assessed by real-time PCR. Following bile duct ligation, alphaSMA and collagen alpha1(I) transgenes were differentially expressed by peribiliary, parenchymal and vascular fibrogenic cells. Peribiliary cells preferentially expressed collagen alpha1(I), while parenchymal myofibroblasts expressed both alphaSMA and collagen alpha1(I). In conclusion, these data demonstrate heterogeneity of gene expression in myofibroblastic cells during active fibrogenesis. These reporter mice provide a useful tool to further characterize fibrogenic cell types and to evaluate antifibrotic drugs. PMID:15389867

Magness, Scott T; Bataller, Ramón; Yang, Liu; Brenner, David A



Firefly luciferase as a reporter of regulated gene expression in higher plants  

Microsoft Academic Search

The firefly luciferase, assayedin vivo with a low-light video camera, acts as a non-invasive, real-time reporter of the temporal and spatial regulation of gene\\u000a expression in single plants. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue\\u000a makes it a particularly useful marker in transient or stable transformation experiments.

Andrew J. Millar; Sharla R. Short; Kazuyuki Hiratsuka; Nam-Hai Chua; Steve A. Kay



Fiber optic detection of in situ lux reporter gene activity in porous media: system design and performance  

Microsoft Academic Search

A luminescence detection system is described that couples a genetically engineered bioluminescent reporter organism and fiber optic technology for monitoring in situ reporter gene activity in porous media under dynamic conditions. The reporter bacterium used was Pseudomonasputida RB1353, which carries plasmids NAH7 and pUTK9 that encode genes for salicylate degradation (nah) and luminescence (lux) that are regulated by the same

Irfan Yolcubal; Joseph J. Piatt; Shelley A. Pierce; Mark L. Brusseau; Raina M. Maier



Generation of a genomic reporter assay system for analysis of ?- and ?-globin gene regulation.  


A greater understanding of the regulatory mechanisms that govern ?-globin expression in humans, especially the switching from ?- to ?-globin, which occurs after birth, would help to identify new therapeutic targets for patients with ?-hemoglobinopathy. To further elucidate the mechanisms involved in ?-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human ?-globin locus under the control of (G)?- or (A)?-globin promoter and ?-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a ?-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with ?-globin gene suppression. PMID:22267339

Chan, Kasey S K; Xu, Jian; Wardan, Hady; McColl, Bradley; Orkin, Stuart; Vadolas, Jim



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

SciTech Connect

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.

Estelle, Mark



Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation.  


The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino-terminal moieties of the Escherichia coli lacZ gene carrying a multiple-cloning site flanked by phage-specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC-like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning-dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ-phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ alpha-complementation-based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ-phoC fusion-based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles. PMID:9548775

Thaller, M C; Berlutti, F; Schippa, S; Selan, L; Rossolini, G M


Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes.  

PubMed Central

trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS. Images

Bahner, I; Zhou, C; Yu, X J; Hao, Q L; Guatelli, J C; Kohn, D B



Conditional mutagenesis of the murine serum response factor gene blocks cardiogenesis and the transcription of downstream gene targets.  


Serum response factor (SRF) homozygous-null embryos from our backcross of SRF(LacZ/)(+) "knock-in" mice failed to gastrulate and form mesoderm, similar to the findings of an earlier study (Arsenian, S., Weinhold, B., Oelgeschlager, M., Ruther, U., and Nordheim, A. (1998) EMBO J. 17, 6289-6299). Our use of embryonic stem cells provided a model system that could be used to investigate the specification of multiple embryonic lineages, including cardiac myocytes. We observed the absence of myogenic alpha-actins, SM22alpha, and myocardin expression and the failure to form beating cardiac myocytes in aggregated SRF null embryonic stem cells, whereas the appearance of transcription factors Nkx2-5 and GATA4 were unaffected. To study the role of SRF during heart organogenesis, we then performed cardiac-specific ablation of SRF by crossing the transgenic alpha-myosin heavy chain Cre recombinase line with SRF LoxP-engineered mice. Cardiac-specific ablation of SRF resulted in embryonic lethality due to cardiac insufficiency during chamber maturation. Conditional ablation of SRF also reduced cell survival concomitant with increased apoptosis and reduced cellularity. Significant reductions in SRF (> or =95%), atrial naturetic factor (> or =80%), and cardiac (> or =60%), skeletal (> or =90%), and smooth muscle (> or =75%) alpha-actin transcripts were also observed in the cardiac-conditional knock-out heart. This was consistent with the idea that SRF directs de novo cardiac and smooth muscle gene activities. Finally, quantitation of the knock-in LacZ reporter gene transcripts in the hearts of cardiac-conditional knock-out embryos revealed an approximately 30% reduction in gene activity, indicating SRF gene autoregulation during cardiogenesis. PMID:15929941

Niu, Zhiyv; Yu, Wei; Zhang, Shu Xing; Barron, Matthew; Belaguli, Narasimhaswamy S; Schneider, Michael D; Parmacek, Michael; Nordheim, Alfred; Schwartz, Robert J



Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting  

Microsoft Academic Search

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced

Jakub Tolar; Jennifer E Adair; Michael Antoniou; Cynthia C Bartholomae; Pamela S Becker; Bruce R Blazar; Juan Bueren; Thomas Carroll; Marina Cavazzana-Calvo; D Wade Clapp; Robert Dalgleish; Anne Galy; H Bobby Gaspar; Helmut Hanenberg; Christof Von Kalle; Hans-Peter Kiem; Dirk Lindeman; Luigi Naldini; Susana Navarro; Raffaele Renella; Paula Rio; Julián Sevilla; Manfred Schmidt; Els Verhoeyen; John E Wagner; David A Williams; Adrian J Thrasher



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

SciTech Connect

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.

VanEtten, H.



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

PubMed Central

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.



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  

Microsoft Academic Search

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

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



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

SciTech Connect

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 d

Braam, Janet



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

Microsoft Academic Search

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

Bertrand Plesse; Marie-Claire Criqui; Andrée Durr; Yves Parmentier; Jacqueline Fleck; Pascal Genschik



X-linked agammaglobulinemia caused by new mutation in BTK gene: A case report.  


AIM: Primary immunodeficiencies (PID) are becoming a recognized public health problem worldwide. The most important subgroup of these disorders are the antibody deficiencies. X-linked agammaglobulinaemia was the first described entity of this group and is characterised by early onset of recurrent bacterial infections, profound deficiency of all immunoglobulin isotypes and markedly reduced number of peripheral B-lymphocytes. CASE REPORT: We report the case of a 10-year old boy with X-linked agammaglobulinaemia caused by a previously non-described mutation in BTK gene with typical clinical presentation but delayed diagnosis. Following diagnosis, substitution therapy with intravenous immunoglobulins was started and the clinical status of the patient improved. CONCLUSION: We reported a case of X-linked agammaglobulinaemia with delayed diagnosis despite the typical anamnestic signs for primary humoral immunodeficiency. The disease was caused by a previously non-reported mutation in the BTK gene. Measurement of serum immunoglobulins should be performed in all children with recurrent, complicated respiratory infections as a screening test for humoral immunodeficiencies. PMID:23549506

Havlicekova, Zuzana; Jesenak, Milos; Freiberger, Tomas; Banovcin, Peter



Naringenin Regulates Expression of Genes Involved in Cell Wall Synthesis in Herbaspirillum seropedicae?  

PubMed Central

Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.

Tadra-Sfeir, M. Z.; Souza, E. M.; Faoro, H.; Muller-Santos, M.; Baura, V. A.; Tuleski, T. R.; Rigo, L. U.; Yates, M. G.; Wassem, R.; Pedrosa, F. O.; Monteiro, R. A.



Complementation of two related tumour cell classes during experimental metastasis tagged with different histochemical marker genes  

Microsoft Academic Search

Intercellular complementation during tumour development and metastasis was analysed for two different oncogene (ras or sis) transformants of Balb\\/c 3T3 cells, tagged with different histochemical marker genes (lacZ or ALP to generate LZEJ or APSI cells, respectively), by localising them after their co-injection with specific double-staining protocols. This model evaluates whether limited progression of each tumour class can be facilitated

W-C Lin; KL O'Connor; LA Culp



Light and Developmental Regulation of the Gene con-10 of Neurospora crassa  

Microsoft Academic Search

The gene con-10 of Neurospora crassa is expressed preferentially during conidiation and following illumination of vegetative mycelia with blue light. In this study we have examined the segmental locations of the genetic elements associated with con-10 that are responsible for light and developmental expression. A translational fusion was prepared between the initial segment of con-10 and Escherichia coli lacZ. Deletions

Luis M. Corrochano; Frank-Roman Lauter; Daniel J. Ebbole; Charles Yanofsky



Translational regulation of hepatitis B virus polymerase gene by termination-reinitiation of an upstream minicistron in a length-dependent manner.  


Hepatitis B virus (HBV) polymerase (P) gene is translated from the bicistronic pregenomic RNA with the core (C) gene in the first cistron. The P ORF is preceded by the C AUG and three AUG codons within the C region, where a minicistron of 7 amino acids can potentially be translated. Our results indicate that the efficiency of the P gene translation initiation was about 10% of that of the C gene when both genes were fused in-frame to a lacZ reporter in an mRNA similar in structure to the pregenomic RNA. By mutational analysis, about 74% of the translation initiation of HBV P gene was shown to be by ribosomes that reinitiated after terminating translation of this minicistron, while the rest was by two mechanisms: one by ribosomes leaky scanning through every upstream AUG and the other by ribosomal backwards scanning to the P AUG after finishing the translation of the C gene. The efficiency of termination-reinitiation depended on the size of the minicistron, i.e. the reinitiation efficiency decreased about 50% when the size increased from 24 nt to 57 nt. When a 44 nt HBV sequence comprising the minicistron was inserted at the 5' untranslated region of the cat gene, CAT expression was regulated in a similar way to that of the HBV P gene. Moreover, when transfection occurred with an HBV expression plasmid containing an inactivated minicistron, production of virus-like particles dropped to about one-third of the wild-type level, suggesting that the termination-reinitiation mechanism is indeed important for HBV P gene expression. PMID:9747727

Hwang, W L; Su, T S



The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous URA3 as a Reporter Gene in Ganoderma lucidum  

PubMed Central

Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5?-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.

Mu, Dashuai; Shi, Liang; Ren, Ang; Li, Mengjiao; Wu, Fengli; Jiang, Ailiang; Zhao, Mingwen



The usefulness of the gfp reporter gene for monitoring Agrobacterium -mediated transformation of potato dihaploid and tetraploid genotypes  

Microsoft Academic Search

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish\\u000a a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using\\u000a Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin

Elena Rakosy-Tican; Cristian M. Aurori; Camelia Dijkstra; Ramona Thieme; Adriana Aurori; Michael R. Davey



A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells  

PubMed Central

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.

Marc, J; Granger, CL; Brincat, J; Fisher, DD; Kao, Th; McCubbin, AG; Cyr, RJ



Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells.  


The small marine ostracod crustacean, Vargula hilgendorfii, produces a bright blue luminous secretion which is ejected into seawater. The luminescence is due to a simple enzyme-catalyzed reaction involving only luciferase, luciferin (substrate), and molecular oxygen. Thus, V. hilgendorfii luciferase (VL) should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of VL cDNA (vl) linked to the promoters simian virus 40 early region, Rous sarcoma virus long terminal repeat, human elongation factor, or mouse granulocyte colony-stimulating factor were introduced into a series of mammalian cell lines. Following transfection, VL activities in cell extracts and culture media were determined by a rapid light emission assay with V. hilgendorfii luciferin. Parallel experiments were carried out with the chloramphenicol acetyltransferase (CAT)-encoding gene. In all cell lines tested, VL was secreted, allowing the reporter activity to be determined directly from a small aliquot of the culture medium. The results indicate that the secreted VL enzyme is superior to CAT, firefly luciferase, and bacterial luciferase as a convenient and versatile indicator of gene expression in mammalian cells. PMID:2269435

Thompson, E M; Nagata, S; Tsuji, F I



Inhibition of measles virus minireplicon-encoded reporter gene expression by V protein.  


Measles virus V protein is a Cys-rich polypeptide that is dispensable for virus propagation in continuous cell lines, but necessary for efficient viral replication in animals. Those functions modulating virus propagation in vivo are not understood completely, although V protein is known to interfere with the host interferon response and control of viral gene expression. The ability to modulate gene expression was investigated further with a minireplicon transient expression system in which V protein was found to repress reporter activity. Two regions of the polypeptide contributed to this repressive effect including the carboxy-terminus and a region conserved in morbillivirus V proteins located between amino acids 110-131, whereas domains known to mediate the interaction between V and the nucleocapsid (N) protein were not essential. Accumulation of encapsidated minigenome in transfected cells was inhibited by V protein suggesting that it acted as a repressor of genome replication thereby limiting availability of template for reporter gene mRNA transcription. PMID:16445957

Witko, Susan E; Kotash, Cheryl; Sidhu, Mohinderjit S; Udem, Stephen A; Parks, Christopher L



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


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 ?(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

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



Deletion of a single-copy DAAM1 gene in congenital heart defect: a case report  

PubMed Central

Background With an increasing incidence of congenital heart defects (CHDs) in recent years, genotype-phenotype correlation and array-based methods have contributed to the genome-wide analysis and understanding of genetic variations in the CHD population. Here, we report a copy number deletion of chromosomal 14q23.1 in a female fetus with complex congenital heart defects. This is the first description of DAAM1 gene deletion associated with congenital heart anomalies. Case Presentation Compared with the control population, one CHD fetus showed a unique copy number deletion of 14q23.1, a region that harbored DAAM1 and KIAA0666 genes. Conclusions Results suggest that the copy number deletion on chromosome 14q23.1 may be critical for cardiogenesis. However, the exact relationship and mechanism of how DAAM1 and KIAA0666 deletion contributes to the onset of CHD is yet to be determined.



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

PubMed Central

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.

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



Inducible gene expression in fetal thymic epithelium: A new BAC transgenic model.  


The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium. genesis 51:717-724. © 2013 Wiley Periodicals, Inc. PMID:23832856

Fiorini, Emma; Ferrero, Isabel; Poisson, Caroline; Scarpellino, Leonardo; Luther, Sanjiv Andreas; Macdonald, H Robson



Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.  

PubMed Central

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid. Images Fig. 2 Fig. 5

Lawrence, M S; Ho, D Y; Dash, R; Sapolsky, R M



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

SciTech Connect

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.




Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery  

Microsoft Academic Search

Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI\\/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology.

Erh-Hsuan Lin; Michelle Keramidas; Claire Rome; Wen-Ta Chiu; Cheng-Wen Wu; Jean-Luc Coll; Win-Ping Deng



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

SciTech Connect

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.

Kuchka, M.R.



Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report  

PubMed Central

Background Melanoma of the anal region is a very uncommon disease, accounting for only 0.2-0.3% of all melanoma cases. Mutations of the BRAF gene are usually absent in melanomas occurring in this region as well as in other sun-protected regions. The development of a tumour in a longstanding perianal fistula is also extremely rare. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process, although we have found only two cases of fistula-generating melanomas reported in the literature. Case Presentation Here we report the case of a 38-year-old male who presented with a perianal fistula of four years of evolution. Histopathological examination of the fistulous tract confirmed the presence of malignant melanoma. Due to the small size and the central location of the melanoma inside the fistulous tract, we believe the melanoma reported here developed in the epithelium of the fistula once the latter was already formed. Resected sentinel lymph nodes were negative and the patient, after going through a wide local excision, remains disease-free nine years after diagnosis. DNA obtained from melanoma tissue was analysed by automated direct sequencing and the V600E (T1799A) mutation was detected in exon 15 of the BRAF gene. Conclusion Since fistulae experience persistent inflammation, the fact that this melanoma harbours a BRAF mutation strengthens the view that oxidative stress caused by inflammatory processes plays an important role in the genesis of BRAF gene mutations.



Expression of Escherichia coli uvr genes in mammalian cells. Progress report  

SciTech Connect

The goal was to determine if Escherichia coli uvr genes could be expressed in repair deficient mammalian cells (Xeroderma pigmentosum). Progress is reported in the following areas: (1) ATPase activity was assessed in which the release of labelled orthophosphate from ..gamma..-(/sup 32/P)-ATP is effected by damaged DNA and the uvrB protein; (2) the binding of the uvrA protein to damaged DNA was tested; and (3) a double uvrA and uvrC vector was engineered with gpt and the gpt/sup +/ clones isolated. 2 references, 2 figures. (ACR)

Not Available



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

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.

Black, R.C. (Pennsylvania State Univ., Media (USA)); Labriola, J.; Binns, A.N. (Univ. of Pennsylvania, Philadelphia (USA))



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

|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…

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



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

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…

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



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


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

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



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

PubMed Central

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.

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



Cytochrome P450 2C9 gene polymorphism in phenytoin induced gingival enlargement: A case report.  


Gingival enlargement comprises any clinical condition in which an increase in the size of the gingiva is observed. Among the drugs that induce gingival enlargement, the antiepileptic agent phenytoin has been widely related to this condition. The Cytochrome P450(CYP) superfamily is the most commonly involved enzymes in metabolism of drugs. Common coding region CYP variants that affects drug elimination and response has been studied in great detail. Pharmacogenetic influences on drug metabolism have been widely reviewed and gene polymorphism of cytochrome P450 2C9 appeared to be responsible for much of the interindividual variability on drug elimination. Genetic variation in the CYP2C9 gene can affect metabolism, leading to altered phenotypes. Individuals with poor metaboliser alleles of CYP2C9 gene were shown to have a reduced metabolism of phenytoin compared with wild-type alleles. Thus identification of patients genotype prior to anti-epileptic drug administration could potentially prevent higher serum drug concentrations leading to adverse side effects such as gingival enlargement. This case report addresses the influence of CYP2C9 genetic polymorphism on Phenytoin drug metabolism thereby causing gingival enlargement. PMID:24082701

Babu, S P K Kennedy; Ramesh, V; Samidorai, Agila; Charles, N S C



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

SciTech Connect

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.

Fields, C.A.



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

SciTech Connect

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)

Ts`o, P.O.P.



The application of reporter gene assays for the detection of endocrine disruptors in sport supplements.  


The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal. PMID:21742114

Plotan, Monika; Elliott, Christopher T; Scippo, Marie Louise; Muller, Marc; Antignac, Jean-Philippe; Malone, Edward; Bovee, Toine F H; Mitchell, Samuel; Connolly, Lisa



Expression of ADAM28 and IGFBP-3 genes in patients with colorectal cancer - a preliminary report.  


Adamalisynes (ADAMs) play an important role in inter-membrane interactions, cell adhesion and fusion processes and protein shedding from the cell surface. Many reports indicate that members of the ADAMs family are overexpressed in human cancer. The aim of the present study was to evaluate ADAM28 and Insulin Like Growth Factor Binding Protein-3 (IGFBP-3)) gene expression in colorectal carcinoma tissues with regard to the overweight or obese status of the patients using an oligonucleotide microarray technique. Fresh tissue specimens were obtained from colorectal cancer patients during surgical treatment. Eighteen specimens from tumour and 18 normal tissue specimens from colorectal cancer patients at clinical stages III and IV were analysed. The examined patients were divided into two groups; those with BMI greater than or equal to 25 and those with normal BMI. The control group consisted of 18 specimens of non-neoplastic colon tissues, which were divided between overweight/obese and normal body weight patients. The gene transcriptional activity from the specimens was analysed using an oligonucleotide microarray technique. Microarrays and rinsing and marking solutions were prepared according to the procedure in the Gene Expression Analysis Technical Manual. The following conclusions were made: i) change of ADAM28 and IGFBP-3 genes expression are present in the normal tissue in overweight/obese patients with colorectal cancer only; ii) the observed molecular variability of ADAM28 and IGFBP-3 expression may be an initial process of cancer proliferation; iii) the histopathologically normal surgical margin in this group of patients was not equal to the molecular margin. PMID:23527725

Nowakowska-Zajdel, E; Mazurek, U; Wierzgon, J; Kokot, T; Fatyga, E; Ziolko, E; Klakla, K; Blazelonis, A; Waniczek, D; Glogowski, L; Kozowicz, A; Niedworok, E; Muc-Wierzgon, M


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


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

Sřlvsten, Christina; Nielsen, Anders Lade



A novel reporter gene assay for interferons based on CHO-K1 cells.  


Interferons (IFNs) are cytokines playing an important role in the immune response and defence against viruses. They are widely used as biopharmaceuticals. Currently, the anti-viral assay (AVA) is the most commonly used bioassay for determining interferon potency. In the search for rapid and robust but reliable methods, reporter gene assays (RGA) appear to be the most promising approach, therefore we have designed a new reporter cell line, CHO-ISRE-SEAP, suitable for determination of type I interferon potency. Chinese hamster ovary (CHO-K1) cells were stably transfected with secretory alkaline phosphatase (SEAP) gene under the control of interferon stimulated response element (ISRE) promoter. The amount of SEAP in the cell culture medium can be easily measured colorimetrically and has been found to correlate with the amount of IFN added. The new assay is widely applicable for determination of type I IFNs, such as IFN-alpha, IFN-beta and IFN-omega, in research, development of IFN biopharmaceuticals, in batch release, etc. Interestingly, in this assay, IFN-beta shows approximately 6 times higher response than IFN-alpha, which makes it especially appropriate for measuring low levels of IFN-beta. Compared to other known RGAs, the novel CHO-ISRE-SEAP cell line-based RGA appears to have certain advantages with respect to cost and performance. PMID:18295789

Smilovi?, V; Caserman, S; Fonda, I; Gaberc-Porekar, V; Menart, V



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

SciTech Connect

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.

Ge Shimei [College of Biological Sciences and National Key Laboratory for Agrobiotechnology, Key Laboratory of Agro-Microbial and Application, China Agricultural University, Beijing 100094 (China); Xie Baoen [College of Biological Sciences and National Key Laboratory for Agrobiotechnology, Key Laboratory of Agro-Microbial and Application, China Agricultural University, Beijing 100094 (China); Chen Sanfeng [College of Biological Sciences and National Key Laboratory for Agrobiotechnology, Key Laboratory of Agro-Microbial and Application, China Agricultural University, Beijing 100094 (China)]. E-mail:



Structural Consequences of Kcna1 Gene Deletion and Transfer in the Mouse Hippocampus  

PubMed Central

Purpose Mice lacking the Kv1.1 potassium channel ? subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1?/? (“knock-out”) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. Methods Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1?/? animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1?/? brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E.coli lacZ reporter gene. Vector-injected mice were were examined to determine the extent of neuronal infection. Results Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1?/? mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed “ectopic” Kv1.1 ? channel subunit expression. Conclusions Kcna1 deletion in mice results in a seizure disorder that resembles – electrographically and neuropathologically – the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection

Wenzel, H. Jurgen; Vacher, Helene; Clark, Eliana; Trimmer, James S.; Lee, Angela L.; Sapolsky, Robert M.; Tempel, Bruce L; Schwartzkroin, Philip A.



Real-Time Reporting of Circadian-Regulated Gene Expression by Luciferase Imaging in Plants and Mammalian Cells  

Microsoft Academic Search

Luciferase enzymes have been used as reporters of circadian rhythms in organisms as diverse as cyanobacteria, plants, fruit flies, and mice. This article details methodology for real-time reporting of circadian-regulated gene expression by imaging of luciferase bioluminescence in plants and mammalian cells.

David K. Welsh; Takato Imaizumi; Steve A. Kay



Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5  

Microsoft Academic Search

The transcriptional regulation of three distinct alcohol oxidation systems, alcohol dehydrogenase (ADH)-I, ADH-IIB and ADH-IIG, in Pseudomonas putida HK5 was investigated under various induction conditions. The promoter activities of the genes involved in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC, encoding ADH-I and

W. Promden; A. S. Vangnai; H. Toyama; K. Matsushita; P. Pongsawasdi



Cross-species characterization of the ALS2 gene and analysis of its pattern of expression in development and adulthood  

Microsoft Academic Search

Mutations in the ALS2 gene, which encodes alsin, cause autosomal recessive juvenile-onset amyotrophic lateral sclerosis (ALS2) and related conditions. Using both a novel monoclonal antibody and LacZ knock-in mice, we demonstrate that alsin is widely expressed in neurons of the CNS, including the cortex, brain stem and motor neurons of the spinal cord. Interestingly, the highest levels of alsin are

Rebecca S. Devon; Claudia Schwab; Justin D. Topp; Paul C. Orban; Yu-zhou Yang; Terry D. Pape; Jeffrey R. Helm; Tara-Lynne Davidson; Daniel A. Rogers; Francois Gros-Louis; Guy Rouleau; Bruce F. Horazdovsky; Blair R. Leavitt; Michael R. Hayden



Skeletal muscle regeneration after insulin-like growth factor I gene transfer by recombinant Sendai virus vector  

Microsoft Academic Search

We scrutinized the applicability and efficacy of Sendai virus (SeV) vectors expressing either LacZ or human insulin-like growth factor-I (hIGF-I) in gene transfer into skeletal muscle. Seven days after the intramuscular injection of LacZ\\/SeV X-gal labeled myofibers were demonstrated in rat anterior tibialis muscle with\\/without bupivacaine treatment and the transgene expression persisted up to 1 month after injection. Recombinant hIGF-I

A Shiotani; M Fukumura; M Maeda; X Hou; M Inoue; T Kanamori; S Komaba; K Washizawa; S Fujikawa; T Yamamoto; C Kadono; K Watabe; H Fukuda; K Saito; Y Sakai; Y Nagai; J Kanzaki; M Hasegawa



The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.  

PubMed Central

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

Kaouass, M; Audette, M; Ramotar, D; Verma, S; De Montigny, D; Gamache, I; Torossian, K; Poulin, R



The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.  


Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity. PMID:9154797

Kaouass, M; Audette, M; Ramotar, D; Verma, S; De Montigny, D; Gamache, I; Torossian, K; Poulin, R



Mutation in the cobO gene generates auxotrophy for cobalamin and methionine and impairs the symbiotic properties of Sinorhizobium fredii HH103 with soybean and other legumes.  


We report here the isolation of a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 obtained by Tn5-lacZ mutagenesis. Sequence analysis showed that the transposon was inserted into a gene homologous to cobO. This gene codes for a cobalamin adenosyltransferase which is involved in the biosynthesis of vitamin B12. Another HH103 cobO mutant (strain SVQ524), was constructed by the insertion of Omega interposon. Both cobO mutants required the addition of methionine because cobalamin acts as a cofactor of the enzyme MetH, which catalyses the last step of the methionine biosynthesis. Mutant SVQ524 failed to nodulate on Vigna radiate but was able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan, although the total number of nodules formed was highly reduced in comparison with that of plants inoculated with the wild-type strain HH103. The roots of these plants did not seem to secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine auxotrophs in the rhizosphere. In all cases, the phenotype of SVQ524 was nearly overcome by the addition of methionine or cobalamin to the plant growth media or by the presence of a copy of the cobO gene in cosmid pMUS756. PMID:18719891

Medina, Carlos; Crespo-Rivas, Juan Carlos; Moreno, Javier; Espuny, María Rosario; Cubo, María Teresa



Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b: cloning, sequencing, and expression of the tyrosinase gene mepA.  

PubMed Central

Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b (140 MDa). Transfer of this plasmid to GR4-cured derivatives or to Agrobacterium tumefaciens enables these bacteria to produce melanin. Sequence analysis of a 3.5-kb PstI fragment of plasmid pRmeGR4b has revealed the presence of a open reading frame 1,481-bp that codes for a protein whose sequence shows strong homology to two conserved regions involved in copper binding in tyrosinases and hemocyanins. In vitro-coupled transcription-translation experiments showed that this open reading frame codes for a 55-kDa polypeptide. Melanin production in GR4 is not under the control of the RpoN-NifA regulatory system, unlike that in R. leguminosarum bv. phaseoli 8002. The GR4 tyrosinase gene could be expressed in Escherichia coli under the control of the lacZ promoter. For avoiding confusion with mel genes (for melibiose), a change of the name of the previously reported mel genes of R. leguminosarum bv. phaseoli and other organisms to mep genes (for melanin production) is proposed. Images

Mercado-Blanco, J; Garcia, F; Fernandez-Lopez, M; Olivares, J



Homozygous survival motor neuron 2 gene deletion and sporadic lower motor neuron disease in children: case report and literature review.  


A case of lower motor neuron disease with homozygous survival motor neuron 2 (SMN2) gene deletion is reported in this article. A 7-year-old boy was admitted to our hospital with main complaints of lower extremity weakness and difficulty squatting for the past year. SMN gene copies were quantified by multiplex ligation-dependent probe amplification. Exons 7 and 8 of the SMN1 gene were normal, but homozygous deletion of exons 7 and 8 of the SMN2 gene was identified. Homozygous deletion of exons 7 and 8 of the SMN centromeric gene was detected, and exons 7 and 8 of the SMN1 gene were found to be normal in the proband. Two copies of exons 7 and 8 of the SMN1 gene were identified, and zero copies of exons 7 and 8 of the SMN2 gene were found. We consider that this case represents a previously unrecognized type of lower motor neuron disease that resulted from homozygous deletion of the SMN2 gene. PMID:22628217

Liping, Lu; Hongwei, Ma; Lin, Wang



Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia  

PubMed Central

Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (Gast?/?) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1? expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. LacZ reporter mice for Sonic hedgehog (Shh), Gli1, and Gli2 expression bred onto the Gast?/? background revealed reduced Shh and Gli1 expression in the antra compared to wild type controls (WT). Gli2 expression in the Gast?/? corpus was unchanged. However in the hyperplastic Gast?/? antra, Gli2 expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that Gli2 is differentially regulated in the hyperplastic Gast?/? antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1? and Il-11, which promote gastric epithelial proliferation, were increased in the Gast?/? stomach along with Inf?. To test if inflammation could account for elevated epithelial Gli2 expression in the Gast?/? antra, the human gastric cell line AGS was treated with IL-1? and was found to increase GLI2 but decrease GLI1 levels. IL-1? also repressed human GAST gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ?50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the GAST promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed Gast expression but induced Il-1? gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate Il-1? expression, repress Gast gene expression and increase proliferation, leading to antral hyperplasia.

Saqui-Salces, Milena; Coves-Datson, Evelyn; Veniaminova, Natalia A.; Waghray, Meghna; Syu, Li-Jyun; Dlugosz, Andrzej A.; Merchant, Juanita L.



Regulation of Escherichia coli superoxide dismutase genes (sodA and sodB) by oxygen  

Microsoft Academic Search

Two types of superoxide dismutase genes, sodA and sodB, were fused to ß-galactosidase gene (lacZ), in order to quantitatively study the effect of oxygen concentration on the gene expression of sodA and sodB. ß-Galactosidase activity derived from the sodA-lacZ fusion was induced by shifting from anaerobic condition to aerobic condition. Maximum activity (9.4×103 U\\/OD660) was observed when oxygen partial pressure

Yoshinobu Matsumura; Masahiro Takagi; Tadayuki Imanaka



In situ Detection of ? -Galactosidase in Lenses of Transgenic Mice with a ? -Crystallin/lacZ Gene  

NASA Astrophysics Data System (ADS)

Transgenic mice carrying the ? 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of ? -galactosidase activity. These results suggest that ? 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse ? 2-crystallin gene. In a broader context, this study also demonstrates the utility of ? -galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.

Goring, D. R.; Rossant, J.; Clapoff, S.; Breitman, M. L.; Tsui, L.-C.



A novel mutation in the ADA gene causing severe combined immunodeficiency in an Arab patient: a case report  

PubMed Central

Introduction About 20% of the cases of human severe combined immunodeficiency are the result of the child being homozygous for defective genes encoding the enzyme adenosine deaminase. To our knowledge, the mutation pattern in Arab patients with severe combined immunodeficiency has never been reported previously. Case presentation A 14-month-old Arab boy had clinical features typical of severe combined immunodeficiency. His clinical picture and flow cytometric analysis raised the diagnosis of adenosine deaminase deficiency and prompted us to screen the adenosine deaminase gene for mutation(s). We detected a novel mutation in exon 9 of the adenosine deaminase gene (p.Arg282>Gln), which we believe is the cause of the severe combined immunodeficiency phenotype observed in our patient. Conclusion This is the first report of adenosine deaminase mutation in an Arab patient with severe combined immunodeficiency due to a novel pathogenic mutation in the adenosine deaminase gene.



Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942.  

PubMed Central

The impact of promoter modification on the expression of the mosquitocidal Bacillus thuringiensis subsp. israelensis cryIVB gene when used to transform the cyanobacterium Synechococcus sp. strain PCC 7942 has been examined. Maximal transcript and protein abundances were achieved by the addition of the lacZ promoter upstream of the cryIVB sequence. Replacement of the endogenous corresponding Bacillus sequences with the Synechococcus petF1 promoter, ribosome binding site, and initiation codon also resulted in increased expression of the cryIVB gene relative to the expression obtained with the Bacillus promoter alone but decreased expression relative to the expression achieved with the tandem array of the Bacillus and lacZ promoters. Synechococcus cells carrying plasmids in which the expression of the cryIVB gene was regulated by either the lacZ or the petF1 promoter were readily consumed by first-instar Culex restuans larvae and proved to be toxic for these organisms. Images

Soltes-Rak, E; Kushner, D J; Williams, D D; Coleman, J R



Gene-environment interactions in cancer epidemiology: a national cancer institute think tank report.  


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 (0.01 < MAF < 0.05) genetic variants associated with cancer. The marginal effects of most of these variants have been small (odds ratios: 1.1-1.4). There remain unanswered questions on how best to incorporate the joint effects of genes and environment, including gene-environment (G × E) 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 10-11, 2012. The objective of the Think Tank was to facilitate discussions on (1) the state of the science, (2) the goals of G × E 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 G × E 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

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



Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii  

Microsoft Academic Search

In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source and on other environmental factors. We\\u000a have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1\\/ars) into a wall-less strain of C. reinhardtii. A new and sensitive

Marc Ohresser; René F. Matagne; Roland Loppes



The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants  

Microsoft Academic Search

Inter-transformant variability in the expression of introduced genes was studied in the R1 and R2 generations of 10 tobacco transformants, produced by Agrobacterium-mediated transformation. In replicated and physiologically equivalent material, tranformants showed considerable variability in the expression of the reporter gene uidA as shown by transcript levels and ß-glucuronidase (GUS) activity. However, homozygous R2 material could be investigated for seven

Shaun L. A. Hobbs; Pascal Kpodar; Catherine M. O. DeLong



Gene transfer and gene therapy  

Microsoft Academic Search

This book reports the progress in gene transfer that has been made in various species, from Drosophila to higher mammals, including illustrative examples of germline gene transfer and tissue-specific somatic gene regulation in the mouse. Important new information regarding developmental control of gene transcription includes the delineation of distal elements, both cis and trans, controlling specific gene regulation. The book

A. L. Beaudet; R. Mulligan; I. M. Verma



Embryonic expression of an Nkx2-5/Cre gene using ROSA26 reporter mice.  


Nkx2-5, one of the earliest cardiac-specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2-5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2-5 gene activity by beta-galactosidase (beta-gal) activity. beta-gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5-13.5 days postcoitum, dpc), beta-gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2-5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes. PMID:11783008

Moses, K A; DeMayo, F; Braun, R M; Reecy, J L; Schwartz, R J



A Cell-Based ?-Lactamase Reporter Gene Assay for the CREB Signaling Pathway  

PubMed Central

The Cyclic-AMP Response Element Binding (CREB) proteins comprise a family of transcription factors that stimulate or repress the expression of a wide variety of genes by binding to nucleotide sequences known as cAMP Response Elements. CREB-mediated transcription has been implicated in a wide variety of important physiological processes, including long-term memory, and enhancement of CREB signaling has been suggested as an attractive therapeutic strategy for human memory disorders. To identify small molecule compounds that enhance CREB pathway signaling, we have optimized and validated a cell-based ?-lactamase reporter gene CREB pathway assay in 1536-well plate format. The LOPAC library of 1280 compounds was screened in triplicate in this assay on a quantitative high throughput screening (qHTS) platform. A variety of compounds which affect known members of the CREB pathway were identified as active, including twelve known phosphodiesterase (PDE) inhibitors, and forskolin, a known activator of adenylate cyclase, thus validating the assay’s performance. This qHTS platform assay will facilitate identification of novel small molecule CREB signaling enhancers, which will be useful for chemical genetic dissection of the CREB pathway and as starting points for potentially memory-enhancing therapeutics.

Xia, Menghang; Guo, Vicky; Huang, Ruili; Inglese, James; Nirenberg, Marshall; Austin, Christopher P



Reporter gene stimulation by MIDA1 through its DnaJ homology region.  


MIDA1 was reported as a protein that can associate with Id1. Its N-terminus has homology to Z-DNA binding protein, Zuotin, that contains DnaJ motif, considered to interact with Hsp70s, and Id binding domain. In the present study, we found that MIDA1 stimulates the transcription of the co-transfected genes. This stimulation was independent of promoter specificity because it was observed in various transfected genes. MIDA1 enhanced formation of DNA-protein complexes with E-box or TATA box without its direct binding to DNA. Analysis with deletion mutants of MIDA1 showed that the short protein fragment containing DnaJ motif within Zuotin homology region is sufficient for the stimulation of transcription and we demonstrated that MIDA1 associates with Hsp70. These data suggest involvement of MIDA1 in the stimulation of transcription in concert with Hsp70/Hsc70 molecular chaperones, thus providing a link between Hsp70/Hsc70 molecular chaperones and components of the transcriptional machinery. PMID:15465022

Yoshida, Masayoshi; Inoue, Toshiaki; Shoji, Wataru; Ikawa, Shuntaro; Obinata, Masuo



Structure and Function of the Human Metallothionein Gene Family: Final Technical Report.  

National Technical Information Service (NTIS)

The full nucleotide sequence of two additional human metallothionein (hMT) genes has been determined. These genes, hMT-I/sub B/ and hMT-I/sub F/, are located within the MT-I gene cluster we have described originally. The hMT-I/sub F/ gene is the first hMT...

M. Karin



Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.  

PubMed Central

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4

Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S



KIT gene mutation analysis in solid tumours: biology, clincial applications and trends in diagnostic reporting.  


Gain-of-function mutations involving c-kit protein, a cell-surface transmembrane receptor for stem cell factor, have been identified as a key oncogenic driver in a variety of solid tumours. Coupled with the development of tyrosine kinase inhibitors such as imatinib, c-kit has emerged as a viable drug target in what seems to be a validated therapeutic concept. This review will focus on gastrointestinal stromal tumours and melanomas, two types of solid tumours most closely associated with KIT gene mutations. The biology of KIT mutations in both conditions, as well as the value of KIT mutation testing in predicting disease and treatment outcomes are discussed. Since initial response to imatinib is largely influenced by mutation status, genotyping these tumours serves to facilitate personalised oncology. We also summarise our experience with diagnostic reporting of KIT mutation analysis over a period of 3 years, and briefly survey future developments in treatment, which indeed look very promising. PMID:23277171

Tay, Clifton Ming; Ong, Chee Wee; Lee, Victor Kwan Min; Pang, Brendan



Construction of a whole-cell gene reporter for the fluorescent bioassay of nitrate.  


The development of a whole-cell fluorescence-based biosensor for nitrate is reported. The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon (Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP). Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence. The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration. The developed bioassay has established that E. coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine-N-oxide and fumerate, and azide, an inhibitor of redox-active proteins. PMID:15081908

Taylor, Clare J; Bain, Lindsey A; Richardson, David J; Spiro, Stephen; Russell, David A



Comparison of in vitro hormone activities of selected phthalates using reporter gene assays.  


Phthalates are widely used in the plastic industry and food packaging, imparting softness and flexibility to normally rigid plastic medical devices and children's toys. Even though phthalates display low general toxicity, there is increasing concern on the effects of endocrine system induced by some of phthalate compounds. The hormone activity of dibutyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP) were assessed using the luciferase reporter gene assays. The results showed that DBP, MBP and DEHP, not only exhibited potent antiandrogenic activity, with IC(50) value of 1.05x10(-6), 1.22x10(-7)M and exceeding 1x10(-4)M respectively, but also showed the androgenic activity with EC(50) value of 6.17x10(-6), 1.13x10(-5)M and exceeding 1x10(-4)M. We also found that all the three related chemicals possessed thyroid receptor (TR) antagonist activity with IC(50) of 1.31x10(-5), 2.77x10(-6)M and exceeding 1x10(-4)M respectively, and none showed TR agonist activity. These results indicate that TR might be the targets of industrial chemicals. In the ER mediate reporter gene assay, three chemicals showed no agonistic activity except for DBP, which appeared weakly estrogenic at the concentration of 1.0x10(-4)M. Together, the findings demonstrate that the three phthalates could simultaneously disrupt the function of two or more hormonal receptors. Therefore, these phthalates should be considered in risk assessments for human health. PMID:19643168

Shen, Ouxi; Du, Guizhen; Sun, Hong; Wu, Wei; Jiang, Yi; Song, Ling; Wang, Xinru



Differential Gene Expression in Neurospora Crassa Cell Types: Amplification of RRNA Genes. Progress Report, July 1979-30 June 1980.  

National Technical Information Service (NTIS)

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 wit...

S. K. Dutta



Nonessential Genes of Phage ?YeO3-12 Include Genes Involved in Adaptation to Growth on Yersinia enterocolitica Serotype O:3  

PubMed Central

Bacteriophage ?YeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ? gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their ?-galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that ?YeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively.

Kiljunen, Saija; Vilen, Heikki; Pajunen, Maria; Savilahti, Harri; Skurnik, Mikael



Preliminary Report: Missense mutations in the APOL gene family are associated with end stage kidney disease risk previously attributed to the MYH9 gene  

Microsoft Academic Search

MYH9 has been proposed as a major genetic risk locus for a spectrum of non-diabetic end stage kidney disease (ESKD). We use recently released sequences from the 1000 Genomes Project to identify two western African specific missense mutations (S342G and I384M) in the neighbouring APOL1 gene, and demonstrate that these are more strongly associated with ESKD than previously reported MYH9

Shay Tzur; Saharon Rosset; Revital Shemer; Guennady Yudkovsky; Sara Selig; Ayele Tarekegn; Endashaw Bekele; Neil Bradman; Walter G Wasser; Doron M Behar; Karl Skorecki



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

PubMed Central

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.

Gates, J; Thummel, C S



The human p53 negative regulatory domain mediates inhibition of reporter gene transactivation in yeast lacking thioredoxin reductase.  


Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence. A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in delta trr1 yeast lacking the thioredoxin reductase gene. Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase. A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and delta trr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase. Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase. The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription. However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase. The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain. PMID:10397262

Merrill, G F; Dowell, P; Pearson, G D



Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation.  


A currently obscure area of steroid hormone action is where the component factors, including receptor and reporter gene, act. The DNA binding of factors can be precisely defined, but the location and timing of factor binding and action are usually not equivalent. These questions are addressed for several factors (e.g. glucocorticoid receptor (GR), reporter, TIF2, NCoR, NELF-A, sSMRT, and STAMP) using our recently developed competition assay. This assay reveals both the kinetically defined mechanism of factor action and where the above factors act relative to both each other and the equilibrium equivalent to the rate-limiting step, which we call the concentration limiting step (CLS). The utility of this competition assay would be greatly increased if the position of the CLS is invariant and if the factor acting at the CLS is known. Here we report that the exogenous GREtkLUC reporter acts at the CLS as an accelerator for gene induction by GRs in U2OS cells. This mechanism of reporter function at the CLS persists with different reporters, factors, receptors, and cell types. We, therefore, propose that the reporter gene always acts at the CLS during gene induction and constitutes a landmark around which one can order the actions of all other factors. Current data suggest that how and where GR and the short form of SMRT act is also constant. These results validate a novel and rational methodology for identifying distally acting factors that would be attractive targets for pharmaceutical intervention in the treatment of diseases involving GR-regulated genes. PMID:23055525

Blackford, John A; Guo, Chunhua; Zhu, Rong; Dougherty, Edward J; Chow, Carson C; Simons, S Stoney



Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes  

Microsoft Academic Search

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based

Yawei Qiao; Margaret R Spitz; Zhaozheng Guo; Mohammad Hadeyati; Lawrence Grossman; Kenneth H Kraemer; Qingyi Wei



Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion  

Microsoft Academic Search

This study evaluates the utility of Cre-expressing adenovirus for deletion of floxed genes in primary cells using primary murine hepatocytes. Adenovirus infection was very efficient, even at very low MOI (>95% infection at a MOI of 6) and did not reduce viability. High level LacZ expression was cytotoxic to hepatocytes but Cre expression had no effect on viability. Cre-mediated recombination

Sandrine Prost; Sharon Sheahan; Dominic Rannie; David J. Harrison



N-terminal acetyltransferase 3 gene is essential for robust circadian rhythm of bioluminescence reporter in Chlamydomonas reinhardtii.  


Chlamydomonas reinhardtii is a model species of algae for studies on the circadian clock. Previously, we isolated a series of mutants showing defects in the circadian rhythm of a luciferase reporter introduced into the chloroplast genome, and identified the genes responsible for the defective circadian rhythm. However, we were unable to identify the gene responsible for the defective circadian rhythm of the rhythm of chloroplast 97 (roc97) mutant because of a large genomic deletion. Here, we identified the gene responsible for the roc97 mutation through a genetic complementation study. This gene encodes a protein that is homologous to the subunit of N-terminal acetyltransferase (NAT) which catalyzes N-terminal acetylation of proteins. Our results provide the first example of involvement of the protein N-terminal acetyltransferase in the circadian rhythm. PMID:22266323

Matsuo, Takuya; Iida, Takahiro; Ishiura, Masahiro



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

NASA Astrophysics Data System (ADS)

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.

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



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

PubMed Central

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.

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



Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung  

Microsoft Academic Search

Background Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. Methods Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered

Ian A. Pringle; Gerry McLachlan; David D. S. Collie; Stephanie G. Sumner-Jones; Anna E. Lawton; Peter Tennant; Alison Baker; Catherine Gordon; Richard Blundell; Anusha Varathalingam; Lee A. Davies; Ralph A. Schmid; Seng H. Cheng; David J. Porteous; Deborah R. Gill; Stephen C. Hyde



Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992  

Microsoft Academic Search

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




Genetically Marked Male Sterile Genes and Hybrid Seed Production. Final Report on Phase 2.  

National Technical Information Service (NTIS)

The project was intended to make practical the use of nuclear male sterile (ms) genes in hybrid seed production by linking a marker gene to the male fertile allele of the ms gene and selecting for the segregating ms seed by discarding segregants showing t...

A. Morgan



Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery  

Microsoft Academic Search

The development of accurate, safe, and efficient gene delivery remains a major challenge towards the realization of gene therapeutic prevention and treatment of cardiovascular diseases. In this study, we investigated the ability of high-intensity focused ultrasound (HIFU), a form of mechanical wave transmission, to act as a noninvasive tool for the enhancement of in vivo gene transfer into rabbit carotid

P E Huber; M J Mann; L G Melo; A Ehsan; D Kong; L Zhang; M Rezvani; P Peschke; F Jolesz; V J Dzau; K Hynynen



Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models  

Microsoft Academic Search

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania

Gaétan Roy; Carole Dumas; Denis Sereno; Ying Wu; Ajay K. Singh; Michel J. Tremblay; Marc Ouellette; Martin Olivier; Barbara Papadopoulou



Transient reporter gene (GUS) expression in creeping bentgrass ( Agrostis palustris ) is affected by in vivo nucleolytic activity  

Microsoft Academic Search

Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When ß-glucuronidase (GUS) reporter gene activity

Chhandak Basu; Hong Luo; Albert P. Kausch; Joel M. Chandlee



Isolation and Characterization of B-Glucosidase Gene and B-Glucosidase of Trichoderma Viride. Progress Report.  

National Technical Information Service (NTIS)

The goal is to clone and characterize each of the cellulase genes from Trichoderma. This report is principally concerned with B-glucosidase. The induction of the Trichoderma cellulase complex by cellulose and by the soluble inducer, sophorose, has been de...

D. W. Stafford R. L. Lundblad



A novel mutation in the ADA gene causing severe combined immunodeficiency in an Arab patient: a case report  

Microsoft Academic Search

INTRODUCTION: About 20% of the cases of human severe combined immunodeficiency are the result of the child being homozygous for defective genes encoding the enzyme adenosine deaminase. To our knowledge, the mutation pattern in Arab patients with severe combined immunodeficiency has never been reported previously. CASE PRESENTATION: A 14-month-old Arab boy had clinical features typical of severe combined immunodeficiency. His

Ali Hellani; Nidal Almassri; Khaled K Abu-Amero



The involvement of the nif -associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation  

Microsoft Academic Search

The pathway of electron transport to nitrogenase in the endophytic ?-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with

André L. F. Souza; Adriana L. Invitti; Fabiane G. M. Rego; Rose A. Monteiro; Giseli Klassen; Emanuel M. Souza; Leda S. Chubatsu; Fábio O. Pedrosa; Liu U. Rigo



Comparison of prostate cancer cell lines for androgen receptor-mediated reporter gene assays.  


In order to select a better prostate cancer cell model for androgen receptor (AR)-mediated reporter gene assays, we assessed the androgen response characteristics of three cell lines, LNCaP, PC3/AR(+) and 22Rv1, in this study. Both the mRNA and the proteins of AR and glucocorticoid receptor (GR) were expressed in all three cell lines. Among the three cell lines, only in LNCaP cells, DHT concentration-dependently stimulated proliferation. DHT induced the luciferase activity in three cell lines which were transiently transfected with pMMTV-Luc, in a concentration-dependent manner. The maximum induction was 24.0-fold and 13.4-fold in 22Rv1 and in the LNCaP respectively. PC3/AR(+) were more sensitive to respond to DHT at a minimal concentration of 10(-12)M by 14.0-fold induction. The transcriptional activity induced with 10(-8)M DHT was inhibited about 50-75% in the PC3/AR(+) and 22Rv1, and 98% in the LNCaP, by vinclozolin. Dexamethasone concentration-dependently induced the luciferase activity in PC3 and 22Rv1, but not in the LNCaP. However, the response to dexamethasone in 22Rv1 was very weak compared to DHT. The (anti)androgencity of seven pyrethroids was assessed via an AR-mediated luciferase reporter assay. None of them showed the androgenic action in all three cell lines. Permethrin inhibited the DHT induced luciferase activity about 22%, 35.8% and 75.5% in 22Rv1, PC3/AR(+) and LNCaP, respectively. Based on results from in this study and cell line character, 22Rv1 cells seemed to be an appropriate model for the screening of androgenic endocrine disruptors, although it needs further studies with other steroid receptor and thyroid receptor. PMID:16621434

Kim, Hyun-Jung; Park, Young In; Dong, Mi-Sook



Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay.  


A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensitive cell line (GH3.TRE-Luc), which was further optimized into an assay that allowed the detection of Triiodothyronine (T(3)) and Thyroxine (T(4)) concentrations in the picomolar range after only 24 h of exposure. The greater than 20-fold induction of T(3) relative to the solvent control is illustrative of the high responsiveness of the system. The assay was validated by the quantification of the agonistic effect of the natural hormones (T(3) and T(4)), the acetic acid derivatives of T(3) (triiodothyroaceticacid, or Triac) and T(4) (tetraiodothyroacetic acid, or Tetrac), hydroxy polybrominated diphenylethers (OH-PBDEs), hydroxy polychlorinated biphenyls (OH-PCBs) and the antagonistic action of sodium arsenite (NaAsO(2)). The putative antagonist Amiodarone, Bisphenol A (BPA) and its halogenated derivatives (TCBPA and TBBPA) for which effects reported in the literature are not consistent, showed comparable dose-response curves with a slight agonistic effect (5% of T(3)-max) followed by a slight antagonistic effect. The magnitude and reproducibility of the responses to various compounds confirms this assay as a promising tool for the identification and quantification of specific thyroid hormone receptor disrupting potency of compounds. PMID:20732405

Freitas, Jaime; Cano, Patricia; Craig-Veit, Christina; Goodson, Michael L; Furlow, J David; Murk, Albertinka J



Reconstruction of transcriptional dynamics from gene reporter data using differential equations  

PubMed Central

Motivation: Promoter-driven reporter genes, notably luciferase and green fluorescent protein, provide a tool for the generation of a vast array of time-course data sets from living cells and organisms. The aim of this study is to introduce a modeling framework based on stochastic differential equations (SDEs) and ordinary differential equations (ODEs) that addresses the problem of reconstructing transcription time-course profiles and associated degradation rates. The dynamical model is embedded into a Bayesian framework and inference is performed using Markov chain Monte Carlo algorithms. Results: We present three case studies where the methodology is used to reconstruct unobserved transcription profiles and to estimate associated degradation rates. We discuss advantages and limits of fitting either SDEs ODEs and address the problem of parameter identifiability when model variables are unobserved. We also suggest functional forms, such as on/off switches and stimulus response functions to model transcriptional dynamics and present results of fitting these to experimental data. Contact: Supplementary Information: Supplementary data are available at Bioinformatics online.

Finkenstadt, Barbel; Heron, Elizabeth A.; Komorowski, Michal; Edwards, Kieron; Tang, Sanyi; Harper, Claire V.; Davis, Julian R. E.; White, Michael R. H.; Millar, Andrew J.; Rand, David A.



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

|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

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



Case report of a patient with schizophrenia and a mutation in the insulin receptor substrate-4 gene.  


This report deals with a female patient with schizophrenia who was found to have a mutation in the insulin receptor substrate-4 gene that is located on chromosome Xq22.3. Since this mutation is expected to change amino acid coding from histidine to tyrosine and cause an altered insulin receptor substrate-4 protein, and the insulin receptor substrate-4 protein may be involved in neuronal growth and function in the brain, it is possible that it is this insulin receptor substrate-4 gene mutation that underlies this patient's schizophrenia development. PMID:23685414

Melkersson, Kristina



The use of the luxA gene of the bacterial luciferase operon as a reporter gene  

Microsoft Academic Search

Bacterial luciferase can be assayed rapidly and with high sensitivity both in vivo and in vitro. Here we demonstrate that the N-terminal hydrophobic domain of the a catalytic subunit of the luciferase enzyme is indispensable for enzyme activity, although N-terminal translational fusions with full luciferase activity can be obtained. Bacterial luciferase is therefore ideally suited as a reporter enzyme for

Olof Olsson; Csaba Koncz; Aladar A. Szalay



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


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

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



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

PubMed Central

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.

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



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

Microsoft Academic Search

We previously reported that Bacillus thuringiensis strain 407 Cry 32 secretes a zinc-requiring metallopro- tease, 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

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



Two dopamine genes related to reports of childhood retrospective inattention and conduct disorder symptoms  

Microsoft Academic Search

The 7-repeat allele of the dopamine receptor D4 gene (DRD4) and the 10 repeat allele of the dopamine transporter gene (DAT1) have shown association and linkage with symptoms of attention deficit hyperactivity disorder (ADHD) in childhood. The parents of ADHD children (clinic group, n = 80 fathers and 107 mothers) and control children (control group, n = 42 fathers and

D C Rowe; C Stever; D Chase; S Sherman; A Abramowitz; I D Waldman



Expression of Escherichia coli uvr genes in mammalian cells. Progress report  

SciTech Connect

The E. coli uvrA, uvrB and uvrC gene products are required to carry out the incision step in excision repair of damaged DNA. The range of sensitivity to DNA damage in E. coli uvrA uvrB, and uvrC mutants is similar to that of cells derived from patients with xeroderma pigmentosum (XP). Our goal is to introduce the E. coli uvrA, uvrB, and uvrC genes into SV40-transformed XP cells and to examine whether these genes are able to genetically complement the repair deficiency in XP cells. Ecogpt was used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322-SV40 and Ecogpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. We have also constructed vectors in which the uvr gene and the gpt are in tandem and flanked by one set of SV40 regulatory elements. SV40-transformed XP cells of complementation group A (XP12BE) were transfected with pSV2uvrASV2gpt, a vector of the former configuration. Southern gel analysis of the resulting gpt/sup +/ cells has revealed one colony in which the uvrA gene with its SV40 elements has been integrated into the chromosomal DNA intact.

Not Available



Structure and Regulation of Methanogen Genes: Progress Report, March 1, 1988-February 15, 1989.  

National Technical Information Service (NTIS)

The goals of this project were initially to obtain the primary structures of several cloned methanogen genes and subsequently to determine the mechanism(s) by which these genes are transcribed and how their expression is regulated. Studies have focused on...

J. N. Reeve



Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence  

Microsoft Academic Search

Agrobacterium-mediatedinfection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petuniagenes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment

Jen-Chih Chen; Cai-Zhong Jiang; Timothy E. Gookin; Donald A. Hunter; David G. Clark; Michael S. Reid



Misdiagnosing cystic fibrosis in the era of gene analysis: case reports.  


We present a scenario of how gene analysis plays a confusing role in the diagnosis of cystic fibrosis (CF). One of the two siblings we are presenting here was initially misdiagnosed as having CF, based on the two CF gene mutations identified by gene analysis. A CF gene study on the other sibling years later, however, led to further investigation and eventually to a change of diagnosis. As interesting and important as gene analysis is in CF, one must always look at each patient in the big picture. Included in the picture, in addition to the state-of-the-art genotype, is the phenotype, or (to simplify) the back-to-basic clinical manifestations. PMID:15678508

Moser, Chuanpit; Nussbaum, Eliezer; Thompson, Rohan



The Association Between Serotonin Transporter Gene Promoter Polymorphism (5-HTTLPR), Self-Reported Symptoms, and Dental Mercury Exposure  

PubMed Central

The associations between a polymorphism of the serotonin transporter gene (5-HTTLPR), dental mercury exposure, and self-reported symptoms were evaluated among 157 male dentists and 84 female dental assistants. Self-reported symptoms and detailed work histories were obtained by computerized questionnaire. Spot urine samples were collected and analyzed for mercury concentrations to evaluate recent exposures, whereas a chronic mercury exposure index was created from the work histories. 5-HTTLPR polymorphism status was determined using a polymerase chain reaction (PCR)-based assay. Scores for current, recent, and chronic self-reported symptom groups were evaluated with respect to recent and chronic mercury exposure and 5-HTTLPR polymorphism status. Multiple regression analysis controlled for age, socioeconomic status, tobacco and alcohol use, self-reported health problems, and medications. Analyses were restricted to Caucasian subjects due to the highly skewed distribution of the 5-HTTLPR polymorphism. Separate evaluations were conducted for dentists and dental assistants. In contrast to previous reports, no consistent associations were found between either urinary mercury concentration or the chronic index of mercury exposure and any category of symptoms. However, both significant and consistent associations were observed between increased symptoms and the 5-HTTLPR polymorphism involving two copies of the short or “s” allele (full mutation), but not with the polymorphism involving only one copy (heterozygous), demonstrating a gene–dose relationship for symptom reporting. These findings suggest that within this restricted population increased symptoms of depression, anxiety, and memory are associated with the 5-HTTLPR polymorphism among both males and females.

Heyer, Nicholas J.; Echeverria, Diana; Farin, Federico M.; Woods, James S.



Carbon limitation induces sigma(S)-dependent gene expression in Pseudomonas fluorescens in soil.  


Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the sigma(S)-dependent Escherichia coli promoter P(fic) and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of beta-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing beta-galactosidase from P(fic). Changes in cell size and expression of beta-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of beta-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil beta-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted. PMID:11472905

Koch, B; Worm, J; Jensen, L E; Hřjberg, O; Nybroe, O



Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica  

PubMed Central

The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.

Rinaldi, Gabriel; Morales, Maria E.; Cancela, Martin; Castillo, Estela; Brindley, Paul J.; Tort, Jose F.



The murine MHC class I genes, H-2Dq and H-2Lq, are strikingly homologous to each other, H-2Ld, and two genes reported to encode tumor- specific antigens  

PubMed Central

Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.



Transcriptional reporters for genes activated by cell wall stress through a non-catalytic mechanism involving Mpk1 and SBF  

PubMed Central

The Mpk1 MAP kinase of the Cell Wall Integrity (CWI) signaling pathway induces transcription of the FKS2 gene in response to cell wall stress through a non-catalytic mechanism that involves stable association of Mpk1 with the Swi4 transcription factor. This dimeric complex binds to a Swi4 recognition site in the FKS2 promoter. The Swi6 transcription factor is also required to bind this ternary complex for transcription initiation to ensue. In this context, the Mlp1 pseudokinase serves a redundant function with Mpk1. We have identified three additional genes, CHA1, YLR042c, and YKR013w that are induced by cell wall stress through the same mechanism. We report on the behavior of several promoter-lacZ reporter plasmids designed to detect cell wall stress transcription through this pathway.

Kim, Ki-Young; Levin, David E.



Biodistribution of an adenoviral vector carrying the luciferase reporter gene following intravesical or intravenous administration to a mouse  

Microsoft Academic Search

The biodistribution and resulting pattern of transgene expression were determined following intravesical administration of an adenoviral vector carrying the luciferase reporter gene (AdLuc). Female BALB\\/c mice were subjected to intravesical instillation of 1 × 109 or 5 × 109 plaque-forming units of AdLuc. After sacrifice, transgene expression was detected in tissues using luciferase assays; vector DNA was detected by vector-specific

Mark Wood; Paul Perrotte; Eric Onishi; Mary E Harper; Colin Dinney; Lance Pagliaro; Deborah R Wilson



Isolation and characterization of B-glucosidase gene and B-glucosidase of Trichoderma viride. Progress report  

SciTech Connect

The goal is to clone and characterize each of the cellulase genes from Trichoderma. This report is principally concerned with B-glucosidase. The induction of the Trichoderma cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. B-glucosidase has been isolated and purified to homogeneity. The enzyme contains significant amounts of carbohydrate and has a molecular weight greater than bovine serum albumin (68,000). (ACR)

Stafford, D.W.; Lundblad, R.L.



Permanent Neonatal Diabetes Mellitus Due to a C96Y Heterozygous Mutation in the Insulin Gene. A Case Report  

Microsoft Academic Search

Context Neonatal diabetes is a rare disorder with an incidence of 1 in 215,000-500,000 live births with 50% of them having permanent neonatal diabetes mellitus. Case report We present a case of permanent neonatal diabetes mellitus due to a C96Y (c.287G>A; p.Cys96Tyr) heterozygous mutation in the insulin (INS) gene. Both the patient and his father (who had childhood- onset insulin-requiring

Anish Ahamed; Ambika Gopalakrishnan Unnikrishnan; Sanket Sharad Pendsey; Sheela Nampoothiri; Nisha Bhavani; Valliyaparambil Pavithran Praveen; Harish Kumar; Rohinivilasam Vasukutty Jayakumar; Vasantha Nair; Sian Ellard; Emma L Edghill


A Rapid and Sensitive Reporter Gene that Uses Green Fluorescent Protein Expression to Detect Chemicals with Estrogenic Activity  

Microsoft Academic Search

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phospho- glycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a3 *-polyadenylation signal. The plasmid was linearized and trans- fected into MCF-7 cells, a human breast cancer-derived

S. Miller; D. Kennedy; J. Thomson; F. Han; R. Smith; N. Ing; J. Piedrahita; D. Busbee



Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery.  


Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic maintenance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy. PMID:21168204

Lin, Erh-Hsuan; Keramidas, Michelle; Rome, Claire; Chiu, Wen-Ta; Wu, Cheng-Wen; Coll, Jean-Luc; Deng, Win-Ping



Structure and expression of nuclear genes encoding rubisco activase. Progress report.  

National Technical Information Service (NTIS)

Our activities during the past year have centered around two basic aspects of the project: describing more thoroughly the diurnal and light irradiance effects on activase gene expression in barley; and isolating and structurally characterizing cDNA and ge...

R. E. Zielinski



Structure and Expression of Nuclear Genes Encoding Rubisco Activase: Progress Report.  

National Technical Information Service (NTIS)

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 t...



Structure and regulation of methanogen genes. Progress report, July 1, 1987-June 30, 1989.  

National Technical Information Service (NTIS)

Our goals are to determine the mechanisms of gene expression in archaebacterial methanogens and to use molecular biological approaches to investigate methanogenesis. At the outset nothing was known and the basic information gained, detailing the structure...

J. N. Reeve



First Report of the Multidrug Resistance Gene cfr in Enterococcus faecalis of Animal Origin  

PubMed Central

The multiresistance gene cfr was identified for the first time in an Enterococcus faecalis isolate of animal origin. The 32,388-bp plasmid pEF-01, which carried the cfr gene, was sequenced completely. Three copies of the insertion sequence IS1216 were identified in pEF-01, and the detection of a cfr- and IS1216-containing amplicon by inverse PCR suggests that IS1216 may play a role in the dissemination of cfr by a recombination process.

Liu, Yang; Wang, Yang; Wu, Congming; Shen, Zhangqi; Schwarz, Stefan; Du, Xiang-Dang; Dai, Lei; Zhang, Wanjiang



[Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report  

SciTech Connect

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.

Okita, T.W.



Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report  

PubMed Central

Global analysis of gene expression by DNA microarrays is nowadays a widely used tool, especially relevant for cancer research. It helps the understanding of complex biology of cancer tissue, allows identification of novel molecular markers, reveals previously unknown molecular subtypes of cancer that differ by clinical features like drug susceptibility or general prognosis. Our aim was to compare gene expression profiles in breast cancer that develop against a background of inherited predisposing mutations versus sporadic breast cancer. In this preliminary study we analysed seven hereditary, BRCA1 mutation-linked breast cancer tissues and seven sporadic cases that were carefully matched by histopathology and ER status. Additionally, we analysed 6 samples of normal breast tissue. We found that while the difference in gene expression profiles between tumour tissue and normal breast can be easily recognized by unsupervised algorithms, the difference between those two types of tumours is more discrete. However, by supervised methods of data analysis, we were able to select a set of genes that may differentiate between hereditary and sporadic tumours. The most significant difference concerns genes that code for proteins engaged in regulation of transcription, cellular metabolism, signalling, proliferation and cell death. Microarray results for chosen genes (TOB1, SEPHS2) were validated by real-time RT-PCR.



Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993  

SciTech Connect

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.

Parke, D.; Ornston, L.N.



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

SciTech Connect

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.

Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (United States)); Schild, D. (Lawrence Berkeley Lab., CA (United States))



In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes  

PubMed Central

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.

Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan MW



Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene.  


The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis. PMID:11238884

Caruana, G; Bernstein, A



Evidence of Associations between Cytokine Genes and Subjective Reports of Sleep Disturbance in Oncology Patients and Their Family Caregivers  

PubMed Central

The purposes of this study were to identify distinct latent classes of individuals based on subjective reports of sleep disturbance; to examine differences in demographic, clinical, and symptom characteristics between the latent classes; and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. Among 167 oncology outpatients with breast, prostate, lung, or brain cancer and 85 of their FCs, growth mixture modeling (GMM) was used to identify latent classes of individuals based on General Sleep Disturbance Scale (GSDS) obtained prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs) and haplotypes in candidate cytokine genes were interrogated for differences between the two latent classes. Multiple logistic regression was used to assess the effect of phenotypic and genotypic characteristics on GSDS group membership. Two latent classes were identified: lower sleep disturbance (88.5%) and higher sleep disturbance (11.5%). Participants who were younger and had a lower Karnofsky Performance status score were more likely to be in the higher sleep disturbance class. Variation in two cytokine genes (i.e., IL6, NFKB) predicted latent class membership. Evidence was found for latent classes with distinct sleep disturbance trajectories. Unique genetic markers in cytokine genes may partially explain the interindividual heterogeneity characterizing these trajectories.

Miaskowski, Christine; Cooper, Bruce A.; Dhruva, Anand; Dunn, Laura B.; Langford, Dale J.; Cataldo, Janine K.; Baggott, Christina R.; Merriman, John D.; Dodd, Marylin; Lee, Kathryn; West, Claudia; Paul, Steven M.; Aouizerat, Bradley E.



The Sea PansyRenilla reniformisLuciferase Serves as a Sensitive Bioluminescent Reporter for Differential Gene Expression inCandida albicans  

Microsoft Academic Search

The infectious yeast Candida albicans progresses through two developmental programs which involve dif- ferential 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. albicanshas adopted a nontraditional codon strategy that




Renilla luciferase Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals  

Microsoft Academic Search

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the 'humanized' GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in,

Y. Wang; Y. A. Yu; S. Shabahang; G. Wang; A. A. Szalay



Seed-protein genes and the regulation of their expression. Progress report  

SciTech Connect

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.

Larkins, B.A.; Foard, D.E.



CD4+/CD56+ hematodermic neoplasm: report of a rare variant with a T-cell receptor gene rearrangement.  


CD4+/CD56+ hematodermic neoplasm (HN), formerly known as a blastic natural killer (NK) cell lymphoma, is a rare subtype of a cutaneous dendritic cell neoplasm notable for highly aggressive behavior. The characteristic features are: expression of the T-helper/inducer cell marker CD4 and the NK-cell marker CD56 in the absence of other T cell or NK-cell specific markers. In particular, CD3 (surface or cytoplasmic) and CD2 are not expressed. Although T-cell receptor (TCR) genes are generally reported to be in a germline configuration, we present an unusual variant of a CD4+/CD56+ HN with a clonal rearrangement of TCR genes. This feature of a CD4+/CD56+ HN has been only rarely reported. Recognition of the presence of clonal TCR gene rearrangements in a small subset of CD4+/CD56+ HN is important to avoid misdiagnosis of this entity as an unusual variant of a cutaneous T-cell lymphoma. PMID:18005171

Stetsenko, Galina Y; McFarlane, Rob; Kalus, Andrea; Olerud, John; Cherian, Sindhu; Fromm, Jonathan; George, Evan; Argenyi, Zsolt



Development of a Cell-Based Reporter Assay for Screening of Inhibitors of Hypoxia-Inducible Factor 2Induced Gene Expression  

Microsoft Academic Search

Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression

Girma M. Woldemichael; James R. Vasselli; Roberta S. Gardella; Tawnya C. Mckee; W. Marston Linehan; James B. McMahon



DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17.  

PubMed Central

The 3.8-kilobase segment of plasmid DNA that contains the genes required for production of the DNA replication inhibitor microcin B17 was sequenced. The sequence contains four open reading frames which were shown to be translated in vivo by the construction of fusions to lacZ. The location of these open reading frames fits well with the location of the four microcin B17 production genes, mcbABCD, identified previously through genetic complementation. The products of the four genes have been identified, and the observed molecular weights of the proteins agree with those predicted from the nucleotide sequence. The transcription of these genes was studied by using fusions to lacZ and physical mapping of mRNA start sites. Three promoters were identified in this region. The major promoter for all the genes is a growth phase-regulated OmpR-dependent promoter located upstream of mcbA. A second promoter is located within mcbC and is responsible for a low-level basal expression of mcbD. A third promoter, located within mcbD, promotes transcription in the reverse direction starting within mcbD and extending through mcbC. The resulting mRNA appears to be an untranslated antisense transcript that could play a regulatory role in the expression of these genes. Images

Genilloud, O; Moreno, F; Kolter, R



Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana  

PubMed Central

Background Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. Results We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. Conclusion We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation.



Cytoplasmically Retargeted HSV1-tk/GFP Reporter Gene Mutants for Optimization of Noninvasive Molecular-Genetic Imaging  

PubMed Central

Abstract To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a “cryptic” testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improvedmetabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [131I]FIAU and a ?-camera.

Ponomarev, Vladimir; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Balatoni, Julius; Blasberg, Ronald; Tjuvajev, Juri Gelovani



A novel TNFRSF1 gene mutation in a Turkish family: a report of three cases  

Microsoft Academic Search

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autosomal dominantly inherited rare autoinflammatory\\u000a disease. It is caused by mutations in exons 2–3 and 4–5 of the tumor necrosing factor receptor superfamily 1A (TNFRSF1A) gene\\u000a on chromosome 12p13.2. TNFRSF1A gene encodes the 55-kDa receptor for tumor necrosis factor. Attacks are associated with abdominal\\u000a pain, myalgia, erythematous skin rash, conjunctivitis,

Fulya Cosan; Ayten Yazici; Bar?? Y?lmazer; Ahmet Gul; Duran Ustek; Ayse Cefle



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 ...


First report of plasmid-mediated quinolone resistance qnrA1 gene in Klebsiella pneumoniae isolate of animal origin.  


One QnrA1-producing Klebsiella pneumoniae isolate GDKA1 from chicken was detected. The qnrA1 gene on plasmid pGDKA1 was located in a genetic environment similar to that in In36 on plasmid pHSH1 and could be cotransferred to Escherichia coli J53 Az(R) with other resistances by a conjugation experiment. Upstream of the qnrA1 gene, there was a class I integron with the dfrA27 and aadA2 cassettes. Similar genetic environments of qnrA1 in Enterobacteriaceae isolates from both human and animal origin might, to some extent, demonstrate similar mechanisms of qnrA distribution. The presence of qnrA1 in health animal commensal bacteria should be worthy of note. This is the first report of qnrA1 in K. pneumoniae and dfrA27 in an Enterobacteriaceae isolate of animal origin. PMID:21235404

Yue, Lei; Chen, Xueying; Li, Shujuan; Liao, Xiaoping; Zhuang, Na; Zhang, Yue; Liu, Ya-Hong



Feasibility of genome-scale construction of promoter::reporter gene fusions for expression in Caenorhabditis elegans using a multisite gateway recombination system.  


The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology. The capacity for using this system for efficient construction of chimeric genes was explored by constructing promoter::reporter gene fusions with a gfp reporter. The promoters were found to provide appropriate expression of GFP upon introduction into C. elegans, demonstrating that the short Gateway recombination site between the promoter and the reporter did not interfere with transcription or translation. The recombinational cloning involved in the Gateway system, which permits the highly efficient and precise transfer of DNA segments between plasmid vectors, makes this technology ideal for genomics research programs. PMID:15489328

Hope, Ian A; Stevens, Jonathan; Garner, Anna; Hayes, Josie; Cheo, David L; Brasch, Michael A; Vidal, Marc




Microsoft Academic Search

Dr. Harold Varmus, Director, National Institutes of Health (NIH), appointed an ad hoc committee to assess the current status and promise of gene therapy and provide recommendations regarding future NIH-sponsored research in this area. The Panel was asked specifically to comment on how funds and efforts should be distributed among various research areas and what funding mechanisms would be most

Stuart H. Orkin; Arno G. Motulsky


Reporting, Appraising, and Integrating Data on Genotype Prevalence and Gene Disease Associations  

Microsoft Academic Search

The recent completion of the first draft of the human genome sequence and advances in technologies for genomic analysis are generating tremendous opportunities for epidemiologic studies to evaluate the role of genetic variants in human disease. Many methodological issues apply to the investigation of variation in the frequency of allelic variants of human genes, of the possibility that these influence

Julian Little; Linda Bradley; Molly S. Bray; Mindy Clyne; Janice Dorman; Darrell L. Ellsworth; James Hanson; Muin Khoury; Joseph Lau; Thomas R. O'Brien; Nat Rothman; Donna Stroup; Emanuela Taioli; Duncan Thomas; Harri Vainio; Sholom Wacholder; Clarice Weinberg


Characterization of embryo-specific genes. Final report, April 1, 1987--March 31, 1992  

SciTech Connect

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.

Sung, R.



An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa  

Microsoft Academic Search

A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ? allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered

Herbert P. Schweizer; Tung T. Hoang



The promoter of coconut foliar decay-associated circular single-stranded DNA directs phloem-specific reporter gene expression in transgenic tobacco  

Microsoft Academic Search

A full-length double-stranded DNA copy of the single-stranded circular DNA associated with coconut foliar decay virus (CFDV) was constructed. Full-length CFDV DNA and smaller fragments were transcriptionally fused to the ß-glucuronidase reporter gene and examined for promoter activity in vivo. In stably transformed tobacco plants, the CFDV DNA promoter confered a tissue-specific expression pattern in that the reporter gene was

Wolfgang Rohde; Dieter Becker; John W. Randles



Gene transfer and gene therapy  

SciTech Connect

This book reports the progress in gene transfer that has been made in various species, from Drosophila to higher mammals, including illustrative examples of germline gene transfer and tissue-specific somatic gene regulation in the mouse. Important new information regarding developmental control of gene transcription includes the delineation of distal elements, both cis and trans, controlling specific gene regulation. The book also offers an overview of vectors for gene transfer, including retroviral vectors and new retroviral packaging cell lines designed to minimize production of replication-competent virus.

Beaudet, A.L.; Mulligan, R.; Verma, I.M.



Mapping our genes: Federal genome projects: How vast. How fast. Contractor reports, Volume 2  

SciTech Connect

Contractor reports solicited by the Office of Technology Assessment in preparing a briefing report and recommendations to congress on the Federal Role in human genetic mapping are provided. The five reports in this volume are entitled - The mapping and sequencing of genomes: A comparative analysis of methods, benefits and disbenefits; Mapping the human genome: Experimental approaches for cloning and ordering DNA fragments; Mapping and sequencing the human genome in Europe; Application of human genome mapping for the global control of genetic disease; and In search of the ultimate map of the human genome: The Japanese efforts. Each of these reports have been separately indexed and abstracted for the Energy Data Base. (DT)

Not Available



Pheromone-regulated Genes Required for Yeast Mating Differentiation  

PubMed Central

Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for ?-galactosidase (?-gal) expression in the presence and absence of ? factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell–cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: ?-gal and Fig2::?-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

Erdman, Scott; Lin, Li; Malczynski, Michael; Snyder, Michael



[Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report  

SciTech Connect

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.

Guerinot, M.L.



cis-Acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes  

SciTech Connect

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.

Hofmann, A.; Garfinkel, M.D.; Meyerowitz, E.M. (California Inst. of Tech., Pasadena, CA (United States))



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.  


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 ?-galactosidase (?-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 ?-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

Leach, Derrik M; Zacal, Natalie J; Rainbow, Andrew J



Domains involved in osmoregulation of the ompF gene in Escherichia coli.  

PubMed Central

Expression of the ompF gene, which is under the control of the OmpR protein, is regulated by the osmolarity of the medium. To study the mechanism of osmoregulation, plasmids carrying two different types of chimeric genes were constructed. In one type, the coding region of the ompF gene was linked to the trp promoter (trpPO) preceding ompF, and in the other type the ompF upstream region, mostly composed of the region for regulation by OmpR and the promoter region, was linked to the lacZ gene by protein fusion. Expression of beta-galactosidase by the lacZ chimeric gene was OmpR dependent and osmoregulated as sensitively as that of the intact ompF gene. In the ompR20 background the direction of osmoregulation was opposite that of normal osmoregulation, as was the direction of osmoregulation of the intact ompF gene. Osmoregulation was also observed with trpPO-ompF chimeric genes. However, the regulation was not as sensitive to the osmolarity of the medium as was regulation of the intact ompF gene and was independent of OmpR. These results suggest that OmpR-dependent osmoregulation played a primary role in the osmoregulation of ompF expression and that ompR-independent osmoregulation most likely did not play a crucial role. Studies with a series of trpPO-ompF chimeric genes also suggest that the untranslated leader region, about 100 base pairs in length, between the transcription initiation site and the initiation codon was not required for osmoregulation. Images

Inokuchi, K; Itoh, M; Mizushima, S



The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.  


The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation. PMID:20221733

Souza, André L F; Invitti, Adriana L; Rego, Fabiane G M; Monteiro, Rose A; Klassen, Giseli; Souza, Emanuel M; Chubatsu, Leda S; Pedrosa, Fábio O; Rigo, Liu U