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Sample records for lacz reporter gene

  1. The glpD gene is a novel reporter gene for E. coli that is superior to established reporter genes like lacZ and gusA.

    PubMed

    Wegener, Marius; Vogtmann, Kristina; Huber, Madeleine; Laass, Sebastian; Soppa, Jörg

    2016-12-01

    Reporter genes facilitate the characterization of promoter activities, transcript stabilities, translational efficiencies, or intracellular localization. Various reporter genes for Escherichia coli have been established, however, most of them have drawbacks like transcript instability or the inability to be used in genetic selections. Therefore, the glpD gene encoding glycerol-3-phosphate dehydrogenase was introduced as a novel reporter gene for E. coli. The enzymatic assay was optimized, and it was verified that growth on glycerol strictly depends on the presence of GlpD. The 5'-UTRs of three E. coli genes were chosen and cloned upstream of the new reporter gene glpD as well as the established reporter genes lacZ and gusA. Protein and transcript levels were quantified and translational efficiencies were calculated. The lacZ transcript was very unstable and its level highly depended on its translation, compromising its use as a reporter. The results obtained with gusA and glpD were similar, however, only glpD can be used for genetic selections. Therefore, glpD was found to be a superior novel reporter gene compared to the established reporter genes lacZ and gusA.

  2. Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside.

    PubMed

    Lee, Kyung-Han; Byun, Sang Sung; Choi, Joon Hun; Paik, Jin-Young; Choe, Yearn Seong; Kim, Byung-Tae

    2004-03-01

    There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [(123)I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta

  3. An in vitro bioassay for xenobiotics using the SXR-driven human CYP3A4/lacZ reporter gene.

    PubMed

    Lee, Mi R; Kim, Yeon J; Hwang, Dae Y; Kang, Tae S; Hwang, Jin H; Lim, Chae H; Kang, Hyung K; Goo, Jun S; Lim, Hwa J; Ahn, Kwang S; Cho, Jung S; Chae, Kap R; Kim, Yong K

    2003-01-01

    The dose and time effect of nine xenobiotics, including 17beta-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lacZ reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lacZ (hCYP3A4/lacZ) constructs were transiently transfected into HepG2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the HepG2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lacZ transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17beta-estradiol or progesterone. In addition, 17beta-estradiol and progesterone did not change the levels of the lacZ transcripts in the HepG2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the HepG2 cells, did not affect the levels of the lacZ transcript in NIH3T3 cells. These results show that lacZ transcripts can be measured, rapidly and reproducibly, using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the expression of the hCYP3A4/lacZ reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.

  4. 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.

    PubMed

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

    2012-04-01

    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.

  5. Into the blue: the importance of murine lacZ gene expression profiling in understanding and treating human disease.

    PubMed

    Armit, Chris

    2015-11-01

    The International Mouse Phenotyping Consortium (IMPC) is a major international effort to explore the effects of knocking out 20,000 genes in the mouse. A new study by White and colleagues, published in the current issue of Disease Models & Mechanisms, demonstrates the usefulness of lacZ in situ reporter expression patterns in extending our understanding of genotype-phenotype relationships as part of the IMPC high-throughput screen. In situ gene expression profiling is invaluable for evaluating compartment-specific gene expression patterns, and these enrich our understanding of the role of genes in a great number of biological processes in multiple organ systems. Furthermore, the complexity of gene expression patterns informs our understanding of how genes influence lethality. This Editorial aims to highlight ways in which the lacZ expression profiles can impact on biomedical research by uncovering as-yet-unknown genotype-phenotype relationships, and through predicting the role of genes in health and disease.

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

    SciTech Connect

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

    1990-06-01

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

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

    SciTech Connect

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

    2008-03-15

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

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

    PubMed Central

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

    2015-01-01

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

  9. Skeletal Muscle Tissue Clearing for LacZ and Fluorescent Reporters, and Immunofluorescence Staining

    PubMed Central

    Verma, Mayank; Murkonda, Bhavani SR; Asakura, Yoko; Asakura, Atsushi

    2016-01-01

    Skeletal muscle is a highly ordered yet complex tissue containing several cell types that interact with each other in order to maintain structure and homeostasis. It is also a highly regenerative tissue that responds to damage in a highly intricate but stereotypic manner, with distinct spatial and temporal kinetics. Proper examination of this process requires one to look at the three-dimensional orientation of the cellular and subcellular components, which can be accomplished through tissue clearing. While there has been a recent surge of protocols to study biology in whole tissue, it has primarily focused on the nervous system. This chapter describes the workflow for whole mount analysis of murine skeletal muscle for LacZ reporters, fluorescent reporters and immunofluorescence staining. Using this technique, we are able to visualize LacZ reporters more effectively in deep tissue samples, and to perform fluorescent imaging with a depth greater than 1700 μm. PMID:27492170

  10. Radial derivatives of the mouse ventral pallium traced with Dbx1-LacZ reporters.

    PubMed

    Puelles, Luis; Medina, Loreta; Borello, Ugo; Legaz, Isabel; Teissier, Anne; Pierani, Alessandra; Rubenstein, John L R

    2016-09-01

    The progeny of Dbx1-expressing progenitors was studied in the developing mouse pallium, using two transgenic mouse lines: (1) Dbx1(nlslacZ) mice, in which the gene of the β-galactosidase reporter (LacZ) is inserted directly under the control of the Dbx1 promoter, allowing short-term lineage tracing of Dbx1-derived cells; and (2) Dbx1(CRE) mice crossed with a Cre-dependent reporter strain (ROSA26(loxP-stop-loxP-LacZ)), in which the Dbx1-derived cells result permanently labeled (Bielle et al., 2005). We thus examined in detail the derivatives of the postulated longitudinal ventral pallium (VPall) sector, which has been defined among other features by its selective ventricular zone expression of Dbx1 (the recent ascription by Puelles, 2014 of the whole olfactory cortex primordium to the VPall was tested). Earlier notions about a gradiental caudorostral reduction of Dbx1 signal were corroborated, so that virtually no signal was found at the olfactory bulb and the anterior olfactory area. The piriform cortex was increasingly labeled caudalwards. The only endopiriform grisea labeled were the ventral endopiriform nucleus and the bed nucleus of the external capsule. Anterior and basolateral parts of the whole pallial amygdala also were densely marked, in contrast to the negative posterior parts of these pallial amygdalar nuclei (leaving apart medial amygdalar parts ascribed to subpallial or extratelencephalic sources of Dbx1-derived GABAergic and non-GABAergic neurons). Alternative tentative interpretations are discussed to explain the partial labeling obtained of both olfactory and amygdaloid structures. This includes the hypothesis of an as yet undefined part of the pallium, potentially responsible for the posterior amygdala, or the hypothesis that the VPall may not be wholly characterized by Dbx1 expression (this gene not being necessary for VPall molecular distinctness and histogenetic potency), which would leave a dorsal Dbx1-negative VPall subdomain of variable size

  11. A lacZ reporter fusion method for the genetic analysis of regulatory mutations in pathways of fungal secondary metabolism and its application to the Aspergillus nidulans penicillin pathway.

    PubMed Central

    Pérez-Esteban, B; Gómez-Pardo, E; Peñalva, M A

    1995-01-01

    Secondary metabolism, usually superfluous under laboratory conditions, is intrinsically elusive to genetic analysis of its regulation. We describe here a method of analyzing regulatory mutations affecting expression of secondary metabolic genes, with an Aspergillus nidulans penicillin structural gene (ipnA [encoding isopenicillin N-synthase]) as a model. The method is based on a targeted double integration of a lacZ fusion reporter gene in a chromosome different from that containing the penicillin gene cluster. The trans-acting regulatory mutations simultaneously affect lacZ expression and penicillin biosynthesis. One of these mutations (npeE1) has been analyzed in detail. This mutation is recessive, prevents penicillin production and ipnA'::'lacZ expression, and results in very low levels of the ipnA message at certain times of growth. This indicates that npeE positively controls ipnA transcription. We also show that this tandem reporter fusion allows genetic analysis of npeE1 by using the sexual and parasexual cycles and that lacZ expression is an easily scorable phenotype. Haploidization analysis established that npeE is located in chromosome IV, but npeE1 does not show meiotic linkage to a number of known chromosome IV markers. This method might be of general applicability to genetic analysis of regulation of other fungal secondary metabolic pathways. PMID:7592369

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

    PubMed

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

    2013-05-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    1993-01-01

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

  15. Construction of an Escherichia-Pseudomonas shuttle vector containing an aminoglycoside phosphotransferase gene and a lacZ'' Gene for alpha-complementation.

    PubMed

    Lee, Bheong-Uk; Hong, Ja-Heon; Kahng, Hyung-Yeel; Oh, Kye-Heon

    2006-12-01

    A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminoglycoside phosphotransferase gene (aph) from Tn903, a lacZ'' gene for alpha-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DH5alpha and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

  16. Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus

    PubMed Central

    Gordon, Julie; Xiao, Shiyun; Hughes, Bernard; Su, Dong-ming; Navarre, Samuel P; Condie, Brian G; Manley, Nancy R

    2007-01-01

    Background Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. Results We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. Conclusion These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains. PMID:17577402

  17. Effects of vacuum UV and UVC radiation on dry Escherichia coli plasmid pUC19. II. Mutational specificity at the lacZ gene.

    PubMed

    Wehner, J; Horneck, G

    1995-10-01

    The mutational spectra at the lacZ gene, induced either by vacuum UV at 160 nm or UVC at 254 nm in vacuum-dried preparations of Escherichia coli plasmid pUC19 DNA, have been characterized from 72 E. coli-propagated mutants by DNA sequencing. In plasmids irradiated in vacuum, vacuum UV is five times more mutagenic than UVC. In the UV-induced mutants, base substitutions largely predominate, with GC-->AT (G, guanine; C, cytosine; A, adenine; T, thymine) transitions being the most abundant type of base change for vacuum UV (61%) and UVC (47%). Most of the GC-->AT transitions appear to occur at dipyrimidine sites, which are located at the non-transcribed DNA strand. Some, but not all, hot spots for GC-->AT transitions are identical for vacuum UV and UVC. Frameshifts, resulting from a loss of the thymine residue, are specific for UVC (22%), and were not detected after treatment with vacuum UV. They occur predominantly at thymine runs of the transcribed DNA strand. Only a few deletions were detected following irradiation with vacuum UV (7.5%) and UVC (2%); however, their frequency is not enhanced compared with the spontaneous mutation spectrum. The data confirm the important role of base substitution mutations in UV-induced mutagenesis, which is not only valid for the UVC range, but extends towards the vacuum UV range.

  18. Synthesis of radioiodine-labeled 2-phenylethyl 1-thio-beta-D-galactopyranoside for imaging of LacZ gene expression.

    PubMed

    Choi, Joon Hun; Choe, Yearn Seong; Lee, Kyung-Han; Choi, Yong; Kim, Sang Eun; Kim, Byung-Tae

    2003-01-02

    A potent inhibitor of beta-galactosidase (EC 3.2.1.23), 2-phenylethyl 1-thio-beta-D-galactopyranoside (PETG), was radioiodinated for noninvasive imaging of LacZ gene expression. In order to introduce radioiodine to the phenyl ring of PETG, 2-(4-bromophenyl)ethanethiol was prepared and attached to the C-1 position of beta-D-galactose pentaacetate under conditions that resulted in the exclusive formation of the beta anomer. The bromo group of PETG was converted to the tributylstannyl group where radioiododemetallation was carried out. Radioiodine-labeled PETG tetraacetate was purified by HPLC, which can be used as a prodrug for biological evaluation or hydrolyzed to 2-(4-[123I/125I]iodophenyl)ethyl 1-thio-beta-D-galactopyranoside ([123I/125I]7) under basic conditions. The resulting radioiodine-labeled PETG was obtained in overall 62% radiochemical yield (decay-corrected) and with specific activity of 46-74 GBq/micromol.

  19. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

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

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

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

    PubMed Central

    Busuttil, Rita A.; Muñoz, Denise P.; Garcia, Ana Maria; Rodier, Francis; Kim, Woo Ho; Suh, Yousin; Hasty, Paul; Campisi, Judith; Vijg, Jan

    2008-01-01

    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 mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and γH2AX DNA damage foci in Ku80−/− as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements. PMID:18941635

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

    PubMed Central

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

    2015-01-01

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

  2. An improved vector system for constructing transcriptional lacZ fusions: analysis of regulation of the dnaA, dnaN, recF and gyrB genes of Escherichia coli.

    PubMed

    Macián, F; Pérez-Roger, I; Armengod, M E

    1994-07-22

    We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.

  3. Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

    PubMed Central

    Bärtsch, S; Dücker, K; Würgler, F E; Sengstag, C

    1997-01-01

    Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development. PMID:9380517

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  5. Characterisation of Muta™Mouse λgt10-lacZ transgene: evidence for in vivo rearrangements

    PubMed Central

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

    2010-01-01

    The multicopy λgt10-lacZ transgene shuttle vector of Muta™Mouse serves as an important tool for genotoxicity studies. Here, we describe a model for λgt10-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 λgt10 used to construct the transgene and the Muta™Mouse precursor (strain 40.6), we validated the sequence integrity of key λ 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 λgt10-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 λgt10-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. PMID:20724577

  6. A rat 8 kb dentin sialoprotein-phosphophoryn (DSP-PP) promoter directs spatial and temporal LacZ activity in mouse tissues.

    PubMed

    Godovikova, Valentina; Li, Xiu-Rong; Saunders, Thomas L; Ritchie, Helena H

    2006-01-15

    Dentin sialoprotein (DSP) and phosphophoryn (PP) are two major dentin noncollagenous proteins that are encoded on a single DSP-PP transcript whose expression is tightly regulated during tooth dentinogenesis. The recent identification of this gene transcript in other tissues, including inner ear and jaw tissue, suggests that DSP and PP may have pleiotropic effects on other organs besides teeth. To identify candidate regulatory elements that control DSP-PP temporal and spatial expression, we constructed a -5 kb upstream region rat DSP-PP promoter into the beta-galactosidase expression vector pnLacF plasmid and used this construct to prepare DSP-PP-LacZ transgenic mice. Multiple mouse tissues including teeth, bone, and kidney obtained from the six resulting transgenic mouse lines displayed strong LacZ activity. This spatial distribution was confirmed in several of these tissues by in situ hybridization studies. LacZ activity was transiently expressed in preameloblasts and continuously expressed in odontoblasts demonstrating that this -5 kb rat promoter-dependent LacZ expression mimics reported DSP-PP mRNA expression patterns. Interestingly, this -5 kb rat promoter construct drives LacZ expression according to the rat developmental clock. Based on identified transcription factors present in this -5 kb promoter region, we have identified several probable cis-regulatory modules whose interaction with one another could account for the spatial and temporal distribution of DSP-PP transcripts in developing tissues.

  7. Fate Tracing of neurogenin2-Expressing Cells in the Mouse Retina Using CreER™: LacZ

    PubMed Central

    Ma, Wenxin; Wang, Shu-Zhen

    2012-01-01

    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

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

    PubMed

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

    2007-11-30

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

  9. Analysis of a eukaryotic beta-galactosidase gene: the N-terminal end of the yeast Kluyveromyces lactis protein shows homology to the Escherichia coli lacZ gene product.

    PubMed Central

    Breunig, K D; Dahlems, U; Das, S; Hollenberg, C P

    1984-01-01

    The LAC4 gene of Kluyveromyces lactis, encoding the enzyme beta-galactosidase was mapped on a cloned DNA fragment and the sequence of the 5' end was determined. This sequence includes the 5' regulatory region involved in the induction by lactose and the N-terminal end of the protein coding region. Comparison of the deduced amino acid sequence of this eukaryotic enzyme with the N-terminal end of the Escherichia coli beta-galactosidase revealed substantial homology. Two major RNA initiation sites were mapped at -115 and -105. A number of structural peculiarities of the 5'non-coding region are discussed as in comparison to Saccharomyces cerevisiae genes. Images PMID:6324114

  10. An equine herpesvirus 1 mutant with a lacZ insertion between open reading frames 62 and 63 is replication competent and causes disease in the murine respiratory model.

    PubMed

    Csellner, H; Walker, C; Love, D N; Whalley, J M

    1998-01-01

    An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no modification to late gene expression as monitored by synthesis of EHV-1 glycoproteins C and D. The parental EHV-1 isolate HVS25A used here had almost identical nucleotide sequence to that published for isolate Ab4, in a 1200 bp region surrounding the insert, but lacked a HindIII site corresponding to Ab4 position 109,048. The lacZ62/63-EHV-1 caused respiratory disease in BALB/c mice with clinical signs, histopathology and virus titres in lungs throughout days 1-5 post infection similar to those induced by wt EHV-1. X-gal staining for beta-galactosidase expression in murine lungs clearly demonstrated EHV-1 infection in cells of the bronchiolar epithelium and pulmonary parenchyma, with a peak of infection evident at day 2 post infection, when up to 50% of bronchioles demonstrated blue-staining and thus virus-infected epithelial cells. The construction of this replication competent virus carrying a reporter gene identifies a site for insertion of foreign genes and will facilitate studies on the pathogenesis of EHV-1.

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

    PubMed Central

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

    1995-01-01

    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. PMID:8582614

  12. GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling

    PubMed Central

    Rémond, Mathieu C.; Iaffaldano, Grazia; O'Quinn, Michael P.; Mezentseva, Nadejda V.; Garcia, Victor; Harris, Brett S.; Gourdie, Robert G.; Eisenberg, Carol A.

    2011-01-01

    This study examined transgenic mice whose expression of a β-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial β-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states. PMID:21908788

  13. GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling.

    PubMed

    Rémond, Mathieu C; Iaffaldano, Grazia; O'Quinn, Michael P; Mezentseva, Nadejda V; Garcia, Victor; Harris, Brett S; Gourdie, Robert G; Eisenberg, Carol A; Eisenberg, Leonard M

    2011-11-01

    This study examined transgenic mice whose expression of a β-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial β-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states.

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

    PubMed Central

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

    2011-01-01

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

  15. Systemic RNAi Delivery to the Muscles of ROSA26 Mice Reduces lacZ Expression

    PubMed Central

    Wei, Jessica; Chamberlain, Joel R.

    2014-01-01

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

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

    PubMed

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

    2016-03-01

    3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.

  17. Singlet oxygen-induced mutations in M13 lacZ phage DNA.

    PubMed Central

    Decuyper-Debergh, D; Piette, J; Van de Vorst, A

    1987-01-01

    The mutagenic consequences of damages to M13 mp19 RF DNA produced by singlet oxygen have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When the damaged M13 mp19 RF DNA is used to transfect competent E. coli JM105 cells, a 16.6-fold increase in mutation frequency is observed at 5% survivors when measured as a loss of alpha-complementation. The enhanced mutagenicity is largely due to single-nucleotide substitutions, frameshift events and double-mutations. The single-nucleotide substitutions occur in the regulatory and in the structural part of the lacZ gene under the predominant form of a G:C to T:A transversion. The spectrum of mutations detected among the M13 lacZ phages surviving the singlet oxygen treatment is totally different from those appearing spontaneously. SOS induction mediated through u.v.-irradiation of bacteria leads to an increase of the mutation frequency in the M13 surviving to the singlet oxygen treatment. The mutation spectrum in this case is a mixture between those observed with the spontaneous mutants and the mutants induced by singlet oxygen. Lesions introduced in the M13 mp19 RF DNA can be partly repaired by the enzymatic machinery of the bacteria. It turns out that excision-repair and SOS repair are probably involved in the removal of these lesions by singlet oxygen. PMID:3121306

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

    PubMed

    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

    2008-04-18

    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.

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

    PubMed

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

    2013-04-01

    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.

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

    PubMed Central

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

    2016-01-01

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

  1. Efficient expression of gene variants that harbour AGA codons next to the initiation codon

    PubMed Central

    Zamora-Romo, Efraín; Cruz-Vera, Luis Rogelio; Vivanco-Domínguez, Serafín; Magos-Castro, Marco Antonio; Guarneros, Gabriel

    2007-01-01

    In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons. The mixed combinations of AGA and each of the CGN codons usually resulted in efficient rates of lacZ expression independently of the peptidyl-tRNA propensity to dissociate from the ribosome. Thus, the variant harboring the pair of AGA codons was expressed as efficiently as the variant carrying a pair of AAA codons in the same positions, a configuration reported as one of the most common and efficient for gene expression. We explain these results assuming that the presence of adenines in these early positions enhance gene expression. As expected, specific mRNA levels correlated with the intensity of lacZ expression for each variant. However, the induction of lacZ AGA AGA gene in pth cells accumulated peptidyl-tRNAArg4 as well as a short 5′-proximal lacZ mRNA fragment suggesting ribosome stalling due to depletion of aminoacylated-tRNAArg4. PMID:17726048

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

    PubMed Central

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

    2012-01-01

    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 β-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. PMID:22352428

  3. [Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination].

    PubMed

    Li, Shanhu; Shi, Qingguo; Huang, Cuifen; Zhou, Jianguang

    2008-04-01

    To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

  4. An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

    PubMed Central

    Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

    2016-01-01

    The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

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

    PubMed Central

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

    2010-01-01

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

  6. [Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

    PubMed

    Chen, Wei; Yu, Mei; Li, Shan-Hu; Wang, Ming-Gang; Zhou, Jian-Guang

    2005-03-01

    pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

  7. Nonviral gene transfer to surface skin of mid-gestational murine embryos by intraamniotic injection and subsequent electroporation.

    PubMed

    Sato, Masahiro; Tanigawa, Maya; Kikuchi, Natsuko

    2004-11-01

    The surface epithelium of mid-gestational murine embryos is thought to be an attractive target for gene therapy in vivo, due to its visibility and accessibility from the external surface of the maternal uterus. Almost all studies of in utero gene transfer have adopted viral vectors for infection of fetal epithelium, and depended on intraamniotic introduction and simple incubation of vectors, leading to only infection of the surface layer (periderm) of fetal skin. Here we report a simple and convenient method of gene transfer of plasmid DNA into the deeper portion of surface skin of murine mid-gestational fetus. One to two microlitres of a solution containing a lacZ expression plasmid (0.5-1 microg) and trypan blue (0.05%) were placed onto the surface of a fetus (E 14.5) near the eye by a micropipette attached to a mouthpiece. This fetus was immediately electroporated by placing it between tweezer-type electrodes attached to a square-pulse generator. At 1 and 4 days after gene transfer, fetuses were subjected to histochemical staining for lacZ activity in the presence of X-Gal, a substrate for lacZ. Focal reactions were observed in the skin epidermal layers including periderm and basal layer 1 day after DNA introduction. However, lacZ-positive cells were limited to a skin surface layer, the stratum corneum, in the samples obtained 4 days after gene transfer. Similar observation was also made in the transgenic fetuses (carrying a lacZ gene placed immediately downstream of the loxP-flanked sequence) injected with Cre expression vector. These findings suggest rapid movement of fetal epidermal cells toward the surface during late developmental stages. This local gene transfer approach appears to be effective as a method for skin-targeted gene transfer, enabling study of the role of genes of interest and tracing of cell lineage during fetal skin development.

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

    PubMed Central

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

    1992-01-01

    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 PMID:1639062

  9. Posttranscriptional Regulation of the Neurofibromatosis 2 Gene

    DTIC Science & Technology

    2005-08-01

    14 . ABSTRACT Neurofibromatosis type 2 (NF2) is associated with a homozygous inactivation of the neurofibromatosis 2 (NF2) gene. Despite intense...direct β-galactosidase (β-gal) reporter expression as early as embryonic day 5.5 (E5.5). NF2 promoter activity was first detected in the embryonic...8 carrying a 2.4-kb NF2 promoter-driven the lacZ gene with a nuclear localization signal. Whole- mount X-gal staining of embryos at various days

  10. The Chicken Gene Nomenclature Committee report.

    PubMed

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

    2009-07-14

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

  11. Expression of lacZ from the promoter of the Escherichia coli spc operon cloned into vectors carrying the W205 trp-lac fusion.

    PubMed

    Liang, S T; Dennis, P P; Bremer, H

    1998-12-01

    The expression of lacZ has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of lacZ mRNA by hybridization and the amount of beta-galactosidase by standard enzymatic assay. Expression was driven by the promoter, Pspc, of the spc ribosomal protein operon. The vectors contained either the standard W205 trp-lac fusion with the trp operon transcription terminator, trpt, located in the lacZ leader sequence, or a deletion derivative that functionally inactivates trpt. In the presence of trpt, lacZ expression was temperature dependent so that increasing the growth temperature reduced the accumulation of lacZ mRNA and beta-galactosidase activity. The frequency of transcript termination at trpt was estimated to be near zero at 20 degreesC and at about 45% at 37 degreesC. The amount of Pspc-derived lacZ mRNA and the amount of beta-galactosidase produced per lacZ mRNA varied, depending on the mRNA 5' leader sequence between Pspc and lacZ. These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful analysis and interpretation. One particular construct without trpt did not seem to contain fortuitous transcription or translation signals generated at the fusion junction. In this strain, lacZ expression from Pspc was compared at the enzyme activity and mRNA levels with a previously constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the rrnB operon. At any given growth rate, the different activities of beta-galactosidase in these two strains were found to reflect the same differences in their amounts of lacZ mRNA. Assuming that the promoter-lacZ fusions in these strains reflect the properties of the promoters in their normal chromosomal setting, it was possible to estimate the absolute transcription activity of Pspc and the relative translation efficiency of Pspc-lacZ mRNA at different growth rates. Transcription from the spc promoter was found

  12. Mutagenic activity of 4-nitroquinoline-N-oxide in upper aerodigestive tissue in lacZ mice (MutaMouse) and the effects of 1, 4-phenylenebis(methylene)selenocyanate.

    PubMed

    von Pressentin, M M; El-Bayoumy, K; Guttenplan, J B

    2000-03-03

    4-Nitroquinoline-N-oxide (4-NQO) was administered to lacZ mice at a concentration of 20 microg/ml in drinking water for 2 weeks, and the mutagenic fractions in a number of organs were assayed. The mutant fractions in tongue, esophagus and other pooled oral tissues were, respectively, 117+/-26, 73+/-15, and 48+/-15 mutants/10(5) plaque-forming units (pfu) (ca. 15-40xbackground). 4-NQO was not mutagenic in lung, liver or colon at conditions used here. We had previously demonstrated that the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), an established chemopreventive agent, greatly reduced carcinogenicity in 4-NQO in rat tongue, and we observed here that administration of p-XSC (10 ppm se) in the diet for 6 weeks (2 weeks before, during, and 2 weeks after 4-NQO) resulted in a 33% decrease in mutagenesis in oral tissue, a 17% decrease in esophagus, and a slight increase in tongue. Only the decrease in oral tissue reached statistical significance (p<0.04). The results reported here demonstrate that 4-NQO was extremely mutagenic in lacZ mouse tongue, with lower, but highly significant activities in esophagus and other pooled oral tissues. The high activity of 4-NQO in lacZ mouse tongue is consistent with the organ specificity of 4-NQO in the rat. Inhibition of 4-NQO-induced mutagenesis by p-XSC was observed mainly in pooled oral tissues, other than tongue. Possible reasons for the difference between inhibition of mutagenesis and carcinogenesis in tongue are discussed, as well as advantages and disadvantages of in vivo mutagenesis assays as surrogates for carcinogenicity assays in chemoprevention studies.

  13. Extended function of plasmid partition genes: the Sop system of linear phage-plasmid N15 facilitates late gene expression.

    PubMed

    Ravin, Nikolai V; Rech, Jérôme; Lane, David

    2008-05-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    1997-01-01

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

  16. Gene deletion in urothelium by specific expression of Cre recombinase.

    PubMed

    Mo, Lan; Cheng, Jin; Lee, Eva Y-H P; Sun, Tung-Tien; Wu, Xue-Ru

    2005-09-01

    Urothelium that lines almost the entire urinary tract acts as a permeability barrier and is involved in the pathogenesis of major urinary diseases, including urothelial carcinoma, urinary tract infection, and interstitial cystitis. However, investigation of urothelial biology and diseases has been hampered by the lack of tissue-specific approaches. To address this deficiency, we sought to develop a urothelium-specific knockout system using the Cre/loxP strategy. Transgenic mouse lines were generated in which a 3.6-kb mouse uroplakin II (UPII) promoter was used to drive the expression of Cre recombinase (Cre). Among the multiple tissues analyzed, Cre was found to be expressed exclusively in the urothelia of the transgenic mice. Crossing a UPII-Cre transgenic line with a ROSA26-LacZ reporter line, in which LacZ expression depends on Cre-mediated deletion of a floxed "stop" sequence, led to LacZ expression only in the urothelium. Gene recombination was also observed when the UPII-Cre line was crossed to an independent line in which a part of the p53 gene was flanked by the loxP sequences (floxed p53). Truncation of the p53 gene and mRNA was observed exclusively in the urothelia of double transgenic mice harboring both the UPII-Cre transgene and the floxed p53 allele. These results demonstrate for the first time the feasibility and potentially wide applicability of the UPII-Cre transgenic mice to inactivate any genes of interest in the urothelium.

  17. Analysis of the advantages of cis reporters in optimized FACS-Gal.

    PubMed

    Sánchez-Luengo, Miguel Ángel; Rovira, Miguel; Serrano, Manuel; Fernandez-Marcos, Pablo Jose; Martinez, Lola

    2017-04-04

    Flow cytometry is a powerful multiparametric technology, widely used for the identification, quantification, and isolation of defined populations of cells based on the expression of target proteins. It also allows for the use of surrogate reporters, either enzymatic or fluorescent, to indirectly monitor the expression of these target proteins. In this work, we optimised the dissociation protocol for the detection of the enzymatic reporter LacZ using the FACS-Gal detection system with the fluorogenic substrate FDG to compare cis- versus trans-positioned reporters efficiency. Particularly, for the FACS-Gal optimization, we studied lung and haematopoietic tissues, focusing on cell recovery, viability, FDG loading conditions and distribution of cellular populations. Reporter genes such as LacZ can be placed together with the gene of interest in the same polycistronic mRNA (in cis), or in independent alleles (in trans), which can strongly affect the correlation with the reporter readout. To address this issue, we generated a mouse model containing both types of reporters for the same gene, and compared them. Our results clearly indicate that trans-positioned reporters can be misleading, and that using a reporter gene in cis rather than trans is a much more specific method to sort for cells undergoing Cre-mediated recombination. © 2017 International Society for Advancement of Cytometry.

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

    PubMed

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

    2009-05-01

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

  19. Chloroform-free permeabilization for improved detection of β-galactosidase activity in Vibrio cholerae.

    PubMed

    Toulouse, Charlotte; Häse, Claudia C; Steuber, Julia

    2017-03-21

    LacZ (β-galactosidase) is used to monitor the transcription of genes in reporter strains carrying the lacZ gene under the control of a promotor of interest. This protocol for LacZ activity determinations in Vibrio cholerae following detergent lysis results in 2.5-fold increase of LacZ activities compared to lysis with chloroform.

  20. A General Approach for Identifying Distant Regulatory Elements Applied to the Gdf6 Gene

    PubMed Central

    Mortlock, Douglas P.; Guenther, Catherine; Kingsley, David M.

    2003-01-01

    Regulatory sequences in higher genomes can map large distances from gene coding regions, and cannot yet be identified by simple inspection of primary DNA sequence information. Here we describe an efficient method of surveying large genomic regions for gene regulatory information, and subdividing complex sets of distant regulatory elements into smaller intervals for detailed study. The mouse Gdf6 gene is expressed in a number of distinct embryonic locations that are involved in the patterning of skeletal and soft tissues. To identify sequences responsible for Gdf6 regulation, we first isolated a series of overlapping bacterial artificial chromosomes (BACs) that extend varying distances upstream and downstream of the gene. A LacZ reporter cassette was integrated into the Gdf6 transcription unit of each BAC using homologous recombination in bacteria. Each modified BAC was injected into fertilized mouse eggs, and founder transgenic embryos were analyzed for LacZ expression mid-gestation. The overlapping segments defined by the BAC clones revealed five separate regulatory regions that drive LacZ expression in 11 distinct anatomical locations. To further localize sequences that control expression in developing skeletal joints, we created a series of BAC constructs with precise deletions across a putative joint-control region. This approach further narrowed the critical control region to an area containing several stretches of sequence that are highly conserved between mice and humans. A distant 2.9-kilobase fragment containing the highly conserved regions is able to direct very specific expression of a minimal promoter/LacZ reporter in proximal limb joints. These results demonstrate that even distant, complex regulatory sequences can be identified using a combination of BAC scanning, BAC deletion, and comparative sequencing approaches. PMID:12915490

  1. Effects of vacuum UV and UVC radiation on dry E. coli plasmid pUC19. I. Inactivation, lacZ- mutation induction and strand breaks.

    PubMed

    Wehner, J; Horneck, G

    1995-04-01

    Using Escherichia coli plasmid pUC19 as a test system to study the effects of radiation on DNA at the molecular level, the wavelength (160-254 nm) dependence of inactivation (loss of the ability to transform E. coli), mutation induction in the target gene lacZ and induction of single-strand breaks and double-strand breaks was investigated. The same fluences were applied for all endpoints tested. In the UVC range, the cross-sections of inactivation and mutation induction match the DNA absorption curve, whereas the cross-section for single-strand break induction deviates from the DNA curve, especially at 220 nm. In the vacuum UV range, with increasing energy of the photons, the cross-sections of inactivation and single-strand breaks increase sharply (from 190 to 160 nm by more than one order of magnitude), which is not reflected by the DNA curve. In this UV range, the shape of the action spectrum is similar to that of the absorption curve of the sugar phosphate moiety of DNA. Only after irradiation with vacuum UV at 160 nm are double-strand breaks detected. Their induction rate is about one order of magnitude lower than that of single-strand breaks at the same wavelength; however, their induction rate is at least twice that of single-strand breaks at longer wavelengths. Concerning mutation induction, the increment in the vacuum UV range is less well expressed. The data suggest the contribution of different kinds of photochemical injury to inactivation and mutation induction.

  2. Vectors for gene expression and amplification in the yeast Yarrowia lipolytica.

    PubMed

    Juretzek, T; Le Dall, M; Mauersberger, S; Gaillardin, C; Barth, G; Nicaud, J

    2001-01-30

    New vector systems were developed for gene expression in Y. lipolytica. These plasmids contain: (a) as integration target sequences, either a rDNA region or the long terminal repeat zeta of the Y. lipolytica retrotransposon Ylt1; (b) the YlURA3 gene as selection marker for Y. lipolytica, either as the non-defective ura3d1 allele for single integration or the promotor truncated ura3d4 allele for multiple integration; (c) the inducible ICL1 or XPR2 promoters for gene expression; and (d) unique restriction sites for gene insertion. Multiple plasmid integration occurred as inserted tandem-repeats, which are present at 3-39 copies per cell. A correlation between gene copy number and the expressed enzyme activity was demonstrated with Escherichia coli lacZ as reporter gene under the control of the regulated ICL1 promoter. Increases in copy numbers from 5 to 13 for the lacZ expression cassettes resulted in an up to 10-11-fold linear increase of the beta-galactosidase activity in multicopy transformants during their growth on ethanol or glucose, compared with the low-copy replicative plasmid transformants (1.6 plasmid copies). These new tools will enhance the interest in Y. lipolytica as an alternative host for heterologous protein production.

  3. Recombinant Carcinoembryonic Antigen as a Reporter Gene for Molecular Imaging

    PubMed Central

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Braun, Jonathan; Wu, Anna M.

    2009-01-01

    Purpose Reporter genes can provide a way of non-invasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. Methods To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Results Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124I-labeled scFv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4h. MicroPET image quantitation showed tumor activity of 1.8(±0.2), 15.2(±1.3) and 4.6(±1.2) %ID/g at 4h, 20h and 48h, respectively. Biodistribution at 48h, demonstrated tumor uptake of 4.8(±0.8) %ID/g. Conclusion The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. PMID:18719907

  4. Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZ.

    PubMed

    Shimosato, Takeshi; Tohno, Masanori; Sato, Takashi; Nishimura, Junko; Kawai, Yasushi; Saito, Tadao; Kitazawa, Haruki

    2009-10-01

    Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  6. Reporter gene assays for algal-derived toxins.

    PubMed

    Fairey, E R; Ramsdell, J S

    1999-01-01

    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.

  7. Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe.

    PubMed

    Wang, Hongcheng; Wang, Haiyang; Wang, Meng; Zhang, Lei; Wang, Ren; Mei, Yanzhen; Shao, Weilan

    2014-06-01

    Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.

  8. Genetics in methylotrophic bacteria: Appendix. Final report

    SciTech Connect

    Lidstrom, M.E.

    1998-09-01

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

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

    SciTech Connect

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

    2005-01-05

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2013-02-01

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

  12. A 19F NMR Approach using Reporter Molecule Pairs to Assess β-Galactosidase in Human Xenograft Tumors in Vivo

    PubMed Central

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

    2011-01-01

    Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection in the target tissue and the longevity of gene expression. Thus, there is increasing interest in the development of non-invasive in vivo reporter techniques to assay gene expression. We recently demonstrated the ability to detect β-galactosidase activity in stably transfected human prostate tumor xenografts in mice in vivo using 19F NMR. We now extend the studies to human MCF7 breast cancer cells growing as xenografts in nude mice. Moreover, by using two spectrally resolved reporters (o-fluoro-p-nitrophenyl-β-D-galactopyranoside and an isomer) two tumors could be interrogated simultaneously revealing lacZ transgene activity in a stably transfected tumor versus no activity in a wild type tumor. Most significantly hydrolytic activity observed by 19F NMR corresponded with differential activity in lacZ expressing tumors. PMID:18288788

  13. 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...

  14. Replication-Competent Influenza A Viruses Expressing Reporter Genes

    PubMed Central

    Breen, Michael; Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. PMID:27347991

  15. Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

    PubMed

    Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

    2009-02-01

    Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

  16. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    PubMed Central

    Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

    2016-01-01

    DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

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

    DOEpatents

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

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

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

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

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

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

    SciTech Connect

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

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

  20. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    PubMed

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

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

    PubMed

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

    2003-12-01

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

  2. The veA gene is necessary for the negative regulation of the veA expression in Aspergillus nidulans.

    PubMed

    Kim, Hyoun-Young; Han, Kap-Hoon; Lee, Mimi; Oh, Miae; Kim, Hee-Seo; Zhixiong, Xie; Han, Dong-Min; Jahng, Kwang-Yeop; Kim, Jong Hwa; Chae, Keon-Sang

    2009-08-01

    The veA gene is one of the key genes in regulating sexual development of Aspergillus nidulans. During the study on the veA gene, it was observed that the veA expression level is slightly higher in a veA1 mutant than in a wild type at 37 degrees C, suggesting that the wild type veA gene is necessary for the negative regulation of the veA expression. In the veA1 mutant, the veA expression was higher than in a wild type grown at 42 degrees C but equal at 30 degrees C. Furthermore, in a veA deletion mutant having its own promoter and the N-terminus of the VeA ORF, expression of the N-terminus by the veA promoter was highly up-regulated, supporting the possibility that the veA gene is important for the negative regulation of the veA expression. Analyses of the lacZ transcript and the beta-galactosidase activity from the reporter strains in the veA1 background, which were constructed by transformation of the lacZ reporter plasmids containing the lacZ gene under the control of the intact or the truncated veA promoters from the -943 to +262 bp region, showed that the truncated promoters produced more veA transcript and higher beta-galactosidase activity than the intact one at 30 degrees C, but equal at 42 degrees C. In addition, the serial-deletion analysis of the veA promoter identified a crucial region in the promoter from -943 to -740 bp for this derepression of the veA expression. Taken together, these results indicated that the veA gene is necessary for the negative regulation of the veA expression. Moreover, the veA expression was derepressed in the light-illuminated condition, where the VeA protein is hardly transported into the nucleus.

  3. Pitfalls of the CAT reporter gene for analyzing translational regulation in Leishmania.

    PubMed

    Folgueira, Cristina; Requena, Jose M

    2007-10-01

    Heterologous reporter genes are widely used for the characterization of gene expression in many organisms. Particularly, constructs bearing reporter genes have greatly contributed to our understanding of gene regulation in kinetoplastids. In some specific circumstances, however, such heterologous reporter has a risk of resulting in irrelevant observations and conclusions, which are primarily due to the introduction of foreign sequence elements. This communication describes our recent experience using the chloramphenicol acetyltransferase (CAT) gene as a reporter for analysis of the translational regulation of HSP70 genes in Leishmania infantum. We show that chimeric mRNAs consisting of the CAT open reading frame (ORF) and the untranslated regions (UTRs) from HSP70-II genes behave differently as endogenous HSP70-II mRNAs and that this difference is due to the presence of CAT sequences. Thus, the main purpose of this communication is to alert researchers working in gene regulation to be cautious when interpreting results based on heterologous reporter genes.

  4. Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon.

    PubMed

    Smolke, Christina D; Keasling, Jay D

    2002-12-30

    The effects of endoribonuclease sites, secondary structures in mRNA, and gene placement on protein production and mRNA stability and steady-state levels were tested in a dual-gene operon containing the genes encoding beta-galactosidase (lacZ) from Escherichia coli and green fluorescent protein (gfp) from Aequorea victoria. Two previously identified RNase E sites were placed separately between the coding regions to direct cleavage in this area and produce two secondary transcripts, each containing a single-gene coding region. Novel secondary structures were engineered into the 3' and 5' ends of each of the coding regions to protect the transcript from inactivation by endoribonucleases (5' hairpins) and degradation by exoribonucleases (3' hairpins). In addition, the effects of relative gene placement were examined by switching the locations of the two coding regions. Depending on the particular secondary structures and RNase E sites placed between the genes the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 2.5-fold and 4-fold, respectively. By changing gene location and incorporating secondary structures and RNase E sites the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 750-fold, respectively.

  5. Hormonal activity of polycyclic musks evaluated by reporter gene assay.

    PubMed

    Mori, Taiki; Iida, Mitsuru; Ishibashi, Hiroshi; Kohra, Shinya; Takao, Yuji; Takemasa, Takehiro; Arizono, Koji

    2007-01-01

    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

  6. Next generation sequencing of benzo(a)pyrene-induced lacZ mutants identifies a germ cell-specific mutation spectrum

    PubMed Central

    O’Brien, Jason M.; Beal, Marc A.; Yauk, Carole L.; Marchetti, Francesco

    2016-01-01

    De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male germline. We investigated whether male germ cells have unique mechanisms for spontaneous or chemically-induced mutation relative to somatic cells using the MutaMouse model. We recovered lacZ transgenes from sperm 42 days after a 28-day exposure to benzo(a)pyrene (BaP, 100 mg/kg/day) to assess mutations arising in dividing spermatogonia. BaP caused a 3.4-fold increase in lacZ mutant frequency over controls which increased to 4.1-fold after clonal correction. We then used next generation sequencing to compare the spontaneous and BaP-induced mutation spectra in sperm and bone marrow. The spontaneous spectrum in sperm had significantly more G:C to A:T transitions and fewer mutations at A:T basepairs than bone marrow. BaP predominantly induced G:C to T:A transversions in both cell types, and both were enriched for mutations at CpG dinucleotides. However, BaP induced significantly more deletions in sperm, but more G:C to A:T transitions and G:C to C:G transversions in bone marrow. Differences in error-prone translesion DNA synthesis polymerases may underlie the observed spectrum differences between sperm and bone marrow. These findings suggest that mutations in sperm can arise via mechanisms that are unique to male germ cells. PMID:27829668

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

    PubMed Central

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

    2013-01-01

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

  8. [Construction and specificity of porcine bmp15 gene reporter vector].

    PubMed

    Qin, Mingming; Wei, Jianghua; Yu, Xiaoli; Zhang, Jinglong; Liu, Xiaopeng; Ma, Xiaoling; Wang, Huayan

    2014-02-01

    The aim of this study is to identify the express specificity of bone morphogenetic protein 15 (Bmp15) in porcine. The pBMP15-EGFP reporter vector was constructed from the 2.2 kb fragment of porcine bmp15 promoter to trace the differentiation process of stem cells into oocyte-like cells. We used porcine ovary and Chinese Hamster Ovary cell line (CHO), mouse myoblast cell line (C2C12) and porcine amniotic fluid stem cell (pAFSC) to investigate the expression and regulation of this gene via RT-PCR, immunofluorescence, cell transfection, and microinjection methods. We also used single layer cell differentiation to detect the application potential of bmp15. The results show that bmp15 gene was specifically expressed in the porcine ovary and CHO rather than in C2C12 and pAFSC. In addition, the characteristic of tissue-specific of Bmp15 was detected on CHO instead of other cell lines by transient transfection. We also detected the expression of Bmp15 in oocyte at different development stages by immunofluorescence of fixed paraffin-embedded ovary sections. Furthermore, microinjection results show that bmp15 expressed in oocytes at 18 h of maturation in vitro, and continued up to 4-cell stage embryos. Most importantly, we found that the expression of Bmp15 started at day 12 after inducing pAFSC into oocyte-like cells by transfection; green fluorescent was visible in round cell masses. It indicated that bmp15 has the expression specificity and the pBMP15-EGFP reporter vector can be used to trace Bmp15 action in the differentiation of stem cells into germ cells.

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

    PubMed

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

    2013-07-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2006-12-01

    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.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Mishra, N.C.

    1995-01-01

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

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

    SciTech Connect

    Fields, C.A.

    1994-09-01

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

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

    SciTech Connect

    Beachy, R.N.

    1985-01-01

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

  15. [The gene therapy for a patient with ADA deficiency; report of the first gene therapy trial in Japan].

    PubMed

    Ariga, T; Kawamura, N; Sakiyama, Y

    1997-06-01

    Since the first gene therapy clinical trial for an ADA deficient patient was performed in September 1990, 10 ADA deficient patients have been enrolled in gene therapy clinical trial. We have been performing the first gene therapy trial in Japan for a 5 year boy with ADA deficiency since August 1995. Activated T cells from the patient's peripheral mononuclear cells were transduced by a retrovirus vector, LASN, which contained cDNA of human ADA gene, and re-infused to him intravenously after 7-11 days. We have already performed 10 cycles of the therapy for the patient. Here, we report the successful results of the gene therapy with laboratory and clinical evaluation. Furthermore, we overview the results of gene therapy for ADA deficient patients which were recently reported from 4 other groups.

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

    SciTech Connect

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

    1988-09-01

    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.

  17. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

    PubMed

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-07-21

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

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

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

  19. A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing.

    PubMed

    Seum, C; Spierer, A; Delattre, M; Pauli, D; Spierer, P

    2000-11-01

    We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

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

    PubMed

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

    2013-05-17

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

  1. Sequences 5' of the basement membrane laminin beta 1 chain gene (LAMB1) direct the expression of beta-galactosidase during development of the mouse testis and ovary.

    PubMed

    Li, C; Gudas, L J

    1997-12-01

    The murine LAMB1 gene encoding laminin beta 1 is expressed in the developing male and female gonads and mesonephros. To identify the cis-acting elements regulating the expression of LAMB1, murine transgenic lines were generated by fusing regions of the LAMB1 gene to the Eschericia coli lacZ gene. The p3.9LAM beta gal construct contained approximately 4 kb of 5' flanking sequence and directed beta-galactosidase expression in many different organs including the kidney, mammary gland, and the male and female genital systems, the focus of this report. In male embryos, between gestational ages E 14.5 and birth beta-galactosidase was transiently expressed in the prospermatogonia cells of the testis and in the differentiating epithelial cells in the ductus deferens, ductus epididymis, and seminal vesicles. In female embryos, beta-galactosidase was not detected in the ovary until about 1 week after birth; at this time, beta-galactosidase was expressed by oocytes of primary and secondary follicles. In contrast, transgenic mice carrying the first 0.7 kb of LAMB1 fused to the lacZ gene expressed beta-galactosidase only in the prospermatogonia cells of the testis. Thus, the cis-acting element(s) necessary for the expression of the LAMB1 gene in prospermatogonia cells are located in the first 0.7 kb of LAMB1 5' flanking sequence; element(s) required for expression of the LAMB1 gene in oocytes and epithelial cells of the mesonephric ducts, mesonephric tubules, the ductus deferens, ductus epididymis, and seminal vesicles are located with 4 kb 5' of the transcription initiation site.

  2. Molecular analysis of the PHO81 gene of Saccharomyces cerevisiae.

    PubMed Central

    Creasy, C L; Madden, S L; Bergman, L W

    1993-01-01

    The PHO81 gene product is a positive regulatory factor required for the synthesis of the phosphate repressible acid phosphatase (encoded by the PHO5 gene) in Saccharomyces cerevisiae. Genetic analysis has suggested that PHO81 may be the signal acceptor molecule; however, the biochemical function of the PHO81 gene product is not known. We have cloned the PHO81 gene and sequenced the promoter. A PHO81-LacZ fusion was shown to be a valid reporter since its expression is regulated by the level of inorganic phosphate and is controlled by the same regulatory factors that regulate PHO5 expression. To elucidate the mechanism by which PHO81 functions, we have isolated and cloned dominant mutations in the PHO81 gene which confer constitutive synthesis of acid phosphatase. We have demonstrated that overexpression of the negative regulatory factor, PHO80, but not the negative regulatory factor PHO85, partially blocks the constitutive acid phosphatase synthesis in a strain containing a dominant constitutive allele of PHO81. This suggests that PHO81 may function by interacting with PHO80 or that these molecules compete for the same target. Images PMID:8493108

  3. Integration of H-2Z1, a somatosensory cortex-expressed transgene, interferes with the expression of the Satb1 and Tbc1d5 flanking genes and affects the differentiation of a subset of cortical interneurons.

    PubMed

    Narboux-Nême, Nicolas; Goïame, Rosette; Mattéi, Marie-Geneviève; Cohen-Tannoudji, Michel; Wassef, Marion

    2012-05-23

    H-2Z1 is an enhancer trap transgenic mouse line in which the lacZ reporter delineates the somatosensory area of the cerebral cortex where it is expressed in a subset of layer IV neurons. In the search of somatosensory specific genes or regulatory sequences, we mapped the H-2Z1 transgene insertion site to chromosome 17, 100 and 460 kb away from Tbc1d5 and Satb1 flanking genes. We show here that insertion of the H-2Z1 transgene results in three distinct outcomes. First, a genetic background-sensitive expression of lacZ in several brain and body structures. While four genes in a 1 Mb region around the insertion are expressed in the barrel cortex, H-2Z1 expression resembles more that of its two direct neighbors. Moreover, H-2Z1 closely reports most of the body and brain expression sites of the Satb1 chromatin remodeling gene including tooth buds, thymic epithelium, pontine nuclei, fastigial cerebellar nuclei, and cerebral cortex. Second, the H-2Z1 transgene causes insertional mutagenesis of Tbc1d5 and Satb1, leading to a strong decrease in their expressions. Finally, insertion of H-2Z1 affects the differentiation of a subset of cortical GABAergic interneurons, a possible consequence of downregulation of Satb1 expression. Thus, the H-2Z1 "somatosensory" transgene is inserted in the regulatory landscape of two genes highly expressed in the developing somatosensory cortex and reports for a subdomain of their expression profiles. Together, our data suggest that regulation of H-2Z1 expression results from local and remote genetic interactions.

  4. Effects of transcription induction homogeneity and transcript stability on expression of two genes in a constructed operon.

    PubMed

    Smolke, C D; Khlebnikov, A; Keasling, J D

    2001-12-01

    A synthetic operon was constructed using the reporter genes gfp and lacZ and the arabinose-inducible araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 3' and 5' ends of the genes and a putative RNase E site was placed between the genes. These mRNA control elements have been shown to affect transcript processing and decay, resulting in altered protein levels. These constructs were transformed into cells harboring the native arabinose-inducible araE gene encoding the arabinose transport protein and engineered cells harboring a constitutively expressed araE. In the strains with arabinose-dependent transport the linear response in the production of both reporter proteins to inducer concentration occurred over a narrow range of arabinose concentrations. In the strains with constitutive transport the linear range of gene expression occurred over a much larger arabinose concentration range than in strains with the arabinose-inducible transport. Strains with the arabinose-inducible transport harboring different operon constructs produced the two reporter proteins at very different levels at low arabinose concentrations; as inducer concentrations increased, differences in relative expression levels decreased. In contrast, strains with constitutive transport harboring different operon constructs produced the reporter proteins at very different levels across the entire range of inducer concentrations, pointing to the importance of optimizing gene expression control at various levels to control the production of heterologous proteins.

  5. MRI Reporter Genes: Application to Imaging of Cell Survival, Proliferation, Migration, and Differentiation

    PubMed Central

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

    2013-01-01

    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 through the use of 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 never the less be detected, though indirectly, through the use of reporter genes 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 or bioluminescent proteins, or the reaction product of expressed enzymes and exogenous substrates, are examined using in vitro histological methods, or in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raise 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 is for monitoring gene expression patterns in gene therapy and for in vivo imaging of the survival, proliferation, migration, and differentiation of pluripotent or multipotent cells used in cell based regenerative therapies for cancer, myocardial infarction, and neural degeneration. In this review, we characterize the variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, cell migration, and cell differentiation. In particular, we discuss which methods are best suited for translation to clinical use in regenerative therapies. PMID:23225197

  6. An eYFP reporter gene for the yeast two-hybrid system.

    PubMed

    Damon, Coralie; Boxus, Mathieu; Twizere, Jean-Claude; Portetelle, Daniel; Vandenbol, Micheline

    2013-02-01

    The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening.

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

    PubMed Central

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

    2016-01-01

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

  8. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

    PubMed

    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    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 μM during growth and 60 μM 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    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, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

    2011-01-01

    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. PMID:21540837

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

    PubMed

    Takahashi, Junko; Iwahashi, Hitoshi

    2004-01-01

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

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

    PubMed

    Gettemy, J M; Li, D; Alic, M; Gold, M H

    1997-06-01

    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 of two mnp genes, mnp1 and mnp2, encoding individual MnP isozymes. Internal deletions of 234 and 359 bp were made within the coding regions of the mnp1 and mnp2 genes, respectively. The truncated mnp genes were subcloned into the shuttle vector pOGI18, which includes the Schizophylum commune ade5 gene as a selectable marker, and transformed into a P. chrysosporium Ade1 auxotrophic mutant. Northern-blot analysis of purified Ade+ transformants demonstrated that both of the truncated mnp genes were regulated in a manner similar to the endogenous mnp genes with respect to nitrogen limitation and induction by Mn, HS, and H2O2.

  14. Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

    PubMed

    Uliczka, Frank; Pisano, Fabio; Kochut, Annika; Opitz, Wiebke; Herbst, Katharina; Stolz, Tatjana; Dersch, Petra

    2011-01-01

    A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

  15. Structural explanation for allolactose (lac operon inducer) synthesis by lacZ β-galactosidase and the evolutionary relationship between allolactose synthesis and the lac repressor.

    PubMed

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

    2013-05-03

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

  16. Bioluminescent reporters for catabolic gene expression and pollutant bioavailability

    SciTech Connect

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

    1991-01-01

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

  17. Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes simplex virus thymidine kinase/ganciclovir system in a model of colon cancer.

    PubMed

    Kagaya, T; Nakamoto, Y; Sakai, Y; Tsuchiyama, T; Yagita, H; Mukaida, N; Kaneko, S

    2006-04-01

    Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.

  18. Glycerophosphorylcholine regulates Haemophilus influenzae glpQ gene expression.

    PubMed

    Alrousan, Enas; Fan, Xin

    2015-05-01

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

  19. A versatile element for gene addition in bacterial chromosomes

    PubMed Central

    Sibley, Marion H.; Raleigh, Elisabeth A.

    2012-01-01

    The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies. Using lacZ as a reporter, we first show that expression from the rhamnose promoter is tightly regulatable, exhibiting very low leakage of background expression compared with background, and moderate rhamnose-induced expression compared with IPTG-induced expression from lacp. Second, the expression of a DNA methyltransferase was used to show that rhamnose regulation yielded on-off expression of this enzyme, such that a resident high-copy plasmid was either fully sensitive or fully resistant to isoschizomer restriction enzyme cleavage. In both cases, growth medium manipulation allows intermediate levels of expression. The vehicle can also be adapted as an ORF-cloning vector. PMID:22123741

  20. Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

    PubMed

    Armstrong, G A; Cook, D N; Ma, D; Alberti, M; Burke, D H; Hearst, J E

    1993-05-01

    The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

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

    SciTech Connect

    Wolstenholme, D.R.

    1994-06-01

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

  2. Feline immunodeficiency virus and retrovirus-mediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular smooth muscle cells.

    PubMed

    Kankkonen, Hanna M; Turunen, Mikko P; Hiltunen, Mikko O; Lehtolainen, Pauliina; Koponen, Jonna; Leppänen, Pia; Turunen, Anna-Mari; Ylä-Herttuala, Seppo

    2004-03-01

    We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.

  3. Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.

    PubMed

    de la Fuente, A; Palacios, P; Vicente, M

    2001-01-01

    Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flärdh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

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

    SciTech Connect

    Haber, J. E.

    2002-11-30

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

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

    PubMed Central

    2013-01-01

    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. PMID:23937994

  6. The use of whole-mount in situ hybridization to illustrate gene expression regulation.

    PubMed

    Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

    2014-01-01

    In situ hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the experiments, students analyzed the repressive effect of Snail over rhomboid expression using reporter lines containing different constructs of the rhomboid neuroectodermal enhancer fused to the lacZ gene. In the second experiment, the epistatic relationship between the cabut and decapentaplegic genes was analyzed. These simple experiments allowed students to (1) understand the role of transcription factors and cis-regulatory elements over gene expression regulation and (2) practice a widespread laboratory technique, in situ hybridization with nonradioactive labeled probes, to detect gene expression patterns. These experiments required 12 hr and were organized into four daily sessions that included the discussion of the results with students. Examples of the results obtained and their relevance are shown and discussed herein. The methods described in these laboratory exercises can be easily adapted to model organisms other than Drosophila.

  7. Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice.

    PubMed

    Aird, W C; Jahroudi, N; Weiler-Guettler, H; Rayburn, H B; Rosenberg, R D

    1995-05-09

    The present study was undertaken to define the 5' and 3' regulatory sequences of human von Willebrand factor gene that confer tissue-specific expression in vivo. Transgenic mice were generated bearing a chimeric construct that included 487 bp of 5' flanking sequence and the first exon fused in-frame to the Escherichia coli lacZ gene. In situ histochemical analyses in independent lines demonstrated that the von Willebrand factor promoter targeted expression of LacZ to a subpopulation of endothelial cells in the yolk sac and adult brain. LacZ activity was absent in the vascular beds of the spleen, lung, liver, kidney, testes, heart, and aorta, as well as in megakaryocytes. In contrast, in mice containing the lacZ gene targeted to the thrombomodulin locus, the 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside reaction product was detected throughout the vascular tree. These data highlight the existence of regional differences in endothelial cell gene regulation and suggest that the 733-bp von Willebrand factor promoter may be useful as a molecular marker to investigate endothelial cell diversity.

  8. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    PubMed

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  9. Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli

    PubMed Central

    Nakashima, Nobutaka; Tamura, Tomohiro

    2009-01-01

    In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications. PMID:19515932

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

    PubMed

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

    2013-05-01

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

  11. Non-invasive imaging using reporter genes altering cellular water permeability

    NASA Astrophysics Data System (ADS)

    Mukherjee, Arnab; Wu, Di; Davis, Hunter C.; Shapiro, Mikhail G.

    2016-12-01

    Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

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

    SciTech Connect

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

    1998-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. The feasibility of targeted selective gene therapy of the hair follicle.

    PubMed

    Li, L; Hoffman, R M

    1995-07-01

    Loss of hair and hair colour is associated with ageing, and when it involves the scalp hair, it can be distressing to both sexes. Hair loss resulting from cancer chemotherapy is particularly distressing. However, safe, effective therapies directed to hair have only just started to be developed. The hair follicle is a complex skin appendage composed of epidermal and dermal tissue, with specialized keratinocytes, the hair matrix cells, forming the hair shaft. Specific therapy of the hair follicle depends on selective targeting of specific cells of the hair follicle. We have developed the histoculture of intact hair-growing skin on sponge-gel matrices. We have recently found in histocultured skin that liposomes can selectively target hair follicles to deliver both small and large molecules. That liposomes can target the hair follicle for delivery has been confirmed independently. Two decades ago we introduced the technique of entrapping DNA in liposomes for use in gene therapy. In this report we describe the selective targeting of the lacZ reporter gene to the hair follicles in mice after topical application of the gene entrapped in liposomes. These results demonstrate that highly selective, safe gene therapy for the hair process is feasible.

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

    PubMed

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

    1995-05-25

    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.

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

    PubMed Central

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

    2010-01-01

    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

  17. Simultaneous deletion of floxed genes mediated by CaMKIIα-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goα.

    PubMed

    Choi, Chan-Il; Yoon, Sang-Phil; Choi, Jung-Mi; Kim, Sung-Soo; Lee, Young-Don; Birnbaumer, Lutz; Suh-Kim, Haeyoung

    2014-05-02

    The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIα-Cre(+/Cre) mice generated by crossing CaMKIIα-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIα-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIα-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Δ)) without inheriting the Cre transgene. The Gnao(Δ/Δ) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.

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

    PubMed

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

    2015-07-01

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

  19. A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

    PubMed

    Guenther, Catherine A; Wang, Zhen; Li, Emma; Tran, Misha C; Logan, Catriona Y; Nusse, Roel; Pantalena-Filho, Luiz; Yang, George P; Kingsley, David M

    2015-08-01

    Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

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

    SciTech Connect

    Mishra, N.C.

    1994-01-01

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

  1. Gene therapy for trigeminal pain in mice

    PubMed Central

    Tzabazis, Alexander Z.; Klukinov, Michael; Feliciano, David P.; Wilson, Steven P.; Yeomans, David C.

    2014-01-01

    The aim of this study was to test the efficacy of a single direct injection of viral vector encoding for encephalin to induce a widespread expression of the transgene and potential analgesic effect in trigeminal behavioral pain models in mice. After direct injection of HSV-1 based vectors encoding for human preproenkephalin (SHPE) or the lacZ reporter gene (SHZ.1, control virus) into the trigeminal ganglia in mice, we performed an orofacial formalin test and assessed the cumulative nociceptive behavior at different time points after injection of the viral vectors. We observed an analgesic effect on nociceptive behavior that lasted up to 8 weeks after a single injection of SHPE into the trigeminal ganglia. Control virus injected animals showed nociceptive behavior similar to naïve mice. The analgesic effect of SHPE injection was reversed/attenuated by subcutaneous naloxone injections, a μ-opioid receptor antagonist. SHPE injected mice also showed normalization in withdrawal latencies upon thermal noxious stimulation of inflamed ears after subdermal complete Freund’s adjuvans injection indicating widespread expression of the transgene. Quantitative immunohistochemistry of trigeminal ganglia showed expression of human preproenkephalin after SHPE injection. Direct injection of viral vectors proved to be useful for exploring the distinct pathophysiology of the trigeminal system and could also be an interesting addition to the pain therapists’ armamentarium. PMID:24572785

  2. Probing for gene specificity in epithelial development.

    PubMed

    Schüpbach, T; Wieschaus, E

    1998-01-01

    We surveyed a total of 228 random insertions of a P[GawB] element to determine the fraction of regulatory regions in the Drosophila genome that activate gene expression specifically in follicle cells versus producing more complex patterns of expression. We monitored the GAL4 expression encoded by this construct in the ovarian follicle cells by crossing the lines to a strain containing a lacZ reporter construct. Sixty four per cent of the insertions showed ovarian expression. To assess the specificity of this expression, 124 of the 228 lines were crossed to strains containing either an activated form of Armadillo, the Drosophila homolog of beta-catenin, or an activated form of Torpedo/Egfr, the Drosophila homolog of the Epidermal Growth Factor receptor, under the control of GAL4 target sites. The lethality and imaginal disc phenotypes observed in these crosses suggest that most random insertions cause GAL4 expression in a variety of tissues. Very few insertions appear to drive expression only in follicle cells. Although the activated form of Armadillo produced higher frequencies of lethality and disk phenotypes, expression in the follicle cell epithelium at later stages of oogenesis did not lead to a visible phenotype. This contrasts with the dorsalized phenotypes observed in the combination of the same GAL4 lines with the activated Torpedo construct.

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

    PubMed

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

    2015-11-03

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

  4. Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.

    PubMed

    Winbanks, Catherine E; Beyer, Claudia; Qian, Hongwei; Gregorevic, Paul

    2012-01-01

    Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

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

    PubMed Central

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

    2016-01-01

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

  6. Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli.

    PubMed

    Herring, Christopher D; Glasner, Jeremy D; Blattner, Frederick R

    2003-06-05

    We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.

  7. Long Term Non-Invasive Imaging of Embryonic Stem Cells Using Reporter Genes

    PubMed Central

    Sun, Ning; Lee, Andrew; Wu, Joseph C.

    2013-01-01

    Development of non-invasive and accurate methods to track cell fate following delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. Here we describe a protocol for the in vivo monitoring of stem cell survival, proliferation, and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes using lentiviral transduction. We used fluorescence activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography imaging to track them in vivo, and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. PMID:19617890

  8. The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter.

    PubMed

    Tinikul, Ruchanok; Thotsaporn, Kittisak; Thaveekarn, Wichit; Jitrapakdee, Sarawut; Chaiyen, Pimchai

    2012-12-31

    Bacterial luciferase from Vibrio campbellii is a thermostable enzyme with an in vitro thermal inactivation half-life of ~1020 min at 37°C. The enzyme also binds tightly to reduced FMN. In this study, a V. campbellii fusion luciferase construct in which the α and β subunits are linked with a decapeptide was made and characterized. In general, the overall enzymatic properties of the two enzymes are similar. Expression of the enzymes in Escherichia coli demonstrated that the V. campbellii fusion luciferase emits less light than the native luciferase, but still emits a much greater amount of light than native luciferase from Vibrio harveyi and Photobacterium leiognathi TH1. The intensity of light emitted by the V. campbellii fusion luciferase was more than 80-fold greater than that from the V. harveyi native luciferase when expressed at 37°C. Biochemical characterization has shown that the V. campbellii fusion luciferase also retains a high binding affinity for reduced flavin mononucleotide and high thermostability. The levels of bioluminescence emitted by the V. campbellii fusion luciferase expressed in HEK293T cells reached ~1×10(6) Relative Light Units/mg total protein. These findings suggest that the V. campbellii fusion luciferase is a promising candidate for further development as a luciferase-based reporter for eukaryotic systems.

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

    SciTech Connect

    Konisky, J.

    1997-10-07

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

  10. Gene transcription and electromagnetic fields. Final progress report

    SciTech Connect

    Henderson, A.S.

    1992-12-31

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

  11. Gene Therapy of Breast Cancer: Studies of Selective Promoter/Enhancer-Modified Vectors to Deliver Suicide Genes

    DTIC Science & Technology

    1998-09-01

    antigen - expressing tu- 36. Wickham. TJ.. P. Mathias. D.A. Cheresh. and G.R. Nemerow. 1993. In- mor cells in bone marrow aspirates by polymerase chain ...transduction of Escherichia Coi lacZ. Proc." 1992. Detection of tumor cells in bone marrow of patients with primary breast Nail. Acad. Sci. USA. 85:2603-2607...tissue specific /selective promoter or enhancer to direct the expression of a therapeutic gene in the desired target

  12. Adaptation of a Luciferase Gene Reporter and lac Expression System to Borrelia burgdorferi▿ †

    PubMed Central

    Blevins, Jon S.; Revel, Andrew T.; Smith, Alexandra H.; Bachlani, Gulnaz N.; Norgard, Michael V.

    2007-01-01

    The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi. PMID:17220265

  13. Functional analysis of the promoter region of amphioxus β-actin gene: a useful tool for driving gene expression in vivo.

    PubMed

    Feng, Jun; Li, Guang; Liu, Xin; Wang, Jing; Wang, Yi-Quan

    2014-10-01

    Amphioxus is a promising new animal model for developmental biology. To develop molecular tools for this model, we characterized the promoter region of a cytoplasmic β-actin gene (Bb-actin-6-2) from the Chinese amphioxus Branchiostoma belcheri. In situ hybridization and real time-quantitative PCR analyses showed that this gene is expressed in many tissues throughout embryonic development. Cloning of cDNA revealed two isoforms with distinct transcription start sites. Isoform #1 exhibits a similar exon/intron and regulatory element organization to that of vertebrate β-actin, whereas isoform #2 lacks the first exon of isoform #1 and recruits its first intron as a promoter. The activities of upstream promoter regions in the two isoforms were examined using the lacZ reporter system in amphioxus embryos. The proximal promoter of isoform #1 drove reporter gene expression broadly in 58.6 % of injected embryos. That of isoform #2 exhibited much higher activity (91.5 %) than that of isoform #1 or the human EF-1-α gene (38.2 %). We determined the minimal promoter regions of the two isoforms via functional analysis. These two regions, alone or inserted a random DNA fragment upstream, had no detectable activity, but when an upstream enhancer was inserted, the promoters directed reporter gene expression in 61.0 and 93.8 %, respectively, of injected embryos in a tissue-specific manner. Our study not only provides insight into the regulatory mechanism underlying amphioxus Bb-actin-6-2 gene expression, but also identifies two sets of efficient proximal and minimal promoters. These promoters could be used to construct gene expression vectors for transgenic studies using amphioxus as a model.

  14. A systemic lupus erythematosus gene expression array in disease diagnosis and classification: a preliminary report.

    PubMed

    Juang, Y-T; Peoples, C; Kafri, R; Kyttaris, V C; Sunahori, K; Kis-Toth, K; Fitzgerald, L; Ergin, S; Finnell, M; Tsokos, G C

    2011-03-01

    Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease diagnosed on the presence of a constellation of clinical and laboratory findings. At the pathogenetic level, multiple factors using diverse biochemical and molecular pathways have been recognized. Succinct recognition and classification of clinical disease subsets, as well as the availability of disease biomarkers, remains largely unsolved. Based on information produced by the present authors' and other laboratories, a lupus gene expression array consisting of 30 genes, previously claimed to contribute to aberrant function of T cells, was developed. An additional eight genes were included as controls. Peripheral blood was obtained from 10 patients (19 samples) with SLE and six patients with rheumatoid arthritis (RA) as well as 19 healthy controls. T cell mRNA was subjected to reverse transcription and PCR, and the gene expression levels were measured. Conventional statistical analysis was performed along with principal component analysis (PCA) to capture the contribution of all genes to disease diagnosis and clinical parameters. The lupus gene expression array faithfully informed on the expression levels of genes. The recorded changes in expression reflect those reported in the literature by using a relatively small (5 ml) amount of peripheral blood. PCA of gene expression levels placed SLE samples apart from normal and RA samples regardless of disease activity. Individual principal components tended to define specific disease manifestations such as arthritis and proteinuria. Thus, a lupus gene expression array based on genes previously claimed to contribute to immune pathogenesis of SLE may define the disease, and principal components of the expression of 30 genes may define patients with specific disease manifestations.

  15. Integer programming-based approach to allocation of reporter genes for cell array analysis.

    PubMed

    Hayashida, Morihiro; Sun, Fuyan; Aburatani, Sachiyo; Horimoto, Katsuhisa; Akutsu, Tatsuya

    2008-01-01

    In this paper, we consider the problem of selecting the most effective set of reporter genes for analysis of biological networks using cell microarrays. We propose two graph theoretic formulations of the reporter gene allocation problem, and show that both problems are hard to approximate. We propose integer programming-based methods for solving practical instances of these problems optimally. We apply them to apoptosis pathway maps, and discuss the biological significance of the result. We also apply them to artificial networks, the result of which shows that optimal solutions can be obtained within several seconds for networks with 10,000 nodes.

  16. Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

    PubMed Central

    2014-01-01

    Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred

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

    NASA Astrophysics Data System (ADS)

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

    1993-05-01

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

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

    PubMed Central

    Zimmerman, Emily; Maron, Jill L.

    2016-01-01

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

  19. Optical imaging of Renilla luciferase reporter gene expression in living mice

    PubMed Central

    Bhaumik, S.; Gambhir, S. S.

    2002-01-01

    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 for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes. PMID:11752410

  20. Mesoscopic tomography imaging of reporter genes in thick printed tissue constructs

    NASA Astrophysics Data System (ADS)

    Ozturk, Mehmet S.; Lee, Vivian K.; Zhao, Lingling; Dai, Guohoa; Intes, Xavier

    2013-06-01

    We report an application of Mesoscopic Fluorescence Molecular Tomography to 3D tissue engineering construct. Engineered thick tissue was hosting two 3D printed vasculatures. The channels were formed by live cells, expressing GFP and mCherry reporter genes, embedded in 3mm turbid media. Tissue and cells kept in a 3mm thick perfusion chamber during the entire imaging process which took less than 5 minutes.

  1. The Myxococcus xanthus two-component system CorSR regulates expression of a gene cluster involved in maintaining copper tolerance during growth and development.

    PubMed

    Sánchez-Sutil, María Celestina; Pérez, Juana; Gómez-Santos, Nuria; Shimkets, Lawrence J; Moraleda-Muñoz, Aurelio; Muñoz-Dorado, José

    2013-01-01

    Myxococcus xanthus is a soil-dwelling member of the δ-Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains.

  2. The Myxococcus xanthus Two-Component System CorSR Regulates Expression of a Gene Cluster Involved in Maintaining Copper Tolerance during Growth and Development

    PubMed Central

    Sánchez-Sutil, María Celestina; Pérez, Juana; Gómez-Santos, Nuria; Shimkets, Lawrence J.; Moraleda-Muñoz, Aurelio; Muñoz-Dorado, José

    2013-01-01

    Myxococcus xanthus is a soil-dwelling member of the δ–Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains. PMID:23874560

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

    SciTech Connect

    Not Available

    1991-12-31

    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)

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

    SciTech Connect

    Schultz, J.A.; Gremke, L.; Storti, R.V. ); Tansey, T. )

    1991-04-01

    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.

  5. Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts

    PubMed Central

    Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future. PMID:25271765

  6. Construction and characterization of Escherichia coli polA-lacZ gene fusions.

    PubMed Central

    Ward, D F; Murray, N E

    1980-01-01

    The promoter of the polA gene of Escherichia coli K-12 was fused to the lacZ gene by selecting deletions within a lambda lacZ polA transducing phage. Four fusions, deleting varying amounts of the polA gene, were characterized. The polA promoter was found to be approximately 3% as active as the fully induced lac promoter. This figure is compatible with the normal intracellular level of deoxyribonucleic acid polymerase I. No evidence was found for outogenous regulation of transcription from the polA promoter. Expression from this promoter was influenced by neither recA nor mitomycin C, but uvrD and uvrE mutations reduced expression slightly. Images PMID:6445899

  7. Xenoestrogenic gene expression: structural features of active polycyclic aromatic hydrocarbons.

    PubMed

    Schultz, T Wayne; Sinks, Glendon D

    2002-04-01

    Estrogenicity was assessed using the Saccharomyces cerevisiae-based Lac-Z reporter assay and was reported as the logarithm of the inverse of the 50% molar beta-galactosidase activity (log[EC50(-1)]). In an effort to quantify the relationship between molecular structure of polycyclic aromatic hydrocarbons (PAHs) and estrogenic gene expression, a series of PAHs were evaluated. With noted exceptions, the results of these studies indicate that the initial two-dimensional structural warning for estrogenicity, the superpositioning of a hydroxylated aromatic system on the phenolic A-ring of 17-beta-estradiol, can be extended to the PAHs. This two-dimensional-alignment criterion correctly identified estrogenicity of 22 of the 29 PAHs evaluated. Moreover, the estrogenic potency of these compounds was directly related to the size of the hydrophobic backbone. The seven compounds classified incorrectly by this structural feature were either dihydroxylated naphthalenes or aromatic nitrogen-heterocyclic compounds; all such compounds were false positives. Results with dihydroxylated naphthalenes reveal derivatives that were nonestrogenic when superimposed on the phenolic A-ring of 17-beta-estradiol had the second hydroxyl group in the position of the C-ring or were catechol-like in structure. Structural alerts for nitrogen-heterocyclic compounds must take into account the position of the hydroxyl group and the in-ring nitrogen atom; compounds with the hydroxyl group and nitrogen atom involved with the same ring were observed to be nonactive.

  8. Comparison and calibration of different reporters for quantitative analysis of gene expression.

    PubMed

    Garcia, Hernan G; Lee, Heun Jin; Boedicker, James Q; Phillips, Rob

    2011-08-03

    Absolute levels of gene expression in bacteria are observed to vary over as much as six orders of magnitude. Thermodynamic models have been proposed as a tool to describe the expression levels of a given transcriptional circuit. In this context, it is essential to understand both the limitations and linear range of the different methods for measuring gene expression and to determine to what extent measurements from different reporters can be directly compared with one aim being the stringent testing of theoretical descriptions of gene expression. In this article, we compare two protein reporters by measuring both the absolute level of expression and fold-change in expression using the fluorescent protein EYFP and the enzymatic reporter β-galactosidase. We determine their dynamic and linear range and show that they are interchangeable for measuring mean levels of expression over four orders of magnitude. By calibrating these reporters such that they can be interpreted in terms of absolute molecular counts, we establish limits for their applicability: autofluorescence on the lower end of expression for EYFP (at ∼10 molecules per cell) and interference with cellular growth on the high end for β-galactosidase (at ∼20,000 molecules per cell). These qualities make the reporters complementary and necessary when trying to experimentally verify the predictions from the theoretical models.

  9. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    SciTech Connect

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  10. Tandem transcription termination sites in the dnaN gene of Escherichia coli.

    PubMed

    Armengod, M E; García-Sogo, M; Pérez-Roger, I; Macián, F; Navarro-Aviñó, J P

    1991-10-15

    The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

  11. Non-invasive imaging using reporter genes altering cellular water permeability

    PubMed Central

    Mukherjee, Arnab; Wu, Di; Davis, Hunter C.; Shapiro, Mikhail G.

    2016-01-01

    Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging. PMID:28008959

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

    SciTech Connect

    Wessler, S.

    1990-12-31

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

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

    PubMed

    Sergi, Consolato; Gekas, Jean; Kamnasaran, Deepak

    2013-07-01

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

  14. Inducible Gene Manipulations in Brain Serotonergic Neurons of Transgenic Rats

    PubMed Central

    Tews, Björn; Bartsch, Dusan

    2011-01-01

    The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system. PMID:22140568

  15. Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system.

    PubMed

    Ellisor, Debra; Koveal, Dorothy; Hagan, Nellwyn; Brown, Ashly; Zervas, Mark

    2009-10-01

    A long-standing problem in development is understanding how progenitor cells transiently expressing genes contribute to complex anatomical and functional structures. In the developing nervous system an additional level of complexity arises when considering how cells of distinct lineages relate to newly established neural circuits. To address these problems, we used both cumulative marking with Cre/loxP and Genetic Inducible Fate Mapping (GIFM), which permanently and heritably marks small populations of progenitors and their descendants with fine temporal control using CreER/loxP. A key component used in both approaches is a conditional phenotyping allele that has the potential to be expressed in all cell types, but is quiescent because of a loxP flanked Stop sequence, which precedes a reporter allele. Upon recombination, the resulting phenotyping allele is 'turned on' and then constitutively expressed. Thus, the reporter functions as a high fidelity genetic lineage tracer in vivo. Currently there is an array of reporter alleles that can be used in marking strategies, but their recombination efficiency and applicability to a wide array of tissues has not been thoroughly described. To assess the recombination/marking potential of the reporters, we utilized CreER(T) under the control of a Wnt1 transgene (Wnt1-CreER(T)) as well as a cumulative, non-inducible En1(Cre) knock-in line in combination with three different reporters: R26R (LacZ reporter), Z/EG (EGFP reporter), and Tau-Lox-STOP-Lox-mGFP-IRES-NLS-LacZ (membrane-targeted GFP/nuclear LacZ reporter). We marked the Wnt1 lineage using each of the three reporters at embryonic day (E) 8.5 followed by analysis at E10.0, E12.5, and in the adult. We also compared cumulative marking of cells with a history of En1 expression at the same stages. We evaluated the reporters by whole-mount and section analysis and ascertained the strengths and weaknesses of each of the reporters. Comparative analysis with the reporters

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

    SciTech Connect

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

    1995-02-01

    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.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  18. Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.

    PubMed

    Smolke, Christina D; Keasling, Jay D

    2002-05-20

    To study the effect of mRNA stability and DNA copy number on protein production from a dual-gene operon, a synthetic operon containing the reporter genes gfp and lacZ under the control of the araBAD promoter was placed in pMB1-based (approximately 100 copies/cell) and F plasmid-based (approximately 1 copy/cell) vectors. DNA cassettes encoding secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the two genes. Although the copy number of the pMB1-based vectors was approximately 100-fold greater than the copy number of the F plasmid-based vectors, transcript and protein levels from the pMB1-based vector were not 100-fold greater than from the F plasmid-based vectors. In identical plasmid backbones, different combinations of mRNA control elements were used to alter steady-state levels of transcripts. Control elements that amplified the stability of one coding region relative to another amplified the ratio of protein produced from those transcripts. The effects of mRNA stability control elements were greater at low inducer concentrations, where mRNA levels limit protein production, than at high inducer concentrations. Although we can alter mRNA and protein levels through copy number, induction level, and mRNA stability control elements, some aspect of gene expression remains dependent on inherent characteristics of the coding region.

  19. Contribution of individual promoters in the ddlB-ftsZ region to the transcription of the essential cell-division gene ftsZ in Escherichia coli.

    PubMed

    Flärdh, K; Garrido, T; Vicente, M

    1997-06-01

    The essential cell-division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQ, and ftsA genes. The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions. The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ. The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription. An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region. A large proportion of transcription (approximately 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene. Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA. However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.

  20. The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis.

    PubMed

    Tarrío, N; Díaz Prado, S; Cerdán, M E; González Siso, M I

    2005-01-01

    Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

  1. Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma.

    PubMed

    Keu, Khun Visith; Witney, Timothy H; Yaghoubi, Shahriar; Rosenberg, Jarrett; Kurien, Anita; Magnusson, Rachel; Williams, John; Habte, Frezghi; Wagner, Jamie R; Forman, Stephen; Brown, Christine; Allen-Auerbach, Martin; Czernin, Johannes; Tang, Winson; Jensen, Michael C; Badie, Behnam; Gambhir, Sanjiv S

    2017-01-18

    High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8(+) cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [(18)F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer.

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

    SciTech Connect

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

    2002-03-28

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

  3. Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases

    PubMed Central

    Farwell Hagman, Kelly D.; Shinde, Deepali N.; Mroske, Cameron; Smith, Erica; Radtke, Kelly; Shahmirzadi, Layla; El-Khechen, Dima; Powis, Zöe; Chao, Elizabeth C.; Alcaraz, Wendy A.; Helbig, Katherine L.; Sajan, Samin A.; Rossi, Mari; Lu, Hsiao-Mei; Huether, Robert; Li, Shuwei; Wu, Sitao; Nuñes, Mark E.; Tang, Sha

    2017-01-01

    Purpose: Diagnostic exome sequencing (DES) is now a commonly ordered test for individuals with undiagnosed genetic disorders. In addition to providing a diagnosis for characterized diseases, exome sequencing has the capacity to uncover novel candidate genes for disease. Methods: Family-based DES included analysis of both characterized and novel genetic etiologies. To evaluate candidate genes for disease in the clinical setting, we developed a systematic, rule-based classification schema. Results: Testing identified a candidate gene among 7.7% (72/934) of patients referred for DES; 37 (4.0%) and 35 (3.7%) of the genes received evidence scores of “candidate” and “suspected candidate,” respectively. A total of 71 independent candidate genes were reported among the 72 patients, and 38% (27/71) were subsequently corroborated in the peer-reviewed literature. This rate of corroboration increased to 51.9% (27/52) among patients whose gene was reported at least 12 months previously. Conclusions: Herein, we provide transparent, comprehensive, and standardized scoring criteria for the clinical reporting of candidate genes. These results demonstrate that DES is an integral tool for genetic diagnosis, especially for elucidating the molecular basis for both characterized and novel candidate genetic etiologies. Gene discoveries also advance the understanding of normal human biology and more common diseases. Genet Med 19 2, 224–235. PMID:27513193

  4. A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome.

    PubMed

    Koma, Daisuke; Yamanaka, Hayato; Moriyoshi, Kunihiko; Ohmoto, Takashi; Sakai, Kiyofumi

    2012-01-01

    We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF (fbr) and tyrA (fbr) or aroF (fbr) and pheA (fbr)) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.

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

    SciTech Connect

    Estelle, Mark

    2000-12-18

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

  6. Targeted viral delivery of Cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain.

    PubMed

    Sinnayah, Puspha; Lindley, Timothy E; Staber, Patrick D; Davidson, Beverly L; Cassell, Martin D; Davisson, Robin L

    2004-06-17

    The Cre/loxP system has shown promise for investigating genes involved in nervous system function and pathology, although its application for studying central neural regulation of cardiovascular function and disease has not been explored. Here, we report for the first time that recombination of loxP-flanked genes can be achieved in discrete cardiovascular regulatory nuclei of adult mouse brain using targeted delivery of adenovirus (Ad) or feline immunodeficiency virus (FIV) bearing Cre recombinase (Ad-Cre, FIV-Cre). Single stereotaxic microinjections of Ad-Cre or FIV-Cre into specific nuclei along the subfornical organ-hypothalamic-hypophysial and brain stem-parabrachial axes resulted in robust and highly localized gene deletion as early as 7 days and for as long as 3 wk in a reporter mouse model in which Cre recombinase activates beta-galactosidase expression. An even greater selectivity in Cre-mediated gene deletion could be achieved in unique subpopulations of cells, such as vasopressin-synthesizing magnocellular neurons, by delivering Ad-Cre via retrograde transport. Moreover, Ad-Cre and FIV-Cre induced gene recombination in differential cell populations within these cardiovascular nuclei. FIV-Cre infection resulted in LacZ activation selectively in neurons, whereas both neuronal and glial cell types underwent gene recombination upon infection with Ad-Cre. These results establish the feasibility of using a combination of viral and Cre/loxP technologies to target specific cardiovascular nuclei in the brain for conditional gene modification and suggest the potential of this approach for determining the functional role of genes within these sites.

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

    PubMed

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

    2016-01-01

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

  8. Transcriptional regulation of the Drosophila glial gene repo.

    PubMed

    Lee, Bruce P; Jones, Bradley W

    2005-06-01

    reversed polarity (repo) is a putative target gene of glial cells missing (gcm), the primary regulator of glial cell fate in Drosophila. Transient expression of Gcm is followed by maintained expression of repo. Multiple Gcm binding sites are found in repo upstream DNA. However, while repo is expressed in Gcm positive glia, it is not expressed in Gcm positive hemocytes. These observations suggest factors in addition to Gcm are required for repo expression. Here we have undertaken an analysis of the cis-regulatory DNA elements of repo using lacZ reporter activity in transgenic embryos. We have found that a 4.2 kb DNA region upstream of the repo start site drives the wild-type repo expression pattern. We show that expression is dependent on multiple Gcm binding sites. By ectopically expressing Repo, we show that Repo can regulate its own enhancer. Finally, by systematically analyzing fragments of repo upstream DNA, we show that expression is dependent on multiple elements that are responsible for activity in subsets of glia, as well as repressing inappropriate expression in the epidermis. Our results suggest that Gcm acts synergistically with other factors to control repo transcription in glial cells.

  9. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    PubMed

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  10. Development of a facile method for high throughput screening with reporter gene assays.

    PubMed

    Goetz, A S; Andrews, J L; Littleton, T R; Ignar, D M

    2000-10-01

    This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

  11. First report of a de novo germline mutation in the MLH1 gene.

    PubMed

    Stulp, Rein P; Vos, Yvonne J; Mol, Bart; Karrenbeld, Arend; de Raad, Monique; van der Mijle, Huub J C; Sijmons, Rolf H

    2006-02-07

    Hereditary non-polyposis colorectal carcinoma (HNPCC) is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven, or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation (in MSH2) has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient' parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.

  12. Identification of circadian-clock-regulated enhancers and genes of Drosophila melanogaster by transposon mobilization and luciferase reporting of cyclical gene expression.

    PubMed Central

    Stempfl, Thomas; Vogel, Marion; Szabo, Gisela; Wülbeck, Corinna; Liu, Jian; Hall, Jeffrey C; Stanewsky, Ralf

    2002-01-01

    A new way was developed to isolate rhythmically expressed genes in Drosophila by modifying the classic enhancer-trap method. We constructed a P element containing sequences that encode firefly luciferase as a reporter for oscillating gene expression in live flies. After generation of 1176 autosomal insertion lines, bioluminescence screening revealed rhythmic reporter-gene activity in 6% of these strains. Rhythmically fluctuating reporter levels were shown to be altered by clock mutations in genes that specify various circadian transcription factors or repressors. Intriguingly, rhythmic luminescence in certain lines was affected by only a subset of the pacemaker mutations. By isolating genes near 13 of the transposon insertions and determining their temporal mRNA expression pattern, we found that four of the loci adjacent to the trapped enhancers are rhythmically expressed. Therefore, this approach is suitable for identifying genetic loci regulated by the circadian clock. One transposon insert caused a mutation in the rhythmically expressed gene numb. This novel numb allele, as well as previously described ones, was shown to affect the fly's rhythm of locomotor activity. In addition to its known role in cell fate determination, this gene and the phosphotyrosine-binding protein it encodes are likely to function in the circadian system. PMID:11861563

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

    PubMed Central

    Masser, Anna E.; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan

    2016-01-01

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

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

    PubMed

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

    2016-05-01

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

  15. Transcriptional activation of virulence genes of Rhizobium etli.

    PubMed

    Wang, Luyao; Lacroix, Benoît; Guo, Jianhua; Citovsky, Vitaly

    2017-01-09

    Recently, Rhizobium etli has emerged, in addition to Agrobacterium spp., as a prokaryotic species that encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we showed that the signal phenolic molecule acetosyringone (AS) induced R. etli vir gene expression both in R. etli and in A. tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter T-DNA. Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium, but not from non-host plants. Early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside of the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a versus pTiC58 of A. tumefaciens IMPORTANCE: The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into plant cell genome. In this study, we explore the transcriptional regulation and induction of virulence genes in R. etli and show similarities and differences

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

    SciTech Connect

    VanEtten, H.

    1997-06-01

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

  17. Identification of a Cellobiose Utilization Gene Cluster with Cryptic β-Galactosidase Activity in Vibrio fischeri▿

    PubMed Central

    Adin, Dawn M.; Visick, Karen L.; Stabb, Eric V.

    2008-01-01

    Cellobiose utilization is a variable trait that is often used to differentiate members of the family Vibrionaceae. We investigated how Vibrio fischeri ES114 utilizes cellobiose and found a cluster of genes required for growth on this β-1,4-linked glucose disaccharide. This cluster includes genes annotated as a phosphotransferase system II (celA, celB, and celC), a glucokinase (celK), and a glucosidase (celG). Directly downstream of celCBGKA is celI, which encodes a LacI family regulator that represses cel transcription in the absence of cellobiose. When the celCBGKAI gene cluster was transferred to cellobiose-negative strains of Vibrio and Photobacterium, the cluster conferred the ability to utilize cellobiose. Genomic analyses of naturally cellobiose-positive Vibrio species revealed that V. salmonicida has a homolog of the celCBGKAI cluster, but V. vulnificus does not. Moreover, bioinformatic analyses revealed that CelG and CelK share the greatest homology with glucosidases and glucokinases in the phylum Firmicutes. These observations suggest that distinct genes for cellobiose utilization have been acquired by different lineages within the family Vibrionaceae. In addition, the loss of the celI regulator, but not the structural genes, attenuated the ability of V. fischeri to compete for colonization of its natural host, Euprymna scolopes, suggesting that repression of the cel gene cluster is important in this symbiosis. Finally, we show that the V. fischeri cellobioase (CelG) preferentially cleaves β-d-glucose linkages but also cleaves β-d-galactose-linked substrates such as 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal), a finding that has important implications for the use of lacZ as a marker or reporter gene in V. fischeri. PMID:18487409

  18. Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus.

    PubMed Central

    Richaud, P; Colbeau, A; Toussaint, B; Vignais, P M

    1991-01-01

    The hupR1 gene from Rhodobacter capsulatus was cloned and sequenced. It can encode a protein of 53,843 Da which shares significant similarity with several transcriptional regulators and activates transcription of the structural hupSL genes of [NiFe]hydrogenase, as shown by the use of a translational fusion of lacZ with the hupSL promoter. A Hup- mutant having a point mutation in the hupR1 gene is described. PMID:1885559

  19. Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti.

    PubMed

    Summers, M L; Elkins, J G; Elliott, B A; McDermott, T R

    1998-11-01

    Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation. The transposon and flanking DNA were cloned from each psi::Tn5B22 reporter mutant and the junction DNA sequenced. High identity/similarity of the inferred peptides with those in major data bases allowed identification of the following genes: dnaK, expC, pssB, ackA, vipC, and prkA. The prkA homolog was also found to be up-regulated in response to carbon starvation and when nitrate replaced ammonium as the nitrogen source. Through allele replacement techniques, PhoB- mutants were generated for the expC, ackA, vipC, and pssB reporter strains. Loss of a functional PhoB resulted in the absence of Pi-sensitive induction in all four genes. These experiments suggest the Pho regulon in S. meliloti includes genes that presumably are not directly linked to Pi acquisition or assimilation. The psi strains were tested for their symbiotic properties under growth conditions that were Pi-limiting or Pi-nonlimiting for the host plant. All were Nod+ and Fix+ except the reporter strain of dnaK transcription, which was less effective than the wild-type strain under both P treatments, indicating DnaK is required for optimum symbiotic function.

  20. Regulation of Sinorhizobium meliloti 1021 rrnA-reporter gene fusions in response to cold shock.

    PubMed

    Gustafson, Ann M; O'Connell, Kevin P; Thomashow, Michael F

    2002-09-01

    We previously reported that mutants of Sinorhizobium meliloti 1021 carrying luxAB insertions in each of the three 16S rRNA genes exhibited a dramatic (> or = 28-fold) increase in luminescence following a temperature downshift from 30 to 15 degrees C. These results raised the possibility that the rRNA operons (rrn) of S. meliloti were cold shock loci. In testing this possibility, we found that fusion of the S. meliloti 1021 rrnA promoter to two different reporter genes, luxAB and uidA, resulted in hybrid genes that were transiently upregulated (as measured by transcript accumulation) about four- to sixfold in response to a temperature downshift. These results are consistent with the hypothesis that the rrn promoters are transiently upregulated in response to cold shock. However, much of the apparent cold shock regulation of the initial luxAB insertions was due to an unexpected mechanism: an apparent temperature-dependent inhibition of translation. Specifically, the rrnA sequences from +1 to +172 (relative to the start of transcription) were found to greatly decrease the ability of S. meliloti to translate hybrid rrn-luxAB transcripts into active protein at 30 degrees C. This effect, however, was largely eliminated at 15 degrees C. Possible mechanisms for the apparent transient increase in rrnA promoter activity and temperature-dependent inhibition of translation are discussed.

  1. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    PubMed Central

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  2. The first report of the vanC₁ gene in Enterococcus faecium isolated from a human clinical specimen.

    PubMed

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-09-01

    The vanC₁ gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC₁gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC₁ and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC₁ gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC₁gene. However, this study is the first to report the presence of the vanC₁gene in E. faecium of human origin. Additionally, our research showed the vanC₁gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC₁gene from different species.

  3. The color of mice: in the light of GFP-variant reporters.

    PubMed

    Hadjantonakis, A K; Nagy, A

    2001-01-01

    The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility

  4. The urease gene cluster of Vibrio parahaemolyticus does not influence the expression of the thermostable direct hemolysin (TDH) gene or the TDH-related hemolysin gene.

    PubMed

    Nakaguchi, Yoshitsugu; Okuda, Jun; Iida, Tetsuya; Nishibuchi, Mitsuaki

    2003-01-01

    In order to investigate why the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) of Vibrio parahaemolyticus are produced at low levels from urease-positive strains, the effect of the functional urease gene cluster of V. parahaemolyticus on the expression of the tdh and trh genes was examined. Transcriptional lacZ fusions with the tdh1, tdh2, trh1 and trh2 genes representing variants of the tdh and trh genes were integrated into the chromosome of an Escherichia coli strain and a urease-negative V. parahaemolyticus strain. The plasmid-borne urease gene cluster introduced and expressed in these constructs did not affect expression of any of the fusion genes. The amount of TDH produced from a Kanagawa phenomenon-positive V. parahaemolyticus did not change by introduction of the urease gene cluster either. It was concluded therefore that the urease gene cluster is not involved in the regulation of tdh and trh expression.

  5. Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium.

    PubMed

    Yuan, Lei; Janes, Lauren; Beeler, David; Spokes, Katherine C; Smith, Joshua; Li, Dan; Jaminet, Shou-Ching; Oettgen, Peter; Aird, William C

    2013-05-23

    We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human β-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

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

    PubMed Central

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

    2015-01-01

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

  7. The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif.

    PubMed

    Santel, A; Winhauer, T; Blümer, N; Renkawitz-Pohl, R

    1997-06-01

    We identified and characterized the don juan gene (dj) of Drosophila melanogaster. The don juan gene codes for a sperm specific protein component with an unusual repetitive six amino acid motif (DPCKKK) in the carboxy-terminal part of the protein. The expression of Don Juan is limited to male germ cells where transcription of the dj gene is initiated during meiotic prophase. But Western blot experiments indicate that DJ protein occurs just postmeiotically. Examination of transgenic flies bearing a dj-promoter-lacZ reporter construct revealed lacZ mRNA distribution resembling the expression pattern of the endogenous dj mRNA in the adult testes, whereas beta-galactosidase expression is exclusively present in postmeiotic germ cells. Thus, these observations strongly suggest that dj transcripts are under translational repression until in spermiogenesis. To study the function and subcellular distribution of DJ in spermiogenesis we expressed a chimaeric dj-GFP fusion gene in the male germline exhibiting strong GFP fluorescence in the liver testes, where only elongated spermatids are decorated. With regard to the characteristic expression pattern of DJ protein and its conspicuous repeat units possible functional roles are discussed.

  8. The Naphthalene Catabolic (nag) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

    PubMed Central

    Jones, Rheinallt M.; Britt-Compton, Bethan; Williams, Peter A.

    2003-01-01

    In Ralstonia sp. strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter. nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes. A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon. The transcriptional start of the nag operon was mapped, and a putative −10, −35 σ70-type promoter binding site was identified. Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways. Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate. Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon. However, other mutations in the region increased the effectiveness of salicylate as an inducer. PMID:13129957

  9. User-centered design of multi-gene sequencing panel reports for clinicians.

    PubMed

    Cutting, Elizabeth; Banchero, Meghan; Beitelshees, Amber L; Cimino, James J; Fiol, Guilherme Del; Gurses, Ayse P; Hoffman, Mark A; Jeng, Linda Jo Bone; Kawamoto, Kensaku; Kelemen, Mark; Pincus, Harold Alan; Shuldiner, Alan R; Williams, Marc S; Pollin, Toni I; Overby, Casey Lynnette

    2016-10-01

    The objective of this study was to develop a high-fidelity prototype for delivering multi-gene sequencing panel (GS) reports to clinicians that simulates the user experience of a final application. The delivery and use of GS reports can occur within complex and high-paced healthcare environments. We employ a user-centered software design approach in a focus group setting in order to facilitate gathering rich contextual information from a diverse group of stakeholders potentially impacted by the delivery of GS reports relevant to two precision medicine programs at the University of Maryland Medical Center. Responses from focus group sessions were transcribed, coded and analyzed by two team members. Notification mechanisms and information resources preferred by participants from our first phase of focus groups were incorporated into scenarios and the design of a software prototype for delivering GS reports. The goal of our second phase of focus group, to gain input on the prototype software design, was accomplished through conducting task walkthroughs with GS reporting scenarios. Preferences for notification, content and consultation from genetics specialists appeared to depend upon familiarity with scenarios for ordering and delivering GS reports. Despite familiarity with some aspects of the scenarios we proposed, many of our participants agreed that they would likely seek consultation from a genetics specialist after viewing the test reports. In addition, participants offered design and content recommendations. Findings illustrated a need to support customized notification approaches, user-specific information, and access to genetics specialists with GS reports. These design principles can be incorporated into software applications that deliver GS reports. Our user-centered approach to conduct this assessment and the specific input we received from clinicians may also be relevant to others working on similar projects.

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

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

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

  11. First report of multiresistance gene cfr in Enterococcus species casseliflavus and gallinarum of swine origin.

    PubMed

    Liu, Yang; Wang, Yang; Dai, Lei; Wu, Congming; Shen, Jianzhong

    2014-06-04

    The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr in Enterococcus species of swine origin. Twenty-five cfr-carrying Enterococcus isolates were collected from swine in Beijing, Guangzhou, and Shandong, China. The isolates consist of 24 Enterococcus casseliflavus and one Enterococcus gallinarum isolate, and exhibited six SmaI PFGE patterns. The cfr gene was located on plasmids in all isolates except E. casseliflavus En83, in which cfr was located on the chromosomal DNA. The cfr gene environments in most of these isolates contain DNA sequences similar to pEF-01, which was first found in Enterococcus. However, inverse PCR analysis suggested that the cfr-carrying circular forms might be different from pEF-01. The circular forms in Eg51 and its transconjugant, and En23, En10, and En94 are similar to the circular form in pEF-01, except for the truncated IS1216, which is replaced by a transposase of the IS256 family in En24. The cfr circular form could not be detected in either En77 or En83, and the same cfr-carrying segments of ∼ 10 kb had only 3500bp of sequence similar to pEF-01. This is the first report of cfr gene in E. casseliflavus and E. gallinarum. The potential dissemination of the multidrug resistance gene amongst different bacterial species, especially in enterococci of human and animal origins, is concerning and should be closely monitored.

  12. Hazardous effect of organophosphate compound, dichlorvos in transgenic Drosophila melanogaster (hsp70-lacZ): induction of hsp70, anti-oxidant enzymes and inhibition of acetylcholinesterase.

    PubMed

    Gupta, Subash Chandra; Siddique, Hifzur Rahman; Saxena, Daya Krishna; Chowdhuri, Debapratim Kar

    2005-08-30

    We tested a working hypothesis that stress genes and anti-oxidant enzyme machinery are induced by the organophosphate compound dichlorvos in a non-target organism. Third instar larvae of Drosophila melanogaster transgenic for hsp70 were exposed to 0.1 to 100.0 ppb dichlorvos and 5.0 mM CuSO(4) (an inducer of oxidative stress and stress genes) and hsp70, and activities of acetylcholinesterase (AchE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) product were measured. The study was further extended to examine tissue damage, if any, under such conditions. A concentration- and time-dependent increase in hsp70 and anti-oxidant enzymes was observed in the exposed organism as compared to control. A comparison of stress gene expression with SOD, CAT activities and LPO product under similar experimental conditions revealed that induction of hsp70 precedes the anti-oxidant enzyme activities in the exposed organism. Further, concomitant with a significant inhibition of AChE activity, significant induction of hsp70 was observed following chemical exposure. Mild tissue damage was observed in the larvae exposed to 10.0 ppb dichlorvos for 48 h when hsp70 expression reaches plateau. Dichlorvos at 0.1 ppb dietary concentration did not evoke significant hsp70 expression, anti-oxidant enzymes and LPO and AchE inhibition in the exposed organism, and thereby, was found to be non-hazardous to D. melanogaster. Conversely, 1.0 ppb of the test chemical stimulated a significant induction of hsp70 and anti-oxidant enzymes and significant inhibition of AchE; hence this concentration of test chemical was hazardous to the organism. The present study suggests that (a) both stress genes and anti-oxidant enzymes are stimulated as indices of cellular defense against xenobiotic hazard in D. melanogaster with hsp70 being proposed as first-tier bio-indicator of cellular hazard, (b) 0.1 ppb of the test chemical may be regarded as No Observed Adverse Effect Level (NOAEL), and

  13. The haem-uptake gene cluster in Vibrio fischeri is regulated by Fur and contributes to symbiotic colonization.

    PubMed

    Septer, Alecia N; Wang, Yanling; Ruby, Edward G; Stabb, Eric V; Dunn, Anne K

    2011-11-01

    Although it is accepted that bacteria-colonizing host tissues are commonly faced with iron-limiting conditions and that pathogenic bacteria often utilize iron from host-derived haem-based compounds, the mechanisms of iron acquisition by beneficial symbiotic bacteria are less clear. The bacterium Vibrio fischeri mutualistically colonizes the light organ of the squid Euprymna scolopes. Genome sequence analysis of V. fischeri revealed a putative haem-uptake gene cluster, and through mutant analysis we confirmed this cluster is important for haemin use by V. fischeri in culture. LacZ reporter assays demonstrated Fur-dependent transcriptional regulation of cluster promoter activity in culture. GFP-based reporter assays revealed that gene cluster promoter activity is induced in symbiotic V. fischeri as early as 14 h post inoculation, although colonization assays with the haem uptake mutant suggested an inability to uptake haem does not begin to limit colonization until later stages of the symbiosis. Our data indicate that the squid light organ is a low iron environment and that haem-based sources of iron are used by symbiotic V. fischeri cells. These findings provide important additional information on the availability of iron during symbiotic colonization of E. scolopes by V. fischeri, as well as the role of haem uptake in non-pathogenic host-microbe interactions.

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

    SciTech Connect

    2000-02-15

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

  15. Identification of direct serum-response factor gene targets during Me2SO-induced P19 cardiac cell differentiation.

    PubMed

    Zhang, Shu Xing; Garcia-Gras, Eduardo; Wycuff, Diane R; Marriot, Suzanne J; Kadeer, Nijiati; Yu, Wei; Olson, Eric N; Garry, Daniel J; Parmacek, Michael S; Schwartz, Robert J

    2005-05-13

    Serum-response factor (SRF) is an obligatory transcription factor, required for the formation of vertebrate mesoderm leading to the origin of the cardiovascular system. Protein A-TEV-tagged chromatin immunoprecipitation technology was used to collect direct SRF-bound gene targets from pluripotent P19 cells, induced by Me2SO treatment into an enriched cardiac cell population. From 242 sequenced DNA fragments, we identified 188 genomic DNA fragments as potential direct SRF targets that contain CArG boxes and CArG-like boxes. Of the 92 contiguous genes that were identified, a subgroup of 43 SRF targets was then further validated by co-transfection assays with SRF. Expression patterns of representative candidate genes were compared with the LacZ reporter expression activity of the endogenous SRF gene. According to the Unigene data base, 84% of the SRF target candidates were expressed, at least, in the heart. In SRF null embryonic stem cells, 81% of these SRF target candidates were greatly affected by the absence of SRF. Among these SRF-regulated genes, Raf1, Map4k4, and Bicc1 have essential roles in mesoderm formation. The 12 regulated SRF target genes, Mapk10 (JNK3), Txnl2, Azi2, Tera, Sema3a, Lrp4, Actc1, Myl3, Hspg2, Pgm2, Hif3a, and Asb5, have been implicated in cardiovascular formation, and the Ski and Hes6 genes have roles in muscle differentiation. SRF target genes related to cell mitosis and cycle, E2f5, Npm1, Cenpb, Rbbp6, and Scyl1, expressed in the heart tissue were differentially regulated in SRF null ES cells.

  16. Development of a Conditional Transgenic Mouse Model to Test the Fallopian Tube Origin of Serous Ovarian Cancer

    DTIC Science & Technology

    2009-03-01

    transgene plasmid to follow up on this next strategy. KEY RESEARCH ACCOMPLISHMENTS: • molecular cloning of Ovgp1-lacZ reporter gene plasmid...high-expressing 242 line • molecular cloning of Ovgp1-Cre transgene plasmid REPORTABLE OUTCOMES: • generation of two Ovgp1-lacZ transgenic

  17. Complementation Plasmids, Inducible Gene-Expression Systems, and Reporters for Staphylococci.

    PubMed

    Bertram, Ralph

    2016-01-01

    A cornucopia of methods and molecular tools is available for genetic modification of staphylococci, as shown for at least ten different species to date (Prax et al. Microbiology 159:421-435, 2013). This chapter reviews a number of frequently used vectors for complementation purposes that usually replicate in E. coli and staphylococci and differ in parameters including copy number, mode of replication, and sequence length. Systems for the artificial control of gene expression are described that are modulated by low-molecular-weight effectors such as metal cations, carbohydrates, and antibiotics. Finally, the usefulness of reporter proteins that exhibit enzymatic or autofluorescent characteristics in staphylococci is highlighted.

  18. Nonreplicating vaccinia vector efficiently expresses recombinant genes.

    PubMed

    Sutter, G; Moss, B

    1992-11-15

    Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

  19. Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation.

    PubMed

    Morschhäuser, J; Michel, S; Hacker, J

    1998-02-01

    Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

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

    SciTech Connect

    Braam, Janet

    2008-10-28

    The Arabidopsis TCH genes, originally isolated as a consequence of their upregulation in response to the mechanical stimulus of touch, are also upregulated by a variety of seemingly disparate environmental and hormonal stimuli. To gain insight into the complexities of TCH gene regulation, a number of approaches were taken. Regulatory elements responsible for regulation were identified and characteristics of the regulation were evaluated. Reporter genes were used to monitor expression localization and dynamics. Microarray analyses of genome-wide expression behavior indicated that touch-inducible gene expression is more widespread than generally appreciated. Identification of all touch-regulated genes shed light on the types of cellular processes that may be altered in response to mechanical stress perturbations. Expression of the TCH2 gene, also called CML24, encoding a calmodulin (CaM)-like (CML) protein, was evaluated. CML24 shares over 40% amino acid sequence identity with CaM, has 4 EF hands and undergoes a Ca2+-dependent change in migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs and is induced from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress, in regions undergoing growth, in vascular tissues and various floral organs and in stomata, trichomes and hydathodes. CML24 underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4 and MgCl2. These data present evidence that CML24 encodes a potential Ca2+ sensor that may function to enable responses to ABA, day length and presence of various salts. Further

  1. beta-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes.

    PubMed

    Ohta, Toshihiro; Tokishita, Shin-Ichi; Imazuka, Reiko; Mori, Ichiro; Okamura, Jin; Yamagata, Hideo

    2006-07-01

    Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75 degrees C. Because the frequency of DNA damage, such as deamination, depurination and single-strand breaks, increases as the temperature rises, the regulation of expression as well as the specificities and activities of T.thermophilus DNA repair systems are of particular interest. To study those systems, we developed a gene expression vector using the T.thermophilus beta-glucosidase gene (bgl) with host strain JOS9 (Deltabgl) derived from the T.thermophilus wild-type strain HB27. Since HB27 has two putative beta-galactosidase genes, the use of a single bgl gene as a reporter in combination with a Deltabgl host strain permits the study of gene expression against a low background level. We assayed Bgl activity with 2-nitrophenyl-beta-d-glucopyranoside as the substrate at 80 degrees C. We measured the expression of seven genes involved in DNA repair--three nucleotide excision repair genes (uvrA, uvrB and uvrC) and four recombinational repair genes (recA, ruvA, ruvB and ruvC). Expression levels of uvrA and uvrB were about three times those of uvrC, while those of ruvA, ruvB and ruvC were almost equal. Both ruvA and ruvC formed an operon with their adjacent 5'-upstream gene paaG and ftsQAZ, respectively. recA was transcribed as an operon of four genes, amt-cinA-ligT-recA. All seven DNA repair genes were expressed constitutively, and the DNA damaging agent mitomycin C did not increase their expression.

  2. A method to rapidly and accurately compare relative efficacies of non-invasive imaging reporter genes in a mouse model, and its application to luciferase reporters

    PubMed Central

    Gil, Jose S.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2013-01-01

    Purpose Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Procedures Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. Results We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Conclusions Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET). PMID:21850545

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. The Epstein-Barr virus BZLF1 gene product activates the human immunodeficiency virus type 1 5' long terminal repeat.

    PubMed Central

    Mallon, R; Borkowski, J; Albin, R; Pepitoni, S; Schwartz, J; Kieff, E

    1990-01-01

    The Epstein-Barr virus immediate-early gene product BZLF1 transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). The BZLF1 gene product caused an 18-fold increase in beta-galactosidase activity from an HIV-1 LTR lacZ expression vector, whereas the HIV-1 transactivator tat caused a 44-fold increase in beta-galactosidase activity. When cells were transfected with both BZLF1 (pEBV-Z) and tat (pTAT3) expression vectors, as well as HIV-1 LTR lacZ plasmid (pLRON), a 214-fold increase in beta-galactosidase activity was observed. This result suggests a synergistic effect of BZLF1 and tat on HIV-1 LTR-directed lacZ gene expression. Analysis of quantitative BZLF1 and tat requirements for maximal HIV-1 LTR activation indicates that BZLF1 does not reduce the amount of tat required for maximal LTR activation, as would be expected if the BZLF1 synergistic effect was due to increased tat gene expression. Thus, coordinate effects of BZLF1 and tat on the HIV-1 LTR or its transcript are probably responsible for synergistic HIV-1 LTR activation. PMID:2173793

  6. The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenic Xenopus laevis.

    PubMed

    Lim, Wayland; Neff, Eric S; Furlow, J David

    2004-06-17

    Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

  7. Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic?

    PubMed Central

    Di Fruscio, Giuseppina; Garofalo, Arcomaria; Mutarelli, Margherita; Savarese, Marco; Nigro, Vincenzo

    2016-01-01

    Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders. PMID:25898921

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

    PubMed Central

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

    2013-01-01

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

  9. A case report: Autosomal recessive microcephaly caused by a novel mutation in MCPH1 gene.

    PubMed

    Ghafouri-Fard, Soudeh; Fardaei, Majid; Gholami, Milad; Miryounesi, Mohammad

    2015-10-15

    Autosomal Recessive Primary Microcephaly (MCPH-MIM 251200) is distinguished by congenital decrease in occipito-frontal head circumference (OFC) of at least 2 standard deviations (SD) below population average in addition to non-progressive mental retardation, without any prominent neurological disorder. Mutations in MCPH1, which encodes the protein microcephalin have been detected in this disorder. Here we report a consanguineous Iranian family with 2 children affected with microcephaly. Despite the severe mental retardation observed in the male patient, the female patient had normal intelligent with no delay in motor milestones or speech. A novel splice-acceptor site homozygous mutation has been detected in intron 4 of MCPH1 gene (c.322-2A>T) which results in an RNA processing defect with a 15-nucleotide deletion in exon 5 of the mRNA transcript (r.322_336del15, p.R108_Q112del5). This novel mutation has resulted in different phenotypes in affected male and female patients of this family. The sex-specific variations in gene regulation during brain development may partially explain such difference in phenotypes probably in addition to other mechanisms such as modifier genes.

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

    SciTech Connect

    Fields, C.A.

    1996-06-01

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

  11. Sporadic cerebral cavernous malformations: report of further mutations of CCM genes in 40 Italian patients.

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    1988-04-01

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

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

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

    PubMed

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

    2015-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    PubMed Central

    Miller, Amber; Russell, Stephen J

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. Post-transcriptional regulation of gene PA5507 controls PQS concentration in Pseudomonas aeruginosa

    PubMed Central

    Tipton, Kyle A.; Coleman, James P.; Pesci, Everett C.

    2015-01-01

    Summary Pseudomonas aeruginosa can sense and respond to a myriad of environmental signals and utilizes a system of small molecules to communicate through intercellular signaling. The small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal [PQS]) is one of these signals and its synthesis is important for virulence. Previously, we identified an RpiR-type transcriptional regulator, QapR, that positively affects PQS production by repressing the qapR operon. An in-frame deletion of this regulator caused P. aeruginosa to produce a greatly reduced concentration of PQS. Here, we report that QapR translation is linked to the downstream gene PA5507. We found that introduction of a premature stop codon within qapR eliminates transcriptional autorepression of the qapR operon as expected but has no effect on PQS concentration. This was investigated with a series of lacZ reporter fusions which showed that translation of QapR must terminate at, or close to, the native qapR stop codon in order for translation of PA5507 to occur. Also, it was shown that truncation of the 5′ end of the qapR transcript permitted PA5507 translation without translation of QapR. Our findings led us to conclude that PA5507 transcription and translation are both tightly controlled by QapR and this control is important for PQS homeostasis. PMID:25662317

  19. Progressive cerebellar, auditory, and esophageal dysfunction caused by targeted disruption of the frizzled-4 gene.

    PubMed

    Wang, Y; Huso, D; Cahill, H; Ryugo, D; Nathans, J

    2001-07-01

    Wnt signaling has been implicated in the control of cell proliferation and in synapse formation during neural development, and these actions are presumed to be mediated by frizzled receptors. In this paper we report the phenotype of mice carrying a targeted deletion of the frizzled-4 (fz4) gene. fz4(-/-) mice exhibit three distinct defects: (1) progressive cerebellar degeneration associated with severe ataxia, (2) absence of a skeletal muscle sheath around the lower esophagus associated with progressive esophageal distension and dysfunction, and (3) progressive deafness caused by a defect in the peripheral auditory system unaccompanied by loss of hair cells or other auditory neurons. As assayed using a lacZ knock-in reporter, fz4 is widely expressed within the CNS. In particular, fz4 is expressed in cerebellar Purkinje cells, esophageal skeletal muscle, and cochlear inner hair cells, and the absence of Fz4 in these cells is presumed to account for the fz4(-/-) phenotype. In contrast to the early cell proliferation and patterning effects classically ascribed to Wnts, the auditory and cerebellar phenotypes of fz4(-/-) mice implicate Frizzled signaling in maintaining the viability and integrity of the nervous system in later life.

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

    PubMed

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

    2015-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  2. Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum.

    PubMed

    Mesa, Socorro; Velasco, Leonardo; Manzanera, Maximino E; Delgado, María J; Bedmar, Eulogio J

    2002-11-01

    The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

  3. A 15-base-pair element activates the SPS4 gene midway through sporulation in Saccharomyces cerevisiae.

    PubMed Central

    Hepworth, S R; Ebisuzaki, L K; Segall, J

    1995-01-01

    Sporulation of the yeast Saccharomyces cerevisiae represents a simple developmental process in which the events of meiosis and spore wall formation are accompanied by the sequential activation of temporally distinct classes of genes. In this study, we have examined expression of the SPS4 gene, which belongs to a group of genes that is activated midway through sporulation. We mapped the upstream boundary of the regulatory region of SPS4 by monitoring the effect of sequential deletions of 5'-flanking sequence on expression of plasmid-borne versions of SPS4 introduced into a MATa/MAT alpha delta sps4/delta sps4 strain. This analysis indicated that the 5' boundary of the regulatory region was within 50 bp of the putative TATA box of the gene. By testing various oligonucleotides that spanned this boundary and the downstream sequence for their ability to activate expression of a heterologous promoter, we found that a 15-bp sequence sufficed to act as a sporulation-specific upstream activation sequence. This 15-bp fragment, designated UASSPS4, activated expression of a CYC1-lacZ reporter gene midway through sporulation and was equally active in both orientations. Extending the UAS fragment to include the adjacent 14-bp enhanced its activity 10-fold. We show that expression of SPS4 is regulated in a manner distinct from that of early meiotic genes: mutation of UME6 did not lead to vegetative expression of SPS4, and sporulation-specific expression was delayed by mutation of IME2. In vivo and in vitro assays suggested that a factor present in vegetative cells bind to the UASSPS4 element. We speculate that during sporulation this factor is modified to serve as an activator of the SPS4 gene or, alternatively, that it recruits an activator to the promoter. PMID:7791799

  4. CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Hedges, D; Proft, M; Entian, K D

    1995-01-01

    The expression of gluconeogenic fructose-1,6-bisphosphatase (encoded by the FBP1 gene) depends on the carbon source. Analysis of the FBP1 promoter revealed two upstream activating elements, UAS1FBP1 and UAS2FBP1, which confer carbon source-dependent regulation on a heterologous reporter gene. On glucose media neither element was activated, whereas after transfer to ethanol a 100-fold derepression was observed. This gene activation depended on the previously identified derepression genes CAT1 (SNF1) (encoding a protein kinase) and CAT3 (SNF4) (probably encoding a subunit of Cat1p [Snf1p]). Screening for mutations specifically involved in UAS1FBP1 derepression revealed the new recessive derepression mutation cat8. The cat8 mutants also failed to derepress UAS2FBP1, and these mutants were unable to grow on nonfermentable carbon sources. The CAT8 gene encodes a zinc cluster protein related to Saccharomyces cerevisiae Gal4p. Deletion of CAT8 caused a defect in glucose derepression which affected all key gluconeogenic enzymes. Derepression of glucose-repressible invertase and maltase was still normally regulated. A CAT8-lacZ promoter fusion revealed that the CAT8 gene itself is repressed by Cat4p (Mig1p). These results suggest that gluconeogenic genes are derepressed upon binding of Cat8p, whose synthesis depends on the release of Cat4p (Mig1p) from the CAT8 promoter. However, gluconeogenic promoters are still glucose repressed in cat4 mutants, which indicates that in addition to its transcription, the Cat8p protein needs further activation. The observation that multicopy expression of CAT8 reverses the inability of cat1 and cat3 mutants to grow on ethanol indicates that Cat8p might be the substrate of the Cat1p/Cat3p protein kinase. PMID:7891685

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

    SciTech Connect

    Ts`o, P.O.P.

    1992-08-01

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

  6. [Radiation biology of structurally different Drosophila genes. Report 2. The vestigial gene: molecular characteristics of chromosome mutations].

    PubMed

    Afanas'eva, K P; Aleksandrova, M V; Aleksandrov, I D; Korablinova, S V

    2012-01-01

    The results of the PCR-assay of mutation lesions at each of 16 fragments overlapping the entire vestigial (vg) gene of Drosophila melanogaster in 52 gamma-ray-, neutron- and neutron + gamma-ray-induced vg mutants having the inversion or translocation breakpoint within the vg microregion are presented. 4 from 52 mutants studied were found to have large deletions of about 200 kb covering the entire vg gene and adjacent to sca and l(2)C gene-markers as well. 23 mutants from 48 (47.9%) were found to have a wild-type gene structure showing that the exchange breakpoints are located outside of the vg gene. 25 others display the intragenic lesions of different complexity detected by PCR as the absence of(i) either one fragment or (ii) two or more (6-7) adjacent fragments and (iii) simultaneously several (i) or (i) and (ii) types separated by normal gene regions. It is important that 6 from 25 mutants have the breakpoint inside the vg gene and display the (i) or (ii) type of lesions at the gene regions containing the putative break whereas 5 others from 25 with the above lesions have the exchange breakpoint outside the vg gene. Therefore, the breakpoints underlying either inversions or translocations induced by low- and high-LET radiation are likely to be located within and outside the gene under study. Thereby, the formation of exchanges is accompanied by DNA deletions of various sizes at the exchange breakpoints. The molecular model of formation of such exchange-deletion rearrangements is elaborated and presented. Also, conception of the predominately clustered action of both low- and high-LET radiation on the germ cell genome is suggested as the summing-up of the presented results. The ability of ionizing radiation to induce the clusters of genetic alterations in the form of hidden DNA damages as well as gene/chromosome mutations is determined by the track structure and hierarchical organization of the genome. To detect the quality and frequency patterns of all

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

    PubMed Central

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

    2016-01-01

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

  8. Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.

    PubMed Central

    Colbeau, A; Vignais, P M

    1992-01-01

    The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis. PMID:1624420

  9. A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two-component system.

    PubMed

    Mandin, Pierre; Gottesman, Susan

    2009-05-01

    In Escherichia coli, the largest class of small regulatory RNAs binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific mRNAs. While recent studies have shown that some mRNAs could be subject to post-transcriptional regulation by sRNAs (e.g. mRNAs found by co-immunoprecipitation with Hfq), no method has yet been described to identify small RNAs that regulate them. We developed a method to easily make translational fusions of genes of interest to the lacZ reporter gene, under the control of a P(BAD)-inducible promoter. A multicopy plasmid library of the E. coli genome can then be used to screen for small RNAs that affect the activity of the fusion. This screening method was first applied to the dpiB gene from the dpiBA operon, which encodes a two-component signal transduction system involved in the SOS response to beta-lactams. One small RNA, RybC, was found to negatively regulate the expression of dpiB. Using mutants in the dpiB-lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrate that RybC directly base pairs with the dpiBA mRNA.

  10. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    PubMed

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  11. Identification of multiple genetic loci that regulate adenovirus gene therapy.

    PubMed

    Zhang, H-G; Hsu, H-C; Yang, P-A; Yang, X; Wu, Q; Liu, Z; Yi, N; Mountz, J D

    2004-01-01

    A key aspect of the immune response to adenovirus (Ad) gene therapy is the generation of a cytotoxic T-cell (CTL) response. To better understand the genetic network underlying these events, 20 strains of C57BL/6 x DBA/2 (BXD) recombinant inbred (RI) mice were administered with AdLacZ and analyzed at days 7, 21, 30, and 50 for liver beta-galactosidase (LacZ) expression and CTL response. Sera levels of interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were analyzed at different times after AdLacZ. There was a distinct strain-dependent expression of LacZ, which was strongly correlated with the CTL response. Among the five BXD RI strains that exhibited significantly prolonged LacZ expression, four also exhibited a marked defect in the production of Ad-specific CTL. There was a strong correlation between the sera levels of IFN-gamma, TNF-alpha, and IL-6, but cytokine responses were not significantly correlated with LacZ expression or the CTL response. Quantitative trait loci regulating LacZ on day 30 were found on chromosome (Chr) 19 (33 cM) and Chr 15 (42.8 cM). Cytotoxicity mapped to Chr 7 (41.0 and 57.4-65.2 cM), Chr 15 (61.7 cM), and Chr X (27.8 cM). IFN-gamma production mapped to Chr 18 (22, 27, and 32 cM) and Chr 11 (64.0 cM). TNF-alpha and IL-6 production mapped to Chr 6 (91.5 cM) Chr 9 (42.0 cM) and Chr 8 (52 and 73.0 cM). These results indicate that different strains of mice exhibit different pathways for effective clearance of AdLacZ depending on genetic polymorphisms and interactions at multiple genetic loci.

  12. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  13. The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA.

    PubMed

    Kaminski, P A; Desnoues, N; Elmerich, C

    1994-05-24

    We report the characterization of a mutant of Azorhizobium caulinodans, isolated after ethyl methanesulfonate mutagenesis. This Nod+ Nif- Fix- mutant is unable to synthesize 10 of 15 polypeptides normally induced under conditions of nitrogen fixation. By using lacZ fusions it was shown that nifA and nifA-regulated genes were not expressed in this strain. The mutation was complemented by a constitutively expressed nifA gene or by a 1.1-kb DNA fragment from the wild-type strain, whose nucleotide sequence revealed a single open reading frame of 255 bp coding for an 85-amino acid polypeptide. The deduced amino acid sequence is similar to that of HF-I, an RNA-binding protein of Escherichia coli, which is required for replication of bacteriophage Q beta RNA. The similarity can be extended to the function since hfq, the structural gene for HF-I, complemented the A. caulinodans mutant. The corresponding gene in A. caulinodans was termed nrfA (for nif regulatory factor). Inactivation of nrfA in the mutant was due to a missense mutation resulting in the replacement of a cysteine residue by arginine. A null mutant, constructed by disruption of nrfA, exhibited the same phenotype as the missense mutant. Thus, an additional factor can be added to the already complex system of nifA regulation in A. caulinodans.

  14. Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse).

    PubMed

    Suzuki, T; Wang, X; Miyata, Y; Saeki, K; Kohara, A; Kawazoe, Y; Hayashi, M; Sofuni, T

    2000-11-30

    Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.

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

    PubMed Central

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

    1996-01-01

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

  16. Modified mariner transposons for random inducible-expression insertions and transcriptional reporter fusion insertions in Bacillus subtilis.

    PubMed

    Pozsgai, Eric R; Blair, Kris M; Kearns, Daniel B

    2012-02-01

    Transposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA. mariner is a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existing mariner transposon, TnYLB, such that it can easily be genetically manipulated and introduced into Bacillus subtilis. We generate a series of three new mariner derivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterless lacZ gene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted to B. subtilis and should be applicable to any mariner-compatible host organism, provided that in vitro mutagenesis or an in vivo species-specific delivery vector is employed.

  17. Gene-environment interactions in cancer epidemiology: a National Cancer Institute Think Tank report.

    PubMed

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

    2013-11-01

    Cancer risk is determined by a complex interplay of genetic and environmental factors. Genome-wide association studies (GWAS) have identified hundreds of common (minor allele frequency [MAF] > 0.05) and less common (0.01 < 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.

  18. The β-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells.

    PubMed

    Barman, Hirak Kumar; Mohanta, Ramya; Patra, Swagat Kumar; Chakrapani, Vemulawada; Panda, Rudra Prasanna; Nayak, Swapnarani; Jena, Sasmita; Jayasankar, Pallipuram; Nandanpawar, Priyanka

    2015-06-01

    We previously characterized the β-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The β-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of β-actin gene expression across eukaryotes. Furthermore, the isolated β-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.

  19. Global genetic variation of select opiate metabolism genes in self-reported healthy individuals.

    PubMed

    Wendt, F R; Pathak, G; Sajantila, A; Chakraborty, R; Budowle, B

    2017-04-11

    CYP2D6 is a key pharmacogene encoding an enzyme impacting poor, intermediate, extensive and ultrarapid phase I metabolism of many marketed drugs. The pharmacogenetics of opiate drug metabolism is particularly interesting due to the relatively high incidence of addiction and overdose. Recently, trans-acting opiate metabolism and analgesic response enzymes (UGT2B7, ABCB1, OPRM1 and COMT) have been incorporated into pharmacogenetic studies to generate more comprehensive metabolic profiles of patients. With use of massively parallel sequencing, it is possible to identify additional polymorphisms that fine tune, or redefine, previous pharmacogenetic findings, which typically rely on targeted approaches. The 1000 Genomes Project data were analyzed to describe population genetic variation and statistics for these five genes in self-reported healthy individuals in five global super- and 26 sub-populations. Findings on the variation of these genes in various populations expand baseline understanding of pharmacogenetically relevant polymorphisms for future studies of affected cohorts.The Pharmacogenomics Journal advance online publication, 11 April 2017; doi:10.1038/tpj.2017.13.

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

    PubMed

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

    1999-09-01

    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.

  1. [Neonatal diabetes mellitus and KCNJ11 gene mutation: report of a family case].

    PubMed

    Orío Hernández, M; de la Serna Martínez, M; González Casado, I; Lapunzina, P; Gracia Bouthelier, R

    2008-06-01

    Neonatal diabetes mellitus (NDM) is characterized by hyperglycemia within the first month of life and insulin dependence for at least two weeks. There are two types of NDM, transient (TNDM) and permanent (PNDM), which are genetically different. We report the case of two brothers who developed hyperglycemia without ketosis on the 18th day and 2 h of life, respectively. Thyroid function tests, abdominal ultrasound and karyotype where normal and there were no pancreatic antibodies. The first one required insulin therapy for the first 92 days of life and the second for 5 months. The mother developed gestational diabetes during both pregnancies and she was later diagnosed diabetes mellitus (without antibodies). They were studied for mutations in KCNJ11 gene (principally related to the permanent form). The three of them showed the E229K mutation (frequently associated with the transient form). A genetic study is essential in NDM to achieve the most accurate prognosis possible.

  2. Defining a new vision for the retinoblastoma gene: report from the 3rd International Rb Meeting.

    PubMed

    Rubin, Seth M; Sage, Julien

    2013-11-21

    The retinoblastoma tumor suppressor (Rb) pathway is mutated in most, if not all human tumors. In the G0/G1 phase, Rb and its family members p107 and p130 inhibit the E2F family of transcription factors. In response to mitogenic signals, Cyclin-dependent kinases (CDKs) phosphorylate Rb family members, which results in the disruption of complexes between Rb and E2F family members and in the transcription of genes essential for S phase progression. Beyond this role in early cell cycle decisions, Rb family members regulate DNA replication and mitosis, chromatin structure, metabolism, cellular differentiation, and cell death. While the RB pathway has been extensively studied in the past three decades, new investigations continue to provide novel insights into basic mechanisms of cancer development and, beyond cancer, help better understand fundamental cellular processes, from plants to mammals. This meeting report summarizes research presented at the recently held 3rd International Rb Meeting.

  3. Short report: polymorphisms in the chloroquine resistance transporter gene in Plasmodium falciparum isolates from Lombok, Indonesia.

    PubMed

    Huaman, Maria Cecilia; Yoshinaga, Kazumi; Suryanatha, Aan; Suarsana, Nyoman; Kanbara, Hiroji

    2004-07-01

    The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P. falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia. The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied. Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type S(agt)VMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%). Moreover, one P. falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates. Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1999-01-01

    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 PMID:10464070

  6. Treatment of leptomeningeal metastases in a rat model using a recombinant adenovirus containing the HSV-tk gene.

    PubMed

    Vincent, A J; Esandi, M D; van Someren, G; Noteboom, J L; Avezaat, C J; Vecht, C; Smitt, P A; van Bekkum, D W; Valerio, D; Hoogerbrugge, P M; Bout, A

    1996-10-01

    The authors constructed recombinant adenoviral vectors to investigate their potential for gene therapy treatment of leptomeningeal metastases. Several human cell lines that were derived from tumors occurring as leptomeningeal metastases and that were infected in vitro with major late promoter recombinant adenovirus containing the luciferase (luc) gene (IG.Ad.MLP.luc) showed high levels of expression. When these human tumor cell lines were infected in vitro with recombinant adenovirus harboring the herpes simplex virus-thymidine kinase (HSV-tk) gene (IG.Ad.MLP.TK), they were highly sensitive to the killing effects of ganciclovir (GCV). Transduction efficiency of leptomeningeal tumor cells in vivo was assessed by injecting 9-L rat brain tumor cells into the cerebrospinal fluid of Fischer rats via the cisterna magna. After 3 days, recombinant adenovirus containing the lacZ reporter gene (IG.Ad.MLP.lacZ) was injected via the same route. Six days after tumor cell injection, expression of the reporter gene was observed in tumor cells along the total neural axis. Subsequently, rats with leptomeningeal metastases were treated 3 days after tumor cell injection with HSV-tk. Beginning on the next day, GCV was injected intraperitoneally for 10 days. The rats that developed neurological symptoms were killed immediately. The symptom-free latency of every rat was determined. The rats treated with HSV-tk and subsequent GCV had significantly longer (p < 0.01) symptom-free latency than all control groups. This study demonstrates the feasibility and efficacy of this therapeutic approach in a rat model. Clinically, it should be used in the palliative treatment of patients with leptomeningeal metastases.

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

    PubMed

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

    2002-07-03

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

  8. Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.

    PubMed

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Aoki, Miku; Taguchi, Katsutoshi; Tanaka, Masaki

    2016-02-01

    The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

  9. Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression.

    PubMed

    Krinner, Simone; Heitzer, Asli; Asbach, Benedikt; Wagner, Ralf

    2015-12-01

    Successful therapeutic protein production in vitro and in vivo requires efficient and long-term transgene expression supported by optimized vector and transgene cis-regulatory sequence elements. This study provides a comparative analysis of CpG-rich, highly expressed, versus CpG-depleted, poorly expressed green fluorescent protein (GFP) reporter transgenes, transcribed by various promoters in two different cell systems. Long-term GFP expression from a defined locus in stable Chinese hamster ovary cells was clearly influenced by the combination of transgene CpG content and promoter usage, as shown by differential silencing effects on selection pressure removal among the cytomegalovirus (CMV) promoter and elongation factor (EF)-1α promoter. Whereas a high intragenic CpG content promoted local DNA methylation, CpG depletion rather accelerated transgene loss and increased the local chromatin density. On lentiviral transfer of various expression modules into epigenetically sensitive P19 embryonic pluripotent carcinoma cells, CMV promoter usage led to rapid gene silencing irrespective of the intragenic CpG content. In contrast, EF-1α promoter-controlled constructs showed delayed silencing activity and high-level transgene expression, in particular when the CpG-rich GFP reporter was used. Notably, GFP silencing in P19 cells could be prevented completely by the bidirectional, dual divergently transcribed A2UCOE (ubiquitously acting chromatin-opening element derived from the human HNRPA2B1-CBX3 locus) promoter. Because the level of GFP expression by the A2UCOE promoter was entirely unaffected by the intragenic CpG level, we suggest that A2UCOE can overcome chromatin compaction resulting from intragenic CpG depletion due to its ascribed chromatin-opening abilities. Our analyses provide insights into the interplay of the intragenic CpG content with promoter sequences and regulatory sequence elements, thus contributing toward the design of therapeutic transgene expression

  10. Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

    PubMed Central

    2011-01-01

    Introduction Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes) and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. Case presentation A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. Conclusion In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion. PMID:22011321

  11. Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma: improvement of cell sensitization to ganciclovir.

    PubMed

    Zerrouqi, A; Rixe, O; Ghoumari, A M; Yarovoi, S V; Mouawad, R; Khayat, D; Soubrane, C

    1996-01-01

    In this study, we investigated whether the regulation and the copy number of the herpes simplex virus thymidine kinase (HSVtk) gene increased the sensitization to ganciclovir (GCV) of glioma cell lines (Rat C6 and human U118-MG) using liposome-mediated gene transfer. Three recombinant plasmids carrying the HSVtk gene driven by the thymidine kinase promoter in single (pAGo) and double copy (pYED) or by the human cytomegalovirus promoter (pCMVtk) were used for the transfection. The DNA delivery was optimized by screening a panel of cationic liposomes using Lac-Z and luciferase as reporter genes. The efficiency of transfection reached 33% to 36% in vitro but only 18.6% in vivo after an intratumoral injection of DNA-liposome complexes. Moreover, after transfection of the three plasmids, the cell-killing effect of GCV was evaluated. A significant enhancement (four- to fivefold) of the cell sensitivity to GCV was shown in pCMVtk and pYED as compared with pAGo-transfected cells in both cell lines. According to the plasmid, the effect of the HSVtk/GCV system was confirmed by in vivo experiments and was objectified by a higher tumor weight reduction with pCMVtk (49%) than pAGo (27%). From these results, we conclude that (1) the gene transfer can be achieved by cationic liposomes both in vitro and in vivo and that (2) using this type of vector, the antitumor effect of the HSVtk/GCV system could be potentiated by the up-regulation of HSVtk gene duplication.

  12. Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors.

    PubMed

    Hakkila, Kaisa; Maksimow, Mikael; Karp, Matti; Virta, Marko

    2002-02-15

    The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.

  13. Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene.

    PubMed

    Esandi, M C; van Someren, G D; Vincent, A J; van Bekkum, D W; Valerio, D; Bout, A; Noteboom, J L

    1997-04-01

    Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local

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

    PubMed Central

    Gates, J; Thummel, C S

    2000-01-01

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

  15. One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis.

    PubMed

    Fischer, H M; Velasco, L; Delgado, M J; Bedmar, E J; Schären, S; Zingg, D; Göttfert, M; Hennecke, H

    2001-02-01

    Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these

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

    PubMed Central

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

    2001-01-01

    Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472–1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN1) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN2), which was then cloned by using hemN1 as a probe. The hemN2 gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN1 and HemN2, respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (≥20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK2. In addition, maximal anaerobic hemN1 expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN1 mutant, strains lacking a functional hemN2 gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c

  17. Identification of the merR gene of R100 by using mer-lac gene and operon fusions.

    PubMed Central

    Foster, T J; Brown, N L

    1985-01-01

    Transcriptional (operon) and translational (gene) fusions between the R100 merR gene and lacZ were constructed in vitro in a pBR322 plasmid carrying the mer genes derived from plasmid R100. The translational fusions were oriented in the opposite direction to and divergently from the merTCAD genes. This shows that the reading frame previously thought to be merR was incorrect. Expression of the gene fusion was repressed in trans by a compatible plasmid carrying the R100 merR+ gene, as was a similarly oriented transcriptional fusion. In contrast, expression of beta-galactosidase by the lac fragment located at the same site but in the opposite orientation was at a lower level and was not repressed by merR+. Images PMID:2993235

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

    PubMed Central

    Sinnott, M L; Smith, P J

    1978-01-01

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

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

    PubMed Central

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

    1995-01-01

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

  20. Post-transcriptional regulation of gene PA5507 controls Pseudomonas quinolone signal concentration in P. aeruginosa.

    PubMed

    Tipton, Kyle A; Coleman, James P; Pesci, Everett C

    2015-05-01

    Pseudomonas aeruginosa can sense and respond to a myriad of environmental signals and utilizes a system of small molecules to communicate through intercellular signaling. The small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal [PQS]) is one of these signals and its synthesis is important for virulence. Previously, we identified an RpiR-type transcriptional regulator, QapR, that positively affects PQS production by repressing the qapR operon. An in-frame deletion of this regulator caused P. aeruginosa to produce a greatly reduced concentration of PQS. Here, we report that QapR translation is linked to the downstream gene PA5507. We found that introduction of a premature stop codon within qapR eliminates transcriptional autorepression of the qapR operon as expected but has no effect on PQS concentration. This was investigated with a series of lacZ reporter fusions which showed that translation of QapR must terminate at, or close to, the native qapR stop codon in order for translation of PA5507 to occur. Also, it was shown that truncation of the 5' end of the qapR transcript permitted PA5507 translation without translation of QapR. Our findings led us to conclude that PA5507 transcription and translation are both tightly controlled by QapR and this control is important for PQS homeostasis.

  1. Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay

    PubMed Central

    Amakura, Yoshiaki; Tsutsumi, Tomoaki; Yoshimura, Morio; Nakamura, Masafumi; Handa, Hiroshi; Matsuda, Rieko; Teshima, Reiko; Watanabe, Takahiro

    2016-01-01

    The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay. PMID:28231110

  2. An extract from Prospects and Risks of Gene Technology: The Report of the Enquete Commission to the Bundestag of the Federal Republic of Germany.

    PubMed

    1988-07-01

    We are pleased to publish an English translation of the crucial section on germ line gene therapy from the Report of the German Enquete Commission. This section of the Report raises basic ethical questions concerning embryo research, as well as more specific questions relating to germ line gene therapy. This form of gene therapy involves genetic modification of the germ cells. Such a modification would be passed on to any descendents of the person whose genes had been altered. The Report had previously dealt with somatic gene therapy, which does not affect the germ line and hence is not passed on to descendents.

  3. Cytotoxic effect of a replication-incompetent adenoviral vector with cytosine deaminase gene driven by L-plastin promoter in hepatocellular carcinoma cells.

    PubMed

    Jung, Kihwa; Kim, Sunja; Lee, Kyumhyang; Kim, Changmin; Chung, Injae

    2007-06-01

    Great expectations are set on gene therapy for the treatment of malignant hepatocellular carcinomas (HCC) in East Asia. Recombinant adenoviral vectors (AV) have been developed in which the L-plastin promoter (LP) regulates the expression of transgenes, in a tumor cell specific manner, resulting in an increase in the therapeutic index. The development of the AdLPCD vector, a replication-incompetent AV, containing a transcription unit of LP and E. coli cytosine deaminase (CD), was reported in our previous work. In the present study, the AdLPCD vector combined with 5-fluorocytosine (5-FC) administration was tested to see if it might have significant utility in the chemosensitization of L-plastin positive HCC. Four HCC cell lines (HepG2, Chang Liver, Huh-7 and SK-Hep-1 cells) were investigated for the expression of LacZ after infecting the cells with the AdLPLacZ vector containing a 2.4 kb fragment of LP and the LacZ gene. Relatively high levels of LP activity were detected in HepG2, followed by Chang Liver cells; whereas, no promoter activity was found in Huh-7 and SK-Hep-1 cells, as determined by AdLPLacZ infection followed by the beta-galactosidase assay. In addition, the results of RT-PCR assays for the detection of endogenous L-plastin mRNA in these cells lines correlated well with those of the beta-galactosidase activity after infection with AdLPLacZ. Based on these data, the cytotoxic effect of AdLPCD/5-FC was evaluated in HepG2 cells. These results indicate that the CD gene delivered by AV could sensitize HepG2 cells to the prodrug, 5-FC. However, the observed effects were insufficient to cause the death of most of cells. This suggests that the screening of patients for an AdLP/5-FC strategy based on AdLPLacZ data might not always guarantee a good therapeutic outcome.

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated

  5. Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae).

    PubMed

    Candido-Silva, Juliana Aparecida; Machado, Maiaro Cabral Rosa; Hartfelder, Klaus Hartmann; de Almeida, Jorge Cury; Paçó-Larson, Maria Luisa; Monesi, Nadia

    2015-03-01

    The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage.

  6. [Radiation biology of structurally different Drosophila melanogaster genes. Report I. The vestigial gene: molecular characteristic of "point" mutations].

    PubMed

    Aleksandrov, I D; Afanas'eva, K P; Aleksandrova, M V; Lapidus, I L

    2012-01-01

    The screening of PCR-detected DNA alterations in 9 spontaneous and 59 gamma-ray-, neutron - or neutron + gamma-ray-induced Drosophila vestigial (vg) gene/"point" mutations was carried out. The detected patterns of existence or absence of either of 16 overlapping fragments into which vg gene (15.1 kb, 8 exons, 7 introns) was divided enable us to subdivide all mutants into 4 classes: (i) PCR+ (40.7%) without the detected changes; (ii) "single-site" (33.9%) with the loss of a single fragment; (iii) partial detections (15.2%) as a loss of 2-9 adjacent fragments and (iv) "cluster" mutants (10.2%) having 2-3 independent changes of(ii) and/or (iii) classes. All spontaneous mutants except one were found to be classified as (ii) whereas radiation-induced mutants are represented by all 4 classes whose interrelation is determined by the dose and radiation quality. In particular, the efficacy of neutrons was found to be nine times as large as that of gamma-rays under the "cluster" mutant induction. Essentially, the distribution of DNA changes along the gene is uneven. CSGE-assay of PCR+-exon 3 revealed DNA heteroduplexes in 5 out of 17 PCR+-mutants studied, 2 of which had small deletions (5 and 11 b) and 3 others made transitions (A --> G) as shown by the sequencing. Therefore, gamma-rays and neutrons seem to be significant environmental agents increasing the SNP risk for the population through their action on the germ cells. The results obtained are also discussed within the framework of the track structure theory and the notion of quite different chromatin organization in somatic and germ cells.

  7. First report of a clinical isolate of Leclercia adecarboxylata harbouring multiple resistance genes in Uruguay and review of the literature.

    PubMed

    García-Fulgueiras, Virginia; Seija, Verónica; Aguerrebere, Paula; Cordeiro, Nicolás F; Vignoli, Rafael

    2014-06-01

    Here we report the detection of a Leclercia adecarboxylata strain, isolated from a case of osteomyelitis, harbouring multiple antibiotic resistance genes encoded on a 450-kb IncHI1/HI2 conjugative plasmid (pLa12). The plasmid carried a complex class 1 integron with the genetic array intI1-aac(6')-Ib-cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1-ISCR1; in addition, a blaDHA-1-like allele linked to ampR-qacEΔ1-sul1 as well as blaSHV-12, blaTEM-1 and qnrB4-like genes were found. To the best of our knowledge, this is the first report of L. adecarboxylata harbouring transferable resistance genes to quinolones, chloramphenicol and rifampicin as well as a plasmidic class C β-lactamase.

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

    PubMed

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

    2016-04-01

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

  9. The Bradyrhizobium japonicum napEDABC genes are controlled by the FixLJ-FixK(2)-NnrR regulatory cascade.

    PubMed

    Robles, E F; Sánchez, C; Bonnard, N; Delgado, M J; Bedmar, E J

    2006-02-01

    Nitrate respiration by the N(2)-fixing symbiotic bacteria Bradyrhizobium japonicum USDA110 is mediated by a Nap (periplasmic nitrate reductase) encoded by the napEDABC genes. Expression of a transcriptional fusion of the nap promoter region to the reporter gene lacZ, P(napE)-lacZ, was very low in aerobically grown cells of USDA110, but expression was induced approx. 3-fold when the cells were cultured under microaerobic conditions, and 12-fold when nitrate was added to the microaerobic incubation medium. The P(napE)-lacZ fusion was not expressed in the fixL 7403, fixJ 7360 and fixK(2) 9043 mutant strains. Microaerobic induction of the P(napE)-lacZ fusion was retained in the nnrR 8678 mutant, but no increase in beta-galactosidase activity was observed upon nitrate addition. Western-blot and Methyl Viologen-dependent nitrate reductase activity assays showed that synthesis and activity of the catalytic NapA subunit in USDA110 was similar to that in the napC 0906 and nirK GRK308 mutant strains incubated microaerobically with nitrate. These results suggest that nitrate and nitrite, which are not reduced by the napC 0906 and nirK GRK308 mutant cells respectively, induced the synthesis and activity of NapA; conversely, formation of endogenous NO was not required for induction of Nap expression.

  10. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity.

    PubMed

    Legler, Juliette; Zeinstra, Laura M; Schuitemaker, Femke; Lanser, Peter H; Bogerd, Jan; Brouwer, Abraham; Vethaak, A Dick; De Voogt, Pim; Murk, Albertinka J; Van der Burg, Bart

    2002-10-15

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.

  11. LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo

    PubMed Central

    Bozcal, Elif; Dagdeviren, Melih; Uzel, Atac

    2017-01-01

    It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice. PMID:28235077

  12. Gene-based association studies report genetic links for clinical subtypes of frontotemporal dementia.

    PubMed

    Mishra, Aniket; Ferrari, Raffaele; Heutink, Peter; Hardy, John; Pijnenburg, Yolande; Posthuma, Danielle

    2017-04-05

    Genome-wide association studies in frontotemporal dementia showed limited success in identifying associated loci. This is possibly due to small sample size, allelic heterogeneity, small effect sizes of single genetic variants, and the necessity to statistically correct for testing millions of genetic variants. To overcome these issues, we performed gene-based association studies on 3348 clinically identified frontotemporal dementia cases and 9390 controls (discovery, replication and joint-cohort analyses). We report association of APOE and TOMM40 with behavioural variant frontotemporal dementia, and ARHGAP35 and SERPINA1 with progressive non-fluent aphasia. Further, we found the ɛ2 and ɛ4 alleles of APOE harbouring protective and risk increasing effects, respectively, in clinical subtypes of frontotemporal dementia against neurologically normal controls. The APOE-locus association with behavioural variant frontotemporal dementia indicates its potential risk-increasing role across different neurodegenerative diseases, whereas the novel genetic associations of ARHGAP35 and SERPINA1 with progressive non-fluent aphasia point towards a potential role of the stress-signalling pathway in its pathophysiology.

  13. Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs

    PubMed Central

    Petrunia, Igor V.; Frolova, Olga Y.; Komarova, Tatiana V.; Kiselev, Sergey L.; Citovsky, Vitaly; Dorokhov, Yuri L.

    2008-01-01

    Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectible levels of genetic transformation of mouse organs. PMID:18523638

  14. Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay.

    PubMed

    Kojima, Mihoko; Fukunaga, Kenji; Sasaki, Mari; Nakamura, Masafumi; Tsuji, Motohiro; Nishiyama, Toshimasa

    2005-08-01

    The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA). This system utilizes human ovarian carcinoma cells (BG1) stably transfected with an estrogen-responsive luciferase reporter gene plasmid. It was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity. Several pesticides are often present in agricultural products. Therefore the estrogenicity of the mixtures of two kinds of pesticides was evaluated. The activity of diazinon/tolclofos-methyl, pyriproxyfen/prothiofos and TBZ/o-phenylphenol (OPP) was increased up to 1.2-5.3 fold. On the other hand, chlorfluazuron, imazalil and chlorfenapyr had anti-estrogenic activity. Further, to evaluate the change in the estrogenic activity of pesticide metabolites, an experimental system was established using a rat S9 mixture. Metabolites of permethrin and OPP had no estrogenic activity, but they had weak activity after the metabolism. On the other hand, the metabolites of TBZ exhibited less estrogenic activity than the original compounds.

  15. In vitro reporter gene transfection via plasmid DNA delivered by metered dose inhaler.

    PubMed

    Bains, Baljinder K; Birchall, James C; Toon, Richard; Taylor, Glyn

    2010-07-01

    Aerosolised DNA administration could potentially advance the treatment of inheritable lung diseases, lung malignancies and provide genetic immunisation against infection. Jet nebulisation, the current standard for introducing DNA formulations into the lung, is inherently inefficient. Pressurised metered dose inhalers (pMDIs) offer a potentially more efficacious and convenient alternative, especially for repeat administration. We aim to modify a novel low-energy nanotechnology process to prepare surfactant-coated pDNA nanoparticles for pulmonary gene delivery via a pMDI. Water-in-oil microemulsions containing green fluorescent protein reporter plasmid were snap-frozen and lyophilised. Lyophilised pDNA, in some cases following a surfactant wash, was incorporated into pMDIs with hydrofluoroalkane 134a (HFA134a) propellant and ethanol as cosolvent. To assess biological functionality, A549 human lung epithelial cells were exposed to aerosolised pDNA particles in the presence of dioleoyl-trimethylammonium propane (DOTAP). Transfection studies demonstrated that pDNA biological functionality was maintained following aerosolisation. In vitro toxicity assays (MTT) showed no significant cell viability loss following aerosolised pDNA treatment. We have demonstrated that pDNA particles can be incorporated into an HFA134a formulation and aerosolised using a standard valve and actuator. Particles prepared by this novel process have potential for stable and efficient delivery of pDNA to the lower respiratory tract via standard pMDI technology.

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

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Zemp, Roger J.

    2012-02-01

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

  17. FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl-induced expression is dependent on pdr3 transcriptional regulator.

    PubMed

    Brôco, N; Tenreiro, S; Viegas, C A; Sá-Correia, I

    1999-11-01

    In this work we report the disruption of a Saccharomyces cerevisiae ORF YBR008c (FLR1 gene) within the context of EUROFAN (EUROpean Functional Analysis Network) six-pack programme, using a PCR-mediated gene replacement protocol as well as the results of the basic phenotypic analysis of a deletant strain and the construction of a disruption cassette for inactivation of this gene in any yeast strain. We also show results extending the knowledge of the range of compounds to which FLR1 gene confers resistance to the antimitotic systemic benzimidazole fungicide benomyl and the antitumor agent methotrexate, reinforcing the concept that the FLR1 gene is a multidrug resistance (MDR) determinant. Our conclusions were based on the higher susceptibility to these compounds of flr1Delta compared with wild-type and on the increased resistance of both flr1Delta and wild-type strains upon increased expression of FLR1 gene from a centromeric plasmid clone. The present study also provides, for the first time, evidence that the adaptation of yeast cells to growth in the presence of benomyl involves the dramatic activation of FLR1 gene expression during benomyl-induced latency (up to 400-fold). Results obtained using a FLR1-lacZ fusion in a plasmid indicate that the activation of FLR1 expression in benomyl-stressed cells is under the control of the transcriptional regulator Pdr3p. Indeed, PDR3 deletion severely reduces benomyl-induced activation of FLR1 gene expression (by 85%), while the homologous Pdr1p transcription factor is apparently not involved in this activation.

  18. An evolutionary conserved region (ECR) in the human dopamine receptor D4 gene supports reporter gene expression in primary cultures derived from the rat cortex

    PubMed Central

    2011-01-01

    Background Detecting functional variants contributing to diversity of behaviour is crucial for dissecting genetics of complex behaviours. At a molecular level, characterisation of variation in exons has been studied as they are easily identified in the current genome annotation although the functional consequences are less well understood; however, it has been difficult to prioritise regions of non-coding DNA in which genetic variation could also have significant functional consequences. Comparison of multiple vertebrate genomes has allowed the identification of non-coding evolutionary conserved regions (ECRs), in which the degree of conservation can be comparable with exonic regions suggesting functional significance. Results We identified ECRs at the dopamine receptor D4 gene locus, an important gene for human behaviours. The most conserved non-coding ECR (D4ECR1) supported high reporter gene expression in primary cultures derived from neonate rat frontal cortex. Computer aided analysis of the sequence of the D4ECR1 indicated the potential transcription factors that could modulate its function. D4ECR1 contained multiple consensus sequences for binding the transcription factor Sp1, a factor previously implicated in DRD4 expression. Co-transfection experiments demonstrated that overexpression of Sp1 significantly decreased the activity of the D4ECR1 in vitro. Conclusion Bioinformatic analysis complemented by functional analysis of the DRD4 gene locus has identified a) a strong enhancer that functions in neurons and b) a transcription factor that may modulate the function of that enhancer. PMID:21599953

  19. Cold shock genes cspA and cspB from Caulobacter crescentus are posttranscriptionally regulated and important for cold adaptation.

    PubMed

    Mazzon, Ricardo R; Lang, Elza A S; Silva, Carolina A P T; Marques, Marilis V

    2012-12-01

    Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

  20. Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish

    PubMed Central

    Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-ichi; Kawahara, Atsuo

    2016-01-01

    The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes. PMID:27725766

  1. Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

    PubMed

    Gao, Peng; Wang, Yanli; Villanueva, Iván; Ho, Pak Leung; Davies, Julian; Kao, Richard Yi Tsun

    2016-01-01

    As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  2. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

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

  3. Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression

    PubMed Central

    Gao, Peng; Wang, Yanli; Villanueva, Iván; Ho, Pak Leung; Davies, Julian; Kao, Richard Yi Tsun

    2016-01-01

    As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm. PMID:27625639

  4. prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme.

    PubMed

    Lu, M F; Cheng, H T; Kern, M J; Potter, S S; Tran, B; Diekwisch, T G; Martin, J F

    1999-02-01

    The paired-related homeobox gene, prx-1, is expressed in the postmigratory cranial mesenchyme of all facial prominences and is required for the formation of proximal first arch derivatives. We introduced lacZ into the prx-1 locus to study the developmental fate of cells destined to express prx-1 in the prx-1 mutant background. lacZ was normally expressed in prx-1(neo); prx-1(lacZ )mutant craniofacial mesenchyme up until 11.5 d.p.c. At later time points, lacZ expression was lost from structures that are defective in the prx-1(neo) mutant mice. A related gene, prx-2, demonstrated overlapping expression with prx-1. To test the idea that prx-1 and prx-2 perform redundant functions, we generated prx-1(neo;)prx-2 compound mutant mice. Double mutant mice had novel phenotypes in which the rostral aspect of the mandible was defective, the mandibular incisor arrested as a single, bud-stage tooth germ and Meckel's cartilage was absent. Expression of two markers for tooth development, pax9 and patched, were downregulated. Using a transgene that marks a subset of prx-1-expressing cells in the craniofacial mesenchyme, we showed that cells within the hyoid arch take on the properties of the first branchial arch. These data suggest that prx-1 and prx-2 coordinately regulate gene expression in cells that contribute to the distal aspects of the mandibular arch mesenchyme and that prx-1 and prx-2 play a role in the maintenance of cell fate within the craniofacial mesenchyme.

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

    SciTech Connect

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

    2001-04-30

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

  6. Xp11 neoplasm with melanocytic differentiation of the prostate harbouring the novel NONO-TFE3 gene fusion: report of a unique case expanding the gene fusion spectrum.

    PubMed

    Wang, Xiao-Tong; Xia, Qiu-Yuan; Ni, Hao; Wang, Zi-Yu; Ye, Sheng-Bing; Li, Rui; Wang, Xuan; Lv, Jing-Huan; Shi, Shan-Shan; Ma, Heng-Hui; Lu, Zhen-Feng; Shen, Qin; Zhou, Xiao-Jun; Rao, Qiu

    2016-09-01

    Recently, an increasing number of TFE3 rearrangement-associated tumours have been reported, such as TFE3 rearrangement-associated perivascular epithelioid cell tumours (PEComas), melanotic Xp11 translocation renal cancers and melanotic Xp11 neoplasms. We have suggested that these tumours belong to a single clinicopathological spectrum. 'Xp11 neoplasm with melanocytic differentiation' or 'melanotic Xp11 neoplasm' have been proposed to designate this unique neoplasm. Herein, we describe the first case of an Xp11 neoplasm with melanocytic differentiation to be described in the prostate, bearing the novel NONO-TFE3 gene fusion. This study both adds to the spectrum regarding melanotic Xp11 neoplasms and expands its gene fusion spectrum. Moreover, we discuss the relationship of these rare tumours to neoplasms such as conventional PEComas, alveolar soft part sarcomas, malignant melanomas, clear cell sarcomas and Xp11 translocation renal cancers.

  7. Site-Specific Recombination-Based Genetic System for Reporting Transient or Low-Level Gene Expression†

    PubMed Central

    Casavant, N. Carol; Beattie, Gwyn A.; Phillips, Gregory J.; Halverson, Larry J.

    2002-01-01

    We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 μM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment. PMID:12089047

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

    PubMed Central

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

    2013-01-01

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

  9. Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster.

    PubMed Central

    Chary, V K; de la Fuente, J L; Liras, P; Martin, J F

    1997-01-01

    Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase. PMID:9251185

  10. Increased liposome-mediated gene transfer into haematopoietic cells grown in adhesion to stromal or fibroblast cell line monolayers.

    PubMed

    Marit, G; Cao, Y; Froussard, P; Ripoche, J; Dupouy, M; Elandaloussi, A; Lacombe, F; Mahon, F X; Keller, H; Pla, M; Reiffers, J; Theze, J

    2000-01-01

    We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.

  11. Abrupt and dynamic changes in gene expression revealed by live cell arrays.

    PubMed

    Walling, Maureen A; Shi, Hua; Shepard, Jason R E

    2012-03-20

    A description of the noise associated with gene expression is presented, based on a simplified form of the combined multistep processes of transcription and translation. These processes are influenced by numerous factors, including the accessibility of promoter regions to the transcriptional machinery, the kinetics of assembly of the transcription complexes, and the synthesis and degradation of both mRNA and proteins, among others. Ultimately, stochasticity in cellular processes results in variation in protein levels. Here we constructed a rationally designed RNA-based transcriptional activator to reduce these variables and provide a cleaner, more detailed portrayal of cellular noise. Functioning at a level comparable to natural transcription activation, this activator is isolated to a lacZ reporter gene in yeast cells to quantitatively describe the efficiency of the combined processes of transcription and translation. By employing single-cell array techniques to monitor individual cells simultaneously and in real time, a statistical approach to investigate noise inherent in gene expression is possible. Live cell arrays enabled cell populations to be characterized temporally at the individual cell level. The array platform allowed for a relative measure of protein production in real time and could characterize protein bursts with variable size and random timing, such that bursts occurred in a temporally indiscriminate fashion. The inherent variability and randomness of these processes is characterized, with almost half (47%) of cells experiencing bursting behavior at least once over the course of the experiment. We demonstrate that cells identified on the upper periphery of activity exhibit behaviors that are substantially different from the majority of the population, and such variable activities within a population will provide a more accurate characterization of the population.

  12. A novel KRAS gene mutation report in sporadic colorectal cancer, from Northwest of Iran.

    PubMed

    Dolatkhah, Roya; Somi, Mohammad Hossein; Kermani, Iraj Asvadi; Farassati, Faris; Dastgiri, Saeed

    2017-03-01

    While the role of KRAS gene mutations has been widely accepted for predicting responses to anti-EGFR therapy in patients with colorectal cancer, although this study was based on observation of a single case it gives hope that some KRAS gene mutation may have favorable prognosis. More studies are required on patients with similar mutation to validate this finding.

  13. Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

    PubMed

    Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

    2010-07-01

    Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

  14. Long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (AAV) vectors.

    PubMed

    Mitchell, M; Jerebtsova, M; Batshaw, M L; Newman, K; Ye, X

    2000-12-01

    We have developed a micro-injection technique to deliver recombinant adenovirus and AAV to mouse fetuses at day 15 after conception. Several routes of delivery, including injections to the amniotic fluid, the front limb, the placenta, the liver, and the retro-orbital venus plexus, were tested using an E1-deleted recombinant adenovirus (Ad.CBlacZ) or a recombinant adeno-associated virus (AAV.CMVlacZ) carrying a beta-galactosidase (lacZ) gene. Injection of Ad.CBlacZ into the amniotic cavity led to transgene expression in the skin and in the digestive tract of the fetuses. Injection of Ad.CBlacZ in the front limb resulted in LacZ expression in all major muscle groups around the injection site and at low levels in the liver. The other three routes of delivery, ie intra-placental, intra-hepatic and retro-orbital injections of Ad.CBlacZ, all led to lacZ expression predominantly in the liver. Further studies revealed a maximal tolerant dose (defined as the highest viral dose with < or =20% mortality in the injected fetuses) of 1 x 10(9) particles per fetus for intra- hepatic injections, 3 x 10(9) particles per fetus for intra-placental injection, 1 x 1010 particles per fetus for retro-orbital and intra-amniotic injections, and 2 x 10(10) particle per fetus for intra-muscular injection. The adenovirus-mediated lacZ expression in liver and muscle persisted for at least 6 weeks. Intra-muscular injection of AAV.CMVlacZ also resulted in lacZ expression in the muscle up to 3 months after birth with no indication of cellular immune response at the injection site. Taken together, our results demonstrated that prolonged transgene expression can be achieved by in utero gene transfer using either adenoviral or AAV vectors. The distribution of virus-mediated gene transfer appeared to determined mostly by the route of viral administration.

  15. Homozygous G320V mutation in the HJV gene causing juvenile hereditary haemochromatosis type A. A case report.

    PubMed

    Militaru, Mariela S; Popp, Radu A; Trifa, Adrian P

    2010-06-01

    While classical hereditary haemochromatosis, usually associated with mutations in the HFE gene, has an adult age onset and a long, progressive evolution, juvenile haemochromatosis, most often associated with mutations in the HJV gene, is a more severe, rapidly progressive condition and has an onset before the age of 30. We report a 26-year old woman with a severe iron overload, affected by hypogonadotropic hypogonadism and moderate dilative cardiomyopathy, in whom the molecular analysis revealed a homozygous genotype for G320V mutation in the HJV gene. As juvenile haemochromatosis is a severe disease, death usually occurring from cardiac involvement, an efficient iron removal from the body strategy should be started as soon as possible, in order to prevent irreversible damage.

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

    PubMed

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

    2001-07-01

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

  17. Identification of Vibrio natriegens uvrA and uvrB genes and analysis of gene regulation using transcriptional reporter plasmids.

    PubMed

    Simons, Keryn L; Thomas, Susan M; Anderson, Peter A

    2010-10-01

    Nucleotide excision repair (NER) rectifies a variety of chemically and structurally distinct DNA lesions. The current model of NER is based upon the enteric bacterium Escherichia coli and there is scarce information about how other bacterial species respond to, and correct, DNA damage. Here we report the isolation and functional analysis of the uvrA and uvrB genes from Vibrio natriegens, a naturally occurring marine bacterium. Genetic studies were completed to assess the repair capabilities of V. natriegens uvrA and uvrB in E. coli uvrA and uvrB mutants. In addition to the genetic studies, transcriptional fusions between the luciferase gene and the 5' regulatory regions of uvrA and uvrB gene of V. natriegens and E. coli were constructed. Luminescent measurements from E. coli transformed with these constructs showed that whilst the response to UV irradiation of either E. coli or V. natriegens uvrA regulatory sequences was similar, both the rate and induction of luminescence detected from the uvrB regulatory regions differed.

  18. Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

    PubMed

    Weiser, J; Buriánková, K; Kalachová, L; Branny, P; Pernodet, J L

    2006-01-01

    A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

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

    PubMed Central

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

    2015-01-01

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

  20. On the Use of Gene Ontology Annotations to Assess Functional Similarity among Orthologs and Paralogs: A Short Report.

    PubMed

    Thomas, Paul D; Wood, Valerie; Mungall, Christopher J; Lewis, Suzanna E; Blake, Judith A

    2012-01-01

    A recent paper (Nehrt et al., PLoS Comput. Biol. 7:e1002073, 2011) has proposed a metric for the "functional similarity" between two genes that uses only the Gene Ontology (GO) annotations directly derived from published experimental results. Applying this metric, the authors concluded that paralogous genes within the mouse genome or the human genome are more functionally similar on average than orthologous genes between these genomes, an unexpected result with broad implications if true. We suggest, based on both theoretical and empirical considerations, that this proposed metric should not be interpreted as a functional similarity, and therefore cannot be used to support any conclusions about the "ortholog conjecture" (or, more properly, the "ortholog functional conservation hypothesis"). First, we reexamine the case studies presented by Nehrt et al. as examples of orthologs with divergent functions, and come to a very different conclusion: they actually exemplify how GO annotations for orthologous genes provide complementary information about conserved biological functions. We then show that there is a global ascertainment bias in the experiment-based GO annotations for human and mouse genes: particular types of experiments tend to be performed in different model organisms. We conclude that the reported statistical differences in annotations between pairs of orthologous genes do not reflect differences in biological function, but rather complementarity in experimental approaches. Our results underscore two general considerations for researchers proposing novel types of analysis based on the GO: 1) that GO annotations are often incomplete, potentially in a biased manner, and subject to an "open world assumption" (absence of an annotation does not imply absence of a function), and 2) that conclusions drawn from a novel, large-scale GO analysis should whenever possible be supported by careful, in-depth examination of examples, to help ensure the conclusions have a

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

    PubMed

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

    2016-02-10

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

  2. Transposon tagging of disease resistance genes. Final report, May 1, 1988--April 30, 1993

    SciTech Connect

    Michelmore, R.

    1994-09-01

    The goal of this project was to develop a transposon mutagenesis system for lettuce and to clone and characterize disease resistance genes by transposon tagging. The majority of studies were conducted with the Ac/Ds System. Researchers made and tested several constructs as well as utilized constructions shown to be functional in other plant species. Researchers demonstrated movement of Ac and DS in lettuce; however, they transposed at much lower frequencies in lettuce than in other plant species. Therefore, further manipulation of the system, particularly for flower specific expression of transposase, is required before a routine transposon system is available for lettuce. Populations of lettuce were generated and screened to test for the stability of resistance genes and several spontaneous mutations were isolated. Researchers also identified a resistance gene mutant in plants transformed with a Ds element and chimeric transposase gene. This is currently being characterized in detail.

  3. The regulatory factor PcRFX1 controls the expression of the three genes of β-lactam biosynthesis in Penicillium chrysogenum.

    PubMed

    Domínguez-Santos, Rebeca; Martín, Juan-Francisco; Kosalková, Katarina; Prieto, Carlos; Ullán, Ricardo V; García-Estrada, Carlos

    2012-11-01

    Penicillin biosynthesis is subjected to a complex regulatory network of signalling molecules that may serve as model for other secondary metabolites. The information provided by the new "omics" era about Penicillium chrysogenum and the advances in the knowledge of molecular mechanisms responsible for improved productivity, make this fungus an excellent model to decipher the mechanisms controlling the penicillin biosynthetic pathway. In this work, we have characterized a novel transcription factor PcRFX1, which is an ortholog of the Acremonium chrysogenum CPCR1 and Penicillium marneffei RfxA regulatory proteins. PcRFX1 DNA binding sequences were found in the promoter region of the pcbAB, pcbC and penDE genes. We show in this article that these motifs control the expression of the β-galactosidase lacZ reporter gene, indicating that they may direct the PcRFX1-mediated regulation of the penicillin biosynthetic genes. By means of Pcrfx1 gene knock-down and overexpression techniques we confirmed that PcRFX1 controls penicillin biosynthesis through the regulation of the pcbAB, pcbC and penDE transcription. Morphology and development seemed not to be controlled by this transcription factor under the conditions studied and only sporulation was slightly reduced after the silencing of the Pcrfx1 gene. A genome-wide analysis of processes putatively regulated by this transcription factor was carried out in P. chrysogenum. Results suggested that PcRFX1, in addition to regulate penicillin biosynthesis, is also involved in the control of several pathways of primary metabolism.

  4. [Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

    SciTech Connect

    Okita, T.W.

    1999-04-01

    Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.

  5. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the {beta}-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate {beta}-carboxy-cis,cis-muconate. {beta}-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for {beta}-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to {beta}-carboxy-cis,cis-muconate.

  6. Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

    PubMed

    Steinberg, Pablo; Behnisch, Peter A; Besselink, Harrie; Brouwer, Abraham A

    2016-12-10

    Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

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

    SciTech Connect

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

    1989-07-01

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

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

    PubMed

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

    2007-05-01

    Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

  9. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    SciTech Connect

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  10. Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992

    SciTech Connect

    Michelmore, R.

    1994-06-01

    Our goal is to clone genes in lettuce determining resistance to downy mildew. One approach involves the mobilization of transposons into resistance genes to mutate and tag the target gene. Because transposons have yet to be isolated and characterized from lettuce, the majority of our experiments have involved Ac from corn as this is increasingly the best characterized transposon. Over the past several years, various labs have contributed to a detailed understanding of the biology of Ac in corn and heterologous plant species. We have collaborated closely with several of these labs, exchanged materials and incorporated their advances into our analysis of transposition in lettuce. The original proposal described the development of a transposon mutagenesis system for lettuce and its subsequent use to tag disease resistance genes. The development phase involved characterization and manipulation of Ac transposition, identification of suitable whole plant selectable markers for the construction of chimeric non-autonomous elements, and investigation of the stability of resistance genes. Investigation of Ac transposition in lettuce has received the majority of our attention. Initially, we made a simple construct with wildtype Ac and introduced it into lettuce. No transposition was observed; although other labs demonstrated that the same construct was functional in tomato. We then focused on assaying for Ac transposition with constructs of increasing sophistication that had been demonstrated by others to be functional in other species. The latest constructs for transposon mutagenesis clearly demonstrated transposition in lettuce. This allowed us to generate seed stocks that we will start to screen for insertional inactivation of resistance genes this year.

  11. Inference of quantitative models of bacterial promoters from time-series reporter gene data.

    PubMed

    Stefan, Diana; Pinel, Corinne; Pinhal, Stéphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for

  12. Inference of Quantitative Models of Bacterial Promoters from Time-Series Reporter Gene Data

    PubMed Central

    Stefan, Diana; Pinel, Corinne; Pinhal, Stéphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for

  13. First Report of the Multidrug Resistance Gene cfr in Enterococcus faecalis of Animal Origin

    PubMed Central

    Liu, Yang; Wang, Yang; Wu, Congming; Shen, Zhangqi; Schwarz, Stefan; Du, Xiang-Dang; Dai, Lei; Zhang, Wanjiang

    2012-01-01

    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. PMID:22203597

  14. Molecular characterization of a maize regulatory gene. Annual progress report, November 1991--October 1992

    SciTech Connect

    Wessler, S.R.

    1994-05-01

    All aspects of this year`s work have converged on the central theme of post-transcriptional control of R gene expression. Unlike transcriptional control, relatively little is known about post-transcriptional regulation, especially in plants. We believe that three levels of post-transcriptional regulation have been identified: control of translation initiation as evidenced by the maize Lc gene; control of nuclear localization as evidenced by the Ds allele r-m9 of maize; and control of nuclear localization through alternative splicing of the rice R homolog.

  15. A Bacterial Artificial Chromosome Reporter System for Expression of the Human FOXP3 Gene in Mouse Regulatory T-Cells

    PubMed Central

    Tsuda, Masato; Tone, Yukiko; Ogawa, Chihiro; Nagaoka, Yoshiko; Katsumata, Makoto; Necula, Andra; Howie, Duncan; Masuda, Esteban; Waldmann, Herman; Tone, Masahide

    2017-01-01

    The transcription factor FOXP3 plays key roles in the development and function of regulatory T cells (Treg) capable of preventing and correcting immunopathology. There has been much interest in exploiting Treg as adoptive cell therapy in man, but issues of lack of nominal antigen-specificity and stability of FoxP3 expression in the face of pro-inflammatory cytokines have been a concern. In order to enable fundamental studies of human FOXP3 (hFOXP3) gene regulation and to provide preclinical tools to guide the selection of drugs that might modulate hFOXP3 expression for therapeutic purposes, we generated hFOXP3/AmCyan bacterial artificial chromosome (BAC) transgenic mice and transfectants, wherein hFOXP3 expression was read out as AmCyan expression. Using the transgenic mice, one can now investigate hFOXP3 gene expression under defined experimental conditions used for mouse Foxp3 (mFoxp3) studies. Here, we demonstrate that hFOXP3 gene expression in BAC transgenic mice is solely restricted to CD4+ T-cells, as for mFoxp3 gene expression, showing that hFOXP3 expression in Treg cells depends on fundamentally similar processes to mFoxp3 expression in these cells. Similarly, hFOXP3 expression could be observed in mouse T-cells through TCR stimulation in the presence of TGF-β. These data suggest that, at least in part, cell type-specific human and mouse foxp3 gene expression is regulated by common regulatory regions which for the human, are located within the 110-kb human FOXP3 BAC DNA. To investigate hFOXP3 gene expression further and to screen potential therapeutics in modulating hFOXP3 gene expression in vitro, we also generated hFOXP3/AmCyan expression reporter cell lines. Using the reporter cells and transcription factor inhibitors, we showed that, just as for mFoxp3 expression, inhibitors of NF-κB, AP1, STAT5, Smad3, and NFAT also block hFOXP3 expression. hFOXP3 induction in the reporter cells was also TGF-β dependent, and substantially enhanced by an m

  16. Evidence of Associations between Cytokine Genes and Subjective Reports of Sleep Disturbance in Oncology Patients and Their Family Caregivers

    PubMed Central

    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.

    2012-01-01

    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. PMID:22844404

  17. The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

    PubMed Central

    Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

    2017-01-01

    Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

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

    PubMed

    Bogani, Debora; Morgan, Marc A J; Nelson, Andrew C; Costello, Ita; McGouran, Joanna F; Kessler, Benedikt M; Robertson, Elizabeth J; Bikoff, Elizabeth K

    2013-10-01

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

  19. A kidney injury molecule‐1 (Kim‐1) gene reporter in a mouse artificial chromosome: the responsiveness to cisplatin toxicity in immortalized mouse kidney S3 cells

    PubMed Central

    Kokura, Kenji; Kuromi, Yasushi; Endo, Takeshi; Anzai, Naohiko; Kazuki, Yasuhiro; Oshimura, Mitsuo

    2016-01-01

    Abstract Background Kidney injury molecule‐1 (Kim‐1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim‐1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim‐1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells, such as human kidney‐2 cells and immortalized proximal tubular epithelial cells. Methods We measured the augmentation of Kim‐1 mRNA expression after the addition of cisplatin using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature‐sensitive large T antigen from Simian virus 40. Results A mouse Kim‐1 gene luciferase reporter in conjunction with an Hprt gene reporter detected cisplatin‐induced nephrotoxicity in S3 cells. These two reporter genes were contained in a mouse artificial chromosome, and two luciferases that emitted different wavelengths were used to monitor the respective gene expression. However, the Kim‐1 reporter gene failed to respond to cisplatin in A9 fibroblast cells that contained the same reporter mouse artificial chromosome, suggesting cell type‐specificity for activation of the reporter. Conclusions We report the feasibility of measuring in vitro cisplatin nephrotoxicity using a Kim‐1 reporter gene in S3 cells. PMID:27591740

  20. Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome

    PubMed Central

    Wang, Shuwen; Zhao, Yuanjun; Leiby, Melanie A.; Zhu, Jiyue

    2009-01-01

    The endogenous human telomerase reverse transcriptase (hTERT) gene is repressed in somatic cells. To study the mechanisms of its repression, we developed a strategy of retrovirus-directed Cre recombinase-mediated BAC targeting, or RMBT, to generate single-copy integrations of BAC at pre-engineered chromosomal sites. This technique involved retroviral transduction of acceptor loci, containing an HSV thymidine kinase marker, and subsequent integration of BAC constructs into the acceptor sites, utilizing the loxP and lox511 sites present in the vector backbones. The BAC reporter, with a Renilla luciferase cassette inserted downstream of the hTERT promoter, was retrofitted with a puromycin marker. Through puromycin selection and ganciclovir counter-selection, a targeting efficiency of over 50% was achieved. We demonstrated that the activity and chromatin structures of the hTERT promoter in chromosomally integrated BAC reporter recapitulated its endogenous counterpart of the host cells. Therefore, we have established a genetically amendable platform to study chromatin and epigenetic regulation of the hTERT gene. The highly efficient and versatile RMBT technique has general applicability for studying largely unexplored chromatin-dependent mechanisms of promoter regulation of various genes. PMID:19528078

  1. First report of a pathogenic mutation on exon 24 of the NOTCH3 gene in a CADASIL family.

    PubMed

    Valenti, Raffaella; Bianchi, Silvia; Pescini, Francesca; D'Eramo, Camilla; Inzitari, Domenico; Dotti, Maria Teresa; Pantoni, Leonardo

    2011-09-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetically transmitted small vessel disease clinically characterized by migraine, recurrent subcortical strokes, and cognitive and mood disorders. Pathogenic mutations are located on any of the exons of the NOTCH3 gene coding for epidermal-growth factor (EGF)-like repeats of the extracellular domain of the NOTCH3 receptor. Because the gene is large and the mutations cluster on some exons, many laboratories restrict the analysis to these exons. We report the first missense mutation involving exon 24 and causing CADASIL in a 64-year-old man. The patient was admitted to the hospital for a loss of consciousness accompanied by profuse sweating. On examination, some parkinsonian features were present. Over the last 4 years, he had developed postural instability and gait disturbances with repeated falls, behavioral disorders, and cognitive impairment. A diagnostic hypothesis of atypical parkinsonism had been advanced. The presence of multiple subcortical lacunar infarcts and leukoencephalopathy extended to the external capsule on cerebral MRI suggested the presence of CADASIL. The diagnosis was confirmed by finding a heterozygous mutation leading to a cysteine substitution on exon 24 of the NOTCH3 gene. One proband's brother, who had progressive gait disturbances, unilateral action tremor and bradykinesia, and an asymptomatic niece also resulted affected. This report underlines that when CADASIL is suspected the genetic analysis should be performed on all the NOTCH3 exons coding for EGF-like repeats including exon 24 and confirms that CADASIL may have heterogeneous phenotypes.

  2. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Singh, Monika

    2009-06-24

    The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.

  3. Evaluating the effectiveness of transferrin receptor-1 (TfR1) as a magnetic resonance reporter gene.

    PubMed

    Pereira, Sofia M; Herrmann, Anne; Moss, Diana; Poptani, Harish; Williams, Steve R; Murray, Patricia; Taylor, Arthur

    2016-05-01

    Magnetic resonance (MR) reporter genes have the potential for tracking the biodistribution and fate of cells in vivo, thus allowing the safety, efficacy and mechanisms of action of cell-based therapies to be comprehensively assessed. In this study, we evaluate the effectiveness of the iron importer transferrin receptor-1 (TfR1) as an MR reporter gene in the model cell line CHO-K1. Overexpression of the TfR1 transgene led to a reduction in the levels of endogenous TfR1 mRNA, but to a 60-fold increase in total TfR1 protein levels. Although the mRNA levels of ferritin heavy chain-1 (Fth1) did not change, Fth1 protein levels increased 13-fold. The concentration of intracellular iron increased significantly, even when cells were cultured in medium that was not supplemented with iron and the amount of iron in the extracellular environment was thus at physiological levels. However, we found that, by supplementing the cell culture medium with ferric citrate, a comparable degree of iron uptake and MR contrast could be achieved in control cells that did not express the TfR1 transgene. Sufficient MR contrast to enable the cells to be detected in vivo following their administration into the midbrain of chick embryos was obtained irrespective of the reporter gene. We conclude that TfR1 is not an effective reporter and that, to track the biodistribution of cells with MR imaging in the short term, it is sufficient to simply culture cells in the presence of ferric citrate. Copyright © 2016 The Authors Contrast Media & Molecular Imaging Published by John Wiley & Sons Ltd.

  4. Transformation of somatic embryos of Prunus incisa ‘February Pink’ with a visible reporter gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An Agrobacterium-mediated transformation system was developed for the ornamental cherry species Prunus incisa. This system uses both an antibiotic resistance gene (NPTII) and a visible selectable marker, the green fluorescent protein (GFP), to select plants. Cells from leaf and root explants were tr...

  5. Phylogenetic analysis of the L and HN gene of ophidian paramyxoviruses. Brief report.

    PubMed

    Kindermann, J; Kübber-Heiss, A; Kerschbaumer, P; Nowotny, N

    2001-01-01

    Two reptilian paramyxoviruses, isolated from a neotropical rattlesnake (neotropical virus, NTV, ATCC VR-1408) and a bush viper (bush viper virus, BVV, ATCC VR- 1409), respectively, were analysed to determine their taxonomic position among other reptilian paramyxoviruses investigated previously by Ahne et al.. A 679 bp long region of the hemagglutinin-neuraminidase (HN) gene and a 627 bp long region of the large (L) gene were reverse transcribed, amplified by polymerase chain reaction (PCR), and sequenced. The deduced amino acid sequences were compared to mammalian paramyxoviruses belonging to the genera Respirovirus and Rubulavirus. The deduced amino acid sequences revealed 58.9 to 62% homology for the partial L protein and 41% to 47.1% homology for the partial HN protein. For phylogenetic analyses, a 518 bp L gene and a 352 bp HN gene fragment were used, both generating similar trees consisting of two distinct main groups, and some intermediate isolates. BVV clustered within group "b" while NTV clustered together with the intermediate ophidian paramyxovirus isolate Crot2-OH90.

  6. Characterization of embryo-specific genes. Final report, April 1, 1987--March 31, 1992

    SciTech Connect

    Sung, R.

    1992-06-12

    The objective of the proposed research is to characterize the function and regulation of a set of embryonic genes which are expressed in the embryos, not in the plants. 22 cDNA clones were isolated from a cDNA library we constructed using mRNAS of -carrot somatic embryos. These cDNA clones identified mRNA species that are present in the somatic and zygotic embryos, but not in adult plants. The sequence of all 22cDNA clones were determined; genomic clones for three cDNA clones, DC8, DC59, and DC49 were isolated and gene sequences determined. DC8, DC49, and several other genes identified by the cDNA sequences belong to the category of late embryogenesis abundant protein genes, Lea. The function of these gens have not yet been determined, but they share common structural features, are regulated by ABA and are speculated to play a role in seed desiccation.

  7. Traits, Genes, Particles and Information: Re-Visiting Students' Understandings of Genetics. Research Report

    ERIC Educational Resources Information Center

    Lewis, Jenny; Kattmann, Ulrich

    2004-01-01

    Findings from a study of 10 German students aged 15-19, using problem-centred interviews, suggest that many students hold an 'everyday' conception of genes as small, trait-bearing, particles. Analysis of this notion identified a number of ways in which such a view might restrict the ability of students to develop an understanding of the scientific…

  8. DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17.

    PubMed Central

    Genilloud, O; Moreno, F; Kolter, R

    1989-01-01

    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 PMID:2644225

  9. Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway

    PubMed Central

    Sun, Yueming K.; Flematti, Gavin R.; Smith, Steven M.; Waters, Mark T.

    2016-01-01

    Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2). We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway. PMID:27994609

  10. In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

    PubMed Central

    Feeney, Kevin A.; Putker, Marrit; Brancaccio, Marco; O’Neill, John S.

    2016-01-01

    Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type. PMID:28112045

  11. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    SciTech Connect

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  12. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-07-16

    We have found that soybean cotyledons could be cultured in vitro and that the storage proteins were formed essentially as on a plant. When methionine was added to the medium, the cotyledons grew faster, and the methionine content of the protein fraction was increased by over 20 percent. The high methionine content of the protein fraction was found to be due to a shift in the relative amounts of the two major storage proteins. The later effect was the result of methionine treatment suppressing the expression of one storage protein subunit gene. The goal was to determine the mechanism by which methionine is able to regulate the expression of the ..beta..-subunit gene.

  13. Unsuccessful switch from insulin to sulfonylurea therapy in permanent neonatal diabetes mellitus due to an R201H mutation in the KCNJ11 gene: a case report.

    PubMed

    Heo, Jeong Won; Kim, Sang-Wook; Cho, Eun-Hee

    2013-04-01

    Mutations in KCNJ11 are a common cause of permanent neonatal diabetes mellitus. Previously, all patients carrying an R201H mutation in the KCNJ11 gene showed successful switches from insulin to sulfonylurea. Here, we report an unsuccessful switch in an 18-year-old patient carrying the common R201H mutation in the KCNJ11 gene.

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  15. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  16. (Structure and expression of nuclear genes encoding rubisco activase): Progress report

    SciTech Connect

    Not Available

    1989-01-01

    Our first year's activities include: (1) completing a survey of the basic characteristics of activase gene expression in barley; and (2) isolating and structurally characterizing cDNA and genomic DNA sequences encoding activase from barley. Our goal was to determine whether activase mRNA and protein accumulation are coordinated with those of the rubisco subunits. We utilized the first leaves of barley as an experimental system for these studies because they can be used in two ways to study the expression of leaf genes: by following the naturally occurring differentiation of leaf cells, which occurs acropetally along the barley leaf; and by following the photomorphogenesis of etiolated barley seedlings. In the acropetal gradient of leaf cell differentiation, activase mRNA and mRNA and polypeptide expression is tightly coordinated with rubisco subunit mRNA and polypeptide expression. Although we have not measured their precise stoichiometry at each stage of leaf differentiation, activase protein is expressed at the level of about one polypeptide per rubisco holoenzyme in mature regions of the leaf. Coordination of the expression of activase mRNAs and polypeptides indicates that in the barley leaf gradient, activase gene expression is largely controlled at the level of transcription. However, translational controls may play a role in regulating activase expression on a short term basis.

  17. [Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report

    SciTech Connect

    Guerinot, M.L.

    1992-06-01

    We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

  18. Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

    PubMed Central

    Sohaskey, C D; Arnold, C; Barbour, A G

    1997-01-01

    A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system. PMID:9352937

  19. Cerebral arteriopathy associated with heterozygous Arg179Cys mutation in the ACTA2 gene: Report in 2 newborn siblings.

    PubMed

    de Grazia, Jose; Delgado, Ignacio; Sanchez-Montanez, Angel; Boronat, Susana; Del Campo, Miguel; Vazquez, Elida

    2017-01-01

    Mutations in the ACTA2 gene lead to a multisystemic smooth muscle dysfunction syndrome that causes vascular disease, congenital mydriasis, and variable presentation of urinary and gastrointestinal problems. The heterozygous Arg179 mutation is associated with a distinctive cerebrovascular phenotype. We report the cases of two newborn siblings with heterozygous ACTA2 Arg179Cys substitution and provide neuroimaging exams that demonstrate the distinctive cerebrovascular phenotype, also associated with variable degree of hypoplasia of the vertebro-basilar circulation as well as hypoxic-ischemic lesions.

  20. Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis.

    PubMed

    Ihara, Masaru; Kitamura, Tomokazu; Kumar, Vimal; Park, Chang-Beom; Ihara, Mariko O; Lee, Sang-Jung; Yamashita, Naoyuki; Miyagawa, Shinichi; Iguchi, Taisen; Okamoto, Seiichiro; Suzuki, Yutaka; Tanaka, Hiroaki

    2015-05-19

    The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ERα reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the following: estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ERα reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17β-estradiol, estriol, and 17α-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ERα reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ERα reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ERα reporter gene assay.

  1. Gene-enzyme telationships in somatic cells and their organismal derivatives in higher plants. Progress report

    SciTech Connect

    Jensen, R. A.

    1980-04-21

    Progress is reported in the following subject areas: (1) chemistry of the arogenate molecule; (2) plant enzymology at the organismal level; (3) isolation of regulatory mutants in tobacco; and (4) stability of the haploid state in Nicotiana sylvestris.

  2. Transcription factors GATA/ELT-2 and forkhead/HNF-3/PHA-4 regulate the tropomyosin gene expression in the pharynx and intestine of Caenorhabditis elegans.

    PubMed

    Anokye-Danso, Frederick; Anyanful, Akwasi; Sakube, Yasuji; Kagawa, Hiroaki

    2008-05-30

    Gene regulation during development is an important biological activity that leads to synthesis of biomolecules at specific locations and specific times. The single tropomyosin gene of Caenorhabditis elegans, tmy-1/lev-11, produces four isoforms of protein: two from the external promoter and two from the internal promoter. We investigated the internal promoter of tropomyosin to identify sequences that regulate expression of tmy-1 in the pharynx and intestine. By promoter deletion of tmy-1 reporters as well as by database analyses, a 100-bp fragment that contained binding sequences for a GATA factor, for a chicken CdxA homolog, and for a forkhead factor was identified. Both the forkhead and CdxA binding sequences contributed to pharyngeal and intestinal expression. In addition, the GATA site also influenced intestinal expression of tmy-1 reporter. We showed that ELT-2 and PHA-4 proteins interact directly with the GATA and forkhead binding sequences, respectively, in gel mobility shift assays. RNA interference knockdown of elt-2 diminished tmy-1::gfp expression in the intestine. In contrast to RNA interference knockdown of pha-4, expression of tmy-1::gfp in pha-4;smg-1 mutants was slightly weaker than that of the wild type. Ectopic expression of PHA-4 and ELT-2 by heat shock was sufficient to elicit widespread expression of tmy-1::lacZ reporter in embryos. We found no indication of a synergistic relation between ELT-2 and PHA-4. Based on our data, PHA-4 and CdxA function as general transcription factors for pharyngeal and intestinal regulation of tmy-1. We present models by which ELT-2, PHA-4, and CdxA orchestrate expression from the internal promoter of tmy-1.

  3. A Novel Homozygous Frameshift Mutation in Exon 2 of LEP Gene Associated with Severe Obesity: A Case Report

    PubMed Central

    Altawil, Ashwaq Shukri; Mawlawi, Horia Ahmad; Alghamdi, Khalid Ateeq; Almijmaj, Faten Fohaid

    2016-01-01

    BACKGROUND Monogenic obesity is a rare type of obesity caused by a mutation in a single gene. Patients with monogenic obesity may develop early onset of obesity and severe metabolic abnormalities. CASE PRESENTATION A two-and-half-year-old girl was presented to our clinic because of excessive weight gain and hyperphagia. She was born at full term, by normal vaginal delivery with birth weight of 2.82 kg and no complications during pregnancy. The patient was the second child of two healthy, non-obese Saudis with known consanguinity. She gained weight rapidly leading to obesity at the age of three months. METHODS The demographic data and clinical features were recorded. Blood samples were collected and tested for endocrine and metabolic characteristics and genetic studies. Mutations of the LEP gene were screened. The coding exons 2 and 3 and the corresponding exon–intron boundaries were amplified by polymerase chain reaction using specific primers, analyzed by direct sequencing using an ABI sequencer 3500 xL GA (Applied Biosystems), and evaluated using the JSI SeqPilot software. The resulting sequence data were compared with the reference MM_0002302. CONCLUSION We report a novel homozygous frameshift mutation c.144delin TAC (G1n49Thrfs*23) in exon 2 of the LEP gene associated with extreme obesity. PMID:27980447

  4. Risk assessment of gene flow from genetically engineered virus resistant cassava to wild relatives in Africa: an expert panel report.

    PubMed

    Hokanson, Karen E; Ellstrand, Norman C; Dixon, Alfred G O; Kulembeka, Heneriko P; Olsen, Kenneth M; Raybould, Alan

    2016-02-01

    The probability and consequences of gene flow to wild relatives is typically considered in the environmental risk assessment of genetically engineered crops. This is a report from a discussion by a group of experts who used a problem formulation approach to consider existing information for risk assessment of gene flow from cassava (Manihot esculenta) genetically engineered for virus resistance to the 'wild' (naturalized) relative M. glaziovii in East Africa. Two environmental harms were considered in this case: (1) loss of genetic diversity in the germplasm pool, and (2) loss of valued species, ecosystem resources, or crop yield and quality due to weediness or invasiveness of wild relatives. Based on existing information, it was concluded that gene flow will occur, but it is not likely that this will reduce the genetic diversity in the germplasm pool. There is little existing information about the impact of the virus in natural populations that could be used to inform a prediction about whether virus resistance would lead to an increase in reproduction or survival, hence abundance of M. glaziovii. However, an increase in the abundance of M. glaziovii should be manageable, and would not necessarily lead to the identified environmental harms.

  5. A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2: A case report

    PubMed Central

    SHI, YUNFANG; LI, XIAOZHOU; JU, DUAN; LI, YAN; ZHANG, XIULING; ZHANG, YING

    2016-01-01

    Waardenburg syndrome (WS) is an autosomal dominant disorder with varying degrees of sensorineural hearing loss, and accumulation of pigmentation in hair, skin and iris. There are four types of WS (WS1–4) with differing characteristics. Mutations in six genes [paired box gene 3 (PAX3), microphthalmia-associated transcription factor (MITF), endothelin 3 (END3), endothelin receptor type B (EDNRB), SRY (sex determining region Y)-box 10 (SOX10) and snail homolog 2 (SNAI2)] have been identified to be associated with the various types. This case report describes the investigation of genetic mutations in three patients with WS2 from a single family. Genomic DNA was extracted, and the six WS-related genes were sequenced using next-generation sequencing technology. In addition to mutations in PAX3, EDNRB and SOX10, a novel heterozygous MITF mutation, p.Δ315Arg (c.944_946delGAA) on exon 8 was identified. This is predicted to be a candidate disease-causing mutation that may affect the structure and function of the enzyme. PMID:27073475

  6. Q289P mutation in the FGFR2 gene: first report in a patient with type 1 Pfeiffer syndrome.

    PubMed

    Piccione, Maria; Antona, Vincenzo; Niceta, Marcello; Fabiano, Carmelo; Martines, Manuela; Bianchi, Alberto; Corsello, Giovanni

    2009-09-01

    When normal development and growth of the calvarial sutures is disrupted, craniosynostosis (premature calvarial suture fusion) may result. Classical craniosynostosis syndromes are autosomal dominant traits and include Apert, Pfeiffer, Crouzon, Jackson-Weiss, and Saethre-Chotzen syndromes. In these conditions, there is premature fusion of skull bones leading to an abnormal head shape, ocular hypertelorism with proptosis, and midface hypoplasia. It is known that mutations in the fibroblast growth factor receptors 1, 2, and 3 cause craniosynostosis. We report on a child with a clinically diagnosed Pfeiffer syndrome that shows the missense point mutation Q289P in exon 8 of the FGFR2 gene. This is a mutation not previously described in the Pfeiffer syndrome but reported in the Crouzon, Jackson-Weiss, and Saethre-Chotzen syndromes. In this paper, we propose the concept that these disorders may represent one genetic condition with phenotypic variability.

  7. Molecular characterization of a maize regulatory gene. Annual progress report, March 1990--November 1991

    SciTech Connect

    Wessler, S.R.

    1991-12-01

    Based on initial bombardment studies we have previously concluded that promoter diversity was responsible for the diversity of naturally occurring R alleles. During this period we have found that R is controlled at the level of translation initiation and intron 1 is alternatively spliced. The experiments described in Sections 1 and 2 sought to quantify these effects and to determine whether they contribute to the tissue specific expression of select R alleles. This study was done because very little is understood about the post-transcriptional regulation of plant genes. Section 3 and 4 describe experiments designed to identify important structural components of the R protein.

  8. Pheromone-regulated genes required for yeast mating differentiation.

    PubMed

    Erdman, S; Lin, L; Malczynski, M; Snyder, M

    1998-02-09

    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 beta-galactosidase (beta-gal) expression in the presence and absence of alpha 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:: beta-gal and Fig2::beta-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.

  9. The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.

    PubMed

    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

    2010-02-01

    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.

  10. The sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter confers high-level expression of the GUS reporter gene in the potato tuber.

    PubMed

    Kim, Tae-Won; Goo, Young-Min; Lee, Cheol-Ho; Lee, Byung-Hyun; Bae, Jung-Myung; Lee, Shin-Woo

    2009-10-01

    Molecular farming refers to the process of creating bioengineered plants with the capability of producing potentially valuable products, such as drugs, vaccines, and chemicals. We have investigated the potential of the sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter and its transit peptide (TP) as an expression system for the mass production of foreign proteins in potato. The ibAGP1 promoter and its TP sequence were transformed into potato along with beta-glucuronidase (GUS) as a reporter gene, and GUS activity was subsequently analyzed in the transgenic potato plants. In tuber tissues, GUS activity in transgenic plants carrying only the ibAGP1 promoter (ibAGP1::GUS) increased up to 15.6-fold compared with that of transgenic plants carrying only the CaMV35S promoter (CaMV35S::GUS). GUS activity in transgenic plants was further enhanced by the addition of the sweetpotato TP to the recombinant vector (ibAGP1::TP::GUS), with tuber tissues showing a 26-fold increase in activity compared with that in the CaMV35S::GUS-transgenic lines. In leaf tissues, the levels of GUS activity found in ibAGP1::GUS-transgenic lines were similar to those in CaMV35S::GUS-lines, but they were significantly enhanced in ibAGP1::TP::GUS-lines. GUS activity gradually increased with increasing tuber diameter in ibAGP1::GUS-transgenic plants, reaching a maximum level when the tuber was 35 mm in diameter. In contrast, extremely elevated levels of GUS activity - up to about 10-fold higher than that found in CaMV35S::GUS-lines - were found in ibAGP1::TP::GUS-transgenic lines at a much earlier stage of tuber development (diameter 4 mm), and these higher levels were maintained throughout the entire tuber developmental stage. These results suggest that the sweetpotato ibAGP1 promoter and its TP are a potentially strong foreign gene expression system that can be used for molecular farming in potato plants.

  11. Report on mutation in exon 15 of the APC gene in a case of brain metastasis.

    PubMed

    Pećina-Slaus, Nives; Majić, Zeljka; Musani, Vesna; Zeljko, Martina; Cupić, Hrvoje

    2010-03-01

    The study analyzes exon 15 of the adenomatous polyposis coli gene (APC) in a 49-year-old male patient with brain metastasis. The primary site was lung carcinoma. PCR method and direct DNA sequencing of the metastasis and autologous lymphocyte samples identified the presence of a somatic mutation. The substitution was at position 5883 G-A in the metastasis tissue. The mutation was confirmed by RFLP analysis using Msp I endonuclease, since the mutation strikes the Msp I restriction site. Immunohistochemical analysis revealed the lack of protein expression of this tumor suppressor gene. The main molecular activator of the wnt pathway, beta-catenin, was expressed, and located in the nucleus. The mutation is a silent mutation that might have consequences in the creation of a new splice site. Different single-base substitutions in APC exons need not only be evaluated by the predicted change in amino acid sequence, but rather at the nucleotide level itself. In our opinion, such silent mutations should also be incorporated in mutation detection rate and validation.

  12. The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

    PubMed

    Durantel, D; Croizier, L; Ayres, M D; Croizier, G; Possee, R D; López-Ferber, M

    1998-03-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

  13. First report of a healthy Indian heterozygous for delta 32 mutant of HIV-1 co-receptor-CCR5 gene.

    PubMed

    Husain, S; Goila, R; Shahi, S; Banerjea, A

    1998-01-30

    The beta-chemokine receptor, CCR5, is a major co-receptor for macrophage tropic non-syncytia-inducing isolates of HIV-1. Recently a 32 bp homozygous deletion in the coding region of CCR5 has been reported in a very small percentage (< 1%) of Caucasian individuals who remain uninfected, despite multiple exposure to the wild-type virus. This mutant allele in the heterozygous form (CCR5/32 ccr5) was readily detected in a normal unrelated Caucasian population of European heritage with varying frequencies (13-20%). However, when a large number of the non-Caucasian population (261 Africans and 423 Asians) were screened for the presence of this deleted allele, not a single case of either homozygous or heterozygous mutant for delta 32 allele of CCR5 was detected. We screened 100 normal individuals and found a single heterozygous case with an identical 32 bp deletion in CCR5 gene reported earlier, the rest possessed wild-type alleles. This deleted gene was inherited in Mendelian fashion among the family members of this individual. Thus, the frequency of this deleted allele in India among unrelated normal individuals is likely to be very low (< 1%). We observed a moderate transdominant effect of this mutant allele in a fusion assay. Finally, we show a significant inhibition of fusion of cell membranes when the 176-bp region of CCR5 was used as an antisense.

  14. Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

    PubMed

    Sommer, Jeffrey R; Alderson, Jon; Laible, Goetz; Petters, Robert M

    2006-06-01

    We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.

  15. Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines.

    PubMed

    Metwally, Abdelkader A; Reelfs, Olivier; Pourzand, Charareh; Blagbrough, Ian S

    2012-07-02

    It is important to obtain structure-activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N(4),N(9)-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N(4),N(9)-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N(4),N(9)-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.

  16. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

    PubMed

    Hu, Y; Cooper, T G; Kohlhaw, G B

    1995-01-01

    The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

  17. A case report of pycnodysostosis with atypical femur fracture diagnosed by next-generation sequencing of candidate genes

    PubMed Central

    Song, Hyung Keun; Sohn, Young Bae; Choi, Yong Jun; Chung, Yoon-Sok; Jang, Ja-Hyun

    2017-01-01

    Abstract Rationale: Pycnodysostosis is a rare autosomal recessive skeletal dysplasia characterized by short stature, craniofacial dysmorphism, acro-osteolysis, osteosclerosis, and brittle bone with poor healing. Pycnodysostosis results from the deficient activity of cathepsin K, a lysosomal cysteine protease that is encoded by CTSK. Patient concerns: We report a Korean adult patient with pycnodysostosis and atypical femur fracture whose diagnosis was confirmed by next-generation sequencing (NGS) of candidate genes. A 41-year-old female patient was presented with a left femur fracture after falling down. Underlying sclerotic bone disease was suspected as a radiographic skeletal survey showed thickened cortical bones, and the total body bone density was increased (T score was 5.3, and Z score was 4.9). Diagnoses: We performed candidate gene sequencing of various sclerotic bone diseases for the differential molecular diagnosis of underlying sclerosing bone disease. Two heterozygous variants of CTSK were detected. One was a frameshift variant in exon 5, c.426delT (p.Phe142Leufs∗19), which was previously reported, and the other was a novel missense variant in exon 6, c.755G>A (p.Ser252Asn). Sanger sequencing of CTSK confirmed the 2 heterozygous variants and thus the patient was diagnosed with pycnodysostosis. Interventions: The patient had emergency surgery for subtrochantic femoral fracture. Outcomes: After 4 months of surgery, the patient had almost a full range of hip and knee movements and radiographs show the substantial bridging callus across the fracture. Lessons: Candidate gene sequencing could be a useful diagnostic tool for the genetically heterogeneous skeletal dysplasia group, especially in cases with a mild or atypical clinical phenotype. PMID:28328823

  18. Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

    PubMed Central

    Leung, Alice; Katz, Anna B.; Lim, Foong-Yen; Habli, Mounira; Jones, Helen N.; Wilson, James M.; Crombleholme, Timothy M.

    2012-01-01

    Background Lung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls. Methodology/Principal Findings Human fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts. Conclusions/Significance Based on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis. PMID:22937069

  19. Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.

    PubMed Central

    Sanders, J W; Leenhouts, K J; Haandrikman, A J; Venema, G; Kok, J

    1995-01-01

    In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures. PMID:7665513

  20. Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine.

    PubMed

    Leach, Derrik M; Zacal, Natalie J; Rainbow, Andrew J

    2013-09-01

    Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β-galactosidase (β-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass.

  1. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    SciTech Connect

    Jirtle, Randy L.

    2012-10-11

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A{sup vy}) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure (<10 cGy) during early gestation. This information is particularly important to ascertain given the increased use of CT scans in disease diagnosis, increased number of people predicted to live and work in space, and the present concern about radiological terrorism. We showed for the first time that LDIR significantly increased DNA methylation at the A{sup vy} locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in

  2. RNA interference screening of interferon-stimulated genes with antiviral activities against classical swine fever virus using a reporter virus.

    PubMed

    Wang, Xiao; Li, Yongfeng; Li, Lian-Feng; Shen, Liang; Zhang, Lingkai; Yu, Jiahui; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2016-04-01

    Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is a highly contagious and often fatal disease of pigs, which leads to significant economic losses in many countries. Viral infection can induce the production of interferons (IFNs), giving rise to the transcription of hundreds of IFN-stimulated genes (ISGs) to exert antiviral effects. Although numerous ISGs have been identified to possess antiviral activities against different viruses, rare anti-CSFV ISGs have been reported to date. In this study, to screen anti-CSFV ISGs, twenty-one ISGs reported previously were individually knocked down using small interfering RNAs (siRNAs) followed by infection with a reporter CSFV expressing Renilla luciferase (Rluc). As a result, four novel anti-CSFV ISGs were identified, including natural-resistance-associated macrophage protein 1 (NRAMP1), cytosolic 5'-nucleotidase III A (NT5C3A), chemokine C-X-C motif ligand 10 (CXCL10), and 2'-5'-oligoadenylate synthetase 1 (OAS1), which were further verified to exhibit antiviral activities against wild-type CSFV. We conclude that the reporter virus is a useful tool for efficient screening anti-CSFV ISGs.

  3. Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

    PubMed

    Cevenini, L; Calabretta, M M; Calabria, D; Roda, A; Michelini, E

    2016-01-01

    : The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples.

  4. The Association Between Serotonin Transporter Gene Promoter Polymorphism (5-HTTLPR), Self-Reported Symptoms, and Dental Mercury Exposure

    PubMed Central

    Heyer, Nicholas J.; Echeverria, Diana; Farin, Federico M.; Woods, James S.

    2008-01-01

    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. PMID:18686203

  5. Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes

    PubMed Central

    Jindal, Sunita; Longchar, Bendangchuchang; Singh, Alka; Gupta, Vikrant

    2015-01-01

    Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants. PMID:26340695

  6. Identification of essential genes in bacteria.

    PubMed

    Hillyard, David R; Redd, Michael J

    2007-01-01

    Essential genes are identified in duplicated regions of the bacterial chromosome. Transposition of a vector that forms operon fusions into a strain carrying a chromosomal duplication allows insertion of the transposon into essential genes because a second copy of the essential gene is present. When the duplication is allowed to segregate, only the segregant that carries the copy of the intact essential gene survives. The transposon insertion in the essential gene is maintained only in the duplication derivatives. A technique is described that uses a Tn10 derivative, Tn10dTc-araC(+), which contains a cloned copy of the Escherichia coli araC(+) gene, as a portable region of homology to generate large duplications of the Salmonella chromosome. The duplication is maintained in the population by growth in the presence of tetracycline. When the lac operon fusion vector, MudJ, is transposed into the duplicated region, removal of tetracycline from the growth media allows segregation of the duplication yielding (Ara(-)) haploid segregants which appear as red colonies or as red/white (Ara(-/+)) sectoring colonies on TTC arabinose indicator plates. However, if the insertion is in an essential gene, only segregants that lose the MudJ insertion in the essential gene survive. In this case, selection for the insertion in the essential gene yields solid white (Ara(+)) colonies in the absence of tetracycline. While the specific design presented uses Mud transposon insertions to generate lac operon (transcriptional) and lacZ gene (translational) fusions to essential genes, this technique can be used to generate transposon insertions of any kind into essential genes.

  7. Codon-optimized Human Sodium Iodide Symporter (opt-hNIS) as a Sensitive Reporter and Efficient Therapeutic Gene

    PubMed Central

    Kim, Young-Hwa; Youn, Hyewon; Na, Juri; Hong, Kee-Jong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6 ~ 2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene. PMID:25553100

  8. Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

    NASA Astrophysics Data System (ADS)

    Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

    2012-03-01

    B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

  9. Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

    PubMed Central

    Hancock, K R; Rockman, E; Young, C A; Pearce, L; Maddox, I S; Scott, D B

    1991-01-01

    A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli. Images PMID:1850729

  10. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  11. NFKBIZ in Psoriasis: Assessing the association with gene polymorphisms and report of a new transcript variant.

    PubMed

    Coto-Segura, Pablo; Gonzalez-Lara, Leire; Gómez, Juan; Eiris, Noemí; Batalla, Ana; Gómez, Celia; Requena, Sheila; Queiro, Rubén; Alonso, Belén; Iglesias, Sara; Coto, Eliecer

    2017-03-01

    The IκBζ protein (NFKBIZ gene) is a nuclear inhibitor of NF-κB and plays an important role in the pathogenesis of Psoriasis (Psor). We sought to determine whether common NFKBIZ variants were associated with the risk of developing Psor. A total of 392 patients and 336 controls were genotyped for a common intron 10 indel that could affect pre-mRNA splicing. We found a significantly higher frequency of the insertion among the cw6-positive patients (p=0.01). Cw6-positive+intron 10 ins/ins were significantly more frequent in the patients (OR=3.61). The analysis of the cDNA from leukocytes showed a NFKBIZ transcript lacking exon 10, present in all the tested samples. This new alternative transcript lacks a domain predicted to interact with the NFKB1/p50 protein. Functional studies to define the effect of this alternative transcript on the regulation of the NF-κB pathway are necessary.

  12. Nonketotic hyperglycinemia: novel mutation in the aminomethyl transferase gene. Case report.

    PubMed

    Gencpinar, Pinar; Çavuşoğlu, Dilek; Özbeyler, Ömer; Kaya, Özge Ö; Baydan, Figen; Olgac Dundar, Nihal

    2016-06-01

    Panton-Valentine leukocidin (PVL) is an exotoxin that is produced by many strains of Staphylococcus aureus, and an important virulence factor. A PVL-positive S. aureus infection leads to rapid and severe infections of soft tissue and necrotizing pneumonia in healthy adolescents, and has a high mortality. This case report included a 12-year-old male patient who admitted for fever, respiratory distress and hip pain and was identified with necrotizing pneumonia with septic pulmonary embolism, psoas abscess, cellulitis and osteomyelitis. The PVL positive methicillin-sensitive S. aureus (MSSA) was isolated in the patient blood culture.

  13. Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains

    PubMed Central

    Buckley, Anthony M.; Jukes, Caitlin; Candlish, Denise; Irvine, June J.; Spencer, Janice; Fagan, Robert P.; Roe, Andrew J.; Christie, John M.; Fairweather, Neil F.; Douce, Gillian R.

    2016-01-01

    The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment. PMID:26996606

  14. [Molecular study of CYP21A2 gene for prenatal diagnosis of congenital adrenal hyperplasia. Report of a family].

    PubMed

    Merino, Paulina; Bachega, Tania; Céspedes, Pablo; Trejo, León; Billerbeck, Ana Elisa; Codner, Ethel

    2007-11-01

    Prenatal treatment of pregnancies at risk of congenital adrenal hyperplasia (CAH) may prevent ambiguous genitalia in female fetuses. We present the prenatal treatment performed in an extended family with two mutations. The proband, a boy with CAH-salt losing form, and his relatives were studied. The proband's paternal uncles/aunts were married to the maternal aunts/uncles, respectively. The relatives had normal basal and stimulated 17OHProgesterone levels, which did not clarify their carrier status. The CYP21A2 gene was sequenced. The proband and the paternal relatives harbored a Q318X, R483W mutation in one allele. The maternal relatives and the proband exhibited an R483 frameshift mutation. Early dexamethasone treatment was given during two pregnancies and stopped when male gender was confirmed by early ultrasonography Both newborns were healthy and had normal 17OHProgesterone levels. This family had three mutations which abolish the 21-hydroxylase activity. Two mutations were detected in codon 483 of CYP21A2 gene, exon 10, which have not been reported previously in Latin-America. The molecular study performed in this family allowed us to give an appropriate genetic counseling and prenatal treatment.

  15. A simple method to overcome the interference of hemoglobin in the detection of reporter genes in vivo.

    PubMed

    Reynier, Philippe; Lesage, Denis; Cao, An; Briane, Dominique; Coudert, Robert; Taillandier, Eliane

    2002-02-15

    The determination of chemiluminescent intensity of reporter gene expression in vivo is generally disturbed by the presence of hemoglobin. Current methods consist in using perfusion to eliminate blood from investigated tumors or organs. In this work we propose a simple method to overcome this difficulty. The method consists in establishing an absorbance-dependence plot of the ratio R% = phi/phi(0) between the chemiluminescent intensities measured when hemoglobin is present or absent. For every measurement of the luminescent intensity phi on sample containing blood, if the absorbance A of the hemoglobin is determined, it allows one to have the intensity ratio R% which in turn gives the corrected intensity phi(0) when the absorption by hemoglobin is eliminated. The method is particularly adapted for comparative measurements of transfection levels in tumors where perfusion cannot be easily performed.

  16. [Double mutant alleles in the EXT1 gene not previously reported in a teenager with hereditary multiple exostoses].

    PubMed

    Cammarata-Scalisi, Francisco; Cozar, Mónica; Grinberg, Daniel; Balcells, Susana; Asteggiano, Carla G; Martínez-Domenech, Gustavo; Bracho, Ana; Sánchez, Yanira; Stock, Frances; Delgado-Luengo, Wilmer; Zara-Chirinos, Carmen; Chacín, José Antonio

    2015-04-01

    Hereditary forms of multiple exostoses, now called EXT1/EXT2-CDG within Congenital Disorders of Glycosylation, are the most common benign bone tumors in humans and clinical description consists of the formation of several cartilage-capped bone tumors, usually benign and localized in the juxta-epiphyseal region of long bones, although wide body dissemination in severe cases is not uncommon. Onset of the disease is variable ranging from 2-3 years up to 13-15 years with an estimated incidence ranging from 1/18,000 to 1/50,000 cases in European countries. We present a double mutant alleles in the EXT1 gene not previously reported in a teenager and her family with hereditary multiple exostoses.

  17. Transcriptionally targeted in vivo gene therapy for carcinoembrionic antigen-producing adenocarcinoma.

    PubMed

    Konishi, F; Maeda, H; Yamanishi, Y; Hiyama, K; Ishioka, S; Yamakido, M

    1999-09-01

    Inoperable adenocarcinoma in colon or lung shows resistance to conventional anti-cancer therapy. For these cancers, the feasibility of transcriptionally targeted killing of carcinoembryonic antigen (CEA)-producing adenocarcinoma cells was investigated. Adenovirus vectors carrying a CEA promoter to express E. coli lacZ (AdCEALacZ) or herpes simplex thymidine kinase (AdCEATK) were made and their in vitro and in vivo tumoricidal effects on CEA-producing or non-producing colon and lung cancer cells were evaluated. In vitro infection with AdCEALacZ showed significantly higher CEA promoter-driven lacZ expression in CEA-producing adenocarcinoma cells including VMRC-LCD and LoVo than in CEA-non-producing cells. AdCEATK-infected LoVo showed higher sensitivity to ganciclovir than control vector-infected LoVo or AdCEATK-infected HeLa both in vitro and in subcutaneously implanted tumors of nude mice. Moreover, total tumor elimination in vivo was achieved by either pre-infection of as few as 30% of cells comprising tumors or by direct in vivo injection of AdCEATK to pre-established LoVo tumors. In addition, CEA promoter-driven lacZ expression in LoVo cells was enhanced by the addition of interleukin-6 (IL-6) in vitro. These results provide a rationale for CEA-promoter-driven, adenovirus-mediated gene therapy for CEA-producing adenocarcinomas in colon and lung with reduced toxicity to normal cells.

  18. Identification of a novel Penicillium chrysogenum rhamnogalacturonan rhamnohydrolase and the first report of a rhamnogalacturonan rhamnohydrolase gene.

    PubMed

    Matsumoto, Shotaro; Yamada, Hiroyuki; Kunishige, Yuika; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

    2017-03-01

    Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78. Pfam analysis revealed that PcRGRH78A contains a bacterial α-l-rhamnosidase (PF05592) domain. PcRGRH78A shows optimal activity at 45°C and pH 5. The specificity of PcRGRH78A toward rhamnose (Rha)-containing substrates was compared with that of a P. chrysogenum α-l-rhamnosidase (PcRHA78B) belonging to glycoside hydrolase family 78. PcRGRH78A specifically hydrolyzes RG oligosaccharides that contain Rha at their nonreducing ends, releasing the Rha, but has no activity toward naringin, hesperidin, or rutin. In contrast, PcRHA78B effectively degrades p-nitrophenyl α-l-rhamnopyranoside and the three flavonoids, but not RG oligosaccharides. When galactosyl RG oligosaccharides were used as the substrate, PcRGRH78A released Rha in 3.5-fold greater amounts in the presence of β-galactosidase than in its absence, indicating that PcRGRH78A preferentially acts on Rha residues without the galactose moiety at nonreducing ends. To our knowledge, this is the first report of a gene encoding a RG rhamnohydrolase.

  19. First report of the bla(OXA-58) gene in a clinical isolate of Acinetobacter baumannii in Rio de Janeiro, Brazil.

    PubMed

    Figueiredo, Deuseli Quaresma de; Santos, Kátia Regina Netto Dos; Pereira, Eliezer Menezes; Schuenck, Ricardo Pinto; Mendonça-Souza, Cláudia Rezende Vieira de; Teixeira, Lúcia Martins; Mondino, Silvia Susana Bona de

    2011-05-01

    Carbapenemase production is an important mechanism of carbapenem resistance among nonfermentative Gram-negative isolates. This study aimed to report the detection of bla(OXA-58) gene in multiresistant clinical isolates of Acinetobacter baumannii recovered from inpatients in a public hospital. Polymerase chain reaction tests were performed to detect the bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like) and bla(OXA-51-like) genes. The bla(OXA-58) and bla(OXA-23) genes were detected in one and three isolates, respectively. Sequencing of the bla(OXA-58-like) amplicon revealed 100% identity with the A. baumannii bla(OXA-58) gene listed in the GenBank database. This is the first report of an OXA-58-producing A. baumannii isolate in Rio de Janeiro, Brazil.

  20. A mRNA-based thermosensor controls expression of rhizobial heat shock genes

    PubMed Central

    Nocker, Andreas; Hausherr, Thomas; Balsiger, Sylvia; Krstulovic, Nila-Pia; Hennecke, Hauke; Narberhaus, Franz

    2001-01-01

    Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species. This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures. We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level. A detailed mutational analysis of ROSE1–hspA translationally fused to lacZ revealed that its highly conserved 3′-half is required for repression at normal temperatures (30°C). The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements. Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent. Base substitutions leading to derepression of the lacZ fusion at 30°C exclusively resided in potential stem structures. Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures. We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site. PMID:11726689

  1. A mRNA-based thermosensor controls expression of rhizobial heat shock genes.

    PubMed

    Nocker, A; Hausherr, T; Balsiger, S; Krstulovic, N P; Hennecke, H; Narberhaus, F

    2001-12-01

    Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species. This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures. We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level. A detailed mutational analysis of ROSE(1)-hspA translationally fused to lacZ revealed that its highly conserved 3'-half is required for repression at normal temperatures (30 degrees C). The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements. Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent. Base substitutions leading to derepression of the lacZ fusion at 30 degrees C exclusively resided in potential stem structures. Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures. We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site.

  2. A Novel, Photosynthesis-Associated Thioredoxin-Like Gene: Final Technical Report

    SciTech Connect

    Collier, Jackie, L

    2005-09-13

    ''. These results are consistent with a role for TxlA in the synthesis of the cytochrome b6f complex, which is required for both photosynthetic and respiratory electron transport in cyanobacteria. In contrast, our PCC 7942 mutants in which the C-terminal domain of TxlA was removed are viable and appear to have normal cytochrome content, but have a subtle pigmentation phenotype (increased content of phycocyanin relative to chlorophyll) that depends on both light and CO2 availability. We have also found that PCC 6803 Sll1980 inactivation mutant merodiploids have a similar pigmentation phenotype to the PCC 7942 C-terminal truncation mutants when grown photoautotrophically. In addition, when grown heterotrophically the PCC 6803 Sll1980 inactivation mutant merodiploids remain green instead of turning a golden color like the wild-type, and they are more sensitive to the b6f complex inhibitor DBMIB than is wild type PCC 6803. That the PCC 6803 Sll1980 inactivation mutant merodiploids have these phenotypes despite the fact that they still contain normal copies of the sll1980 gene suggests that the presence of truncated Sll1980 protein interferes with the function of normal Sll1980 protein. Together, these physiological data suggest that TxlA has an essential redox role in cyanobacteria, perhaps a biosynthetic one, and may also have a nonessential regulatory role reflected in the phenotypes of the PCC 7942 C-terminal truncation mutants and the PCC 6803 Sll1980 inactivation mutant merodiploids.

  3. Validating tyrosinase homologue MelA as a photoacoustic reporter gene for imaging Escherichia coli

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Antibiotic drug resistance is a major worldwide issue. Development of new therapies against pathogenic bacteria requires appropriate research tools for replicating and characterizing infections. Previously fluorescence and bioluminescence modalities have been used to image infectious burden in animal models but scattering significantly limits imaging depth and resolution. We hypothesize that photoacoustic imaging, which has improved depth-toresolution ratio, could be useful for visualizing MelA-expressing bacteria since MelA is a bacterial tyrosinase homologue involved in melanin production. Using an inducible expression system, E. coli expressing MelA were visibly black in liquid culture. Phosphate buffered saline (PBS), MelA-expressing bacteria (at different dilutions in PBS), and chicken embryo blood were injected in plastic tubes which were imaged using a VisualSonics Vevo LAZR system. Photoacoustic imaging at 6 different wavelengths (680, 700, 750, 800, 850 and 900nm) enabled spectral de-mixing to distinguish melanin signals from blood. The signal to noise ratio of 9x diluted MelA bacteria was 55, suggesting that ~20 bacteria cells could be detected with our system. When MelA bacteria were injected as a 100 μL bolus into a chicken embryo, photoacoustic signals from deoxy- and oxy- hemoglobin as well as MelA-expressing bacteria could be separated and overlaid on an ultrasound image, allowing visualization of the bacterial location. Photoacoustic imaging may be a useful tool for visualizing bacterial infections and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  4. Characterization of the regulatory region of the dopa decarboxylase gene in Medaka: an in vivo green fluorescent protein reporter assay combined with a simple TA-cloning method.

    PubMed

    Fujimori, Kazuhiro E

    2009-03-01

    The mechanism by which differentiated cells cooperatively express specific sets of genes in multicellular organisms is a fundamental question for biologists. Currently, the mechanism is primarily attributed to complex regulation of transcriptional machinery. Here, I provide a method for studying spatiotemporal characteristics of promoters in vivo by rapid construction of reporter gene-expression vectors based on simple TA-cloning using an in vivo eGFP reporter assay in Medaka (Oryzias latipes). As an application of this method, I focused on the dopa decarboxylase (Ddc) gene, an essential enzyme for production of neurotransmitters, dopamine, and serotonin. Based on the known structure of the Medaka genome, I predicted and cloned the approximately 3 kbp fragment flanking the Ddc gene. Using an eGFP reporter assay in vivo, I showed that it functions as a promoter, directing reporter gene expression in the brain, retina, epiphysis, and gut, but not in sympathetic ganglia, kidney, or liver. Thus, the procedure presented here provides a useful tool for rapid screening of possible promoter regions and for establishing germ line-transmitted transgenic lines of Medaka.

  5. Familial migraine: Exclusion of the susceptibility gene from the reported locus of familial hemiplegic migraine on 19p

    SciTech Connect

    Hovatta, I.; Peltonen, L.; Kallela, M.; Faerkkilae, M.

    1994-10-01

    Genetic isolates are highly useful in analyses of the molecular background of complex diseases since the enrichment of a limited number of predisposing genes can be predicted in representative families or in specific geographical regions. It has been suggested that the pathophysiology and etiology of familial hemiplegic migraine (FHM) and typical migraine with aura are most probably the same. Recent assignment of FHM locus to chromosome 19p in two French families makes it now possible to test this hypothesis. We report here linkage data on four families with multiple cases of migraine disorder originating from the genetically isolated population of Finland. We were interested to discover whether the migraine in these families would also show linkage to the markers on 19p. We could exclude a region of 50 cM, flanking the reported FHM locus, as a site of migraine locus in our four families. It seems evident that locus heterogeneity exists between different diagnostic classes of migraine spectrum of diseases and also between different ethnic groups. 10 refs., 2 figs., 1 tab.

  6. Estrogen-Responsive Transient Expression Assay Using a Brain Aromatase-Based Reporter Gene in Zebrafish (Danio rerio)

    PubMed Central

    Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

    2009-01-01

    Whereas endogenous estrogens play an important role in the development, maintenance, and function of female and male reproductive organs, xenoestrogens present in the environment disrupt normal endocrine function in humans and wildlife. Various in vivo and in vitro assays have been developed to screen these xenoestrogens. However, traditional in vivo assays are laborious and unsuitable for large-scale screening, and in vitro assays do not necessarily replicate in vivo functioning. To overcome these limitations, we developed a transient expression assay in zebrafish, into which a brain aromatase (cyp19a1b)-based estrogen-responsive reporter gene was introduced. In response to 17β-estradiol (10−6 M) and heptachlor (10−6 M), zebrafish embryos carrying the reporter construct expressed enhanced green fluorescent protein in the olfactory bulb, telencephalon, preoptic area, and mediobasal hypothalamus. This system will serve to model the in vivo conversion and breakdown of estrogenic compounds and thus provide a rapid preliminary screening method to estimate their estrogenicity. PMID:19887024

  7. Construction of a cytosolic firefly luciferase reporter cassette for use in PCR-mediated gene deletion and fusion in Saccharomyces cerevisiae.

    PubMed

    Ainsworth, W B; Rome, C M; Hjortsø, M A; Benton, M G

    2012-12-01

    Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(-SKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern.

  8. Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12.

    PubMed Central

    Pradel, E; Schnaitman, C A

    1991-01-01

    In order to study the regulation of a large block of contiguous genes at the rfa locus of Escherichia coli K-12 which are involved in synthesis and modification of the lipopolysaccharide core, the transposon TnlacZ was used to generate in-frame lacZ fusions to the coding regions of five genes (rfaQ, -G, -P, -B and -J) within this block. The beta-galactosidase activity of strains in which these fusions had been crossed into the chromosomal rfa locus was significantly decreased when the rfaH11 (sfrB11) allele was introduced and was restored to wild-type levels when these strains were lysogenized with a lambda phage carrying wild-type rfaH. This indicates that the positive regulatory function encoded by rfaH is required throughout this block of genes. In addition, expression of the lacZ fusion to rfaJ was reduced by growth at 42 degrees C, and this correlated with a temperature-induced change in the electrophoretic profile of the core lipopolysaccharide. Images FIG. 2 PMID:1655711

  9. Registered report: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.

    PubMed

    Cowley, Dale; Pandya, Kumar; Khan, Israr; Kerwin, John; Owen, Kate; Griner, Erin

    2015-09-03

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'A coding-independent function of gene and pseudogene mRNAs regulates tumour biology' by Poliseno et al. (2010), published in Nature in 2010. The key experiments to be replicated are reported in Figures 1D, 2F-H, and 4A. In these experiments, Poliseno and colleagues report microRNAs miR-19b and miR-20a transcriptionally suppress both PTEN and PTENP1 in prostate cancer cells (Figure 1D; Poliseno et al., 2010). Decreased expression of PTEN and/or PTENP1 resulted in downregulated PTEN protein levels (Figure 2H), downregulation of both mRNAs (Figure 2G), and increased tumor cell proliferation (Figure 2F; Poliseno et al., 2010). Furthermore, overexpression of the PTEN 3' UTR enhanced PTENP1 mRNA abundance limiting tumor cell proliferation, providing additional evidence for the co-regulation of PTEN and PTENP1 (Figure 4A; Poliseno et al., 2010). The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published in eLife.

  10. Effect of vascular endothelial growth factor gene therapy on post-traumatic peripheral nerve regeneration and denervation-related muscle atrophy.

    PubMed

    Moimas, S; Novati, F; Ronchi, G; Zacchigna, S; Fregnan, F; Zentilin, L; Papa, G; Giacca, M; Geuna, S; Perroteau, I; Arnež, Z M; Raimondo, S

    2013-10-01

    Functional recovery after peripheral nerve injury depends on both improvement of nerve regeneration and prevention of denervation-related skeletal muscle atrophy. To reach these goals, in this study we overexpressed vascular endothelial growth factor (VEGF) by means of local gene transfer with adeno-associated virus (AAV). Local gene transfer in the regenerating peripheral nerve was obtained by reconstructing a 1-cm-long rat median nerve defect using a vein segment filled with skeletal muscle fibers that have been previously injected with either AAV2-VEGF or AAV2-LacZ, and the morphofunctional outcome of nerve regeneration was assessed 3 months after surgery. Surprisingly, results showed that overexpression of VEGF in the muscle-vein-combined guide led to a worse nerve regeneration in comparison with AAV-LacZ controls. Local gene transfer in the denervated muscle was obtained by direct injection of either AAV2-VEGF or AAV2-LacZ in the flexor digitorum sublimis muscle after median nerve transection and results showed a significantly lower progression of muscle atrophy in AAV2-VEGF-treated muscles in comparison with muscles treated with AAV2-LacZ. Altogether, our results suggest that local delivery of VEGF by AAV2-VEGF-injected transplanted muscle fibers do not represent a rational approach to promote axonal regeneration along a venous nerve guide. By contrast, AAV2-VEGF direct local injection in denervated skeletal muscle significantly attenuates denervation-related atrophy, thus representing a promising strategy for improving the outcome of post-traumatic neuromuscular recovery after nerve injury and repair.

  11. Clinical and genetic characterization of chanarin-dorfman syndrome patients: first report of large deletions in the ABHD5 gene

    PubMed Central

    2010-01-01

    Background Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disorder characterized by nonbullous congenital ichthyosiform erythroderma (NCIE) and an intracellular accumulation of triacylglycerol (TG) droplets in most tissues. The clinical phenotype involves multiple organs and systems, including liver, eyes, ears, skeletal muscle and central nervous system (CNS). Mutations in ABHD5/CGI58 gene are associated with CDS. Methods Eight CDS patients belonging to six different families from Mediterranean countries were enrolled for genetic study. Molecular analysis of the ABHD5 gene included the sequencing of the 7 coding exons and of the putative 5' regulatory regions, as well as reverse transcript-polymerase chain reaction analysis and sequencing of normal and aberrant ABHD5 cDNAs. Results Five different mutations were identified, four of which were novel, including two splice-site mutations (c.47+1G>A and c.960+5G>A) and two large deletions (c.898_*320del and c.662-1330_773+46del). All the reported mutations are predicted to be pathogenic because they lead to an early stop codon or a frameshift producing a premature termination of translation. While nonsense, missense, frameshift and splice-site mutations have been identified in CDS patients, large genomic deletions have not previously been described. Conclusions These results emphasize the need for an efficient approach for genomic deletion screening to ensure an accurate molecular diagnosis of CDS. Moreover, in spite of intensive molecular screening, no mutations were identified in one patient with a confirmed clinical diagnosis of CDS, appointing to genetic heterogeneity of the syndrome. PMID:21122093

  12. Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene

    PubMed Central

    Shen, Liang; Yang, Yang; Ye, Fei; Liu, Gaoshan; Desforges, Marc

    2016-01-01

    Human coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express the Renilla luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication in vitro, with a 50% inhibitory concentration (IC50) of 0.33 μM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50 of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication. PMID:27381385

  13. Longitudinal Assessment of Lung Cancer Progression in Mice Using the Sodium Iodide Symporter Reporter Gene and SPECT/CT Imaging

    PubMed Central

    Anton, Martina; Kusewitt, Donna F.; Norenberg, Jeffrey P.; MacKenzie, Debra A.; Thompson, Todd A.; Muttil, Pavan

    2016-01-01

    Lung cancer has the highest mortality rate of any tissue-specific cancer in both men and women. Research continues to investigate novel drugs and therapies to mitigate poor treatment efficacy, but the lack of a good descriptive lung cancer animal model for preclinical drug evaluation remains an obstacle. Here we describe the development of an orthotopic lung cancer animal model which utilizes the human sodium iodide symporter gene (hNIS; SLC5A5) as an imaging reporter gene for the purpose of non-invasive, longitudinal tumor quantification. hNIS is a glycoprotein that naturally transports iodide (I-) into thyroid cells and has the ability to symport the radiotracer 99mTc-pertechnetate (99mTcO4-). A549 lung adenocarcinoma cells were genetically modified with plasmid or lentiviral vectors to express hNIS. Modified cells were implanted into athymic nude mice to develop two tumor models: a subcutaneous and an orthotopic xenograft tumor model. Tumor progression was longitudinally imaged using SPECT/CT and quantified by SPECT voxel analysis. hNIS expression in lung tumors was analyzed by quantitative real-time PCR. Additionally, hematoxylin and eosin staining and visual inspection of pulmonary tumors was performed. We observed that lentiviral transduction provided enhanced and stable hNIS expression in A549 cells. Furthermore, 99mTcO4- uptake and accumulation was observed within lung tumors allowing for imaging and quantification of tumor mass at two-time points. This study illustrates the development of an orthotopic lung cancer model that can be longitudinally imaged throughout the experimental timeline thus avoiding inter-animal variability and leading to a reduction in total animal numbers. Furthermore, our orthotopic lung cancer animal model is clinically relevant and the genetic modification of cells for SPECT/CT imaging can be translated to other tissue-specific tumor animal models. PMID:28036366

  14. Associations between oxytocin receptor gene (OXTR) polymorphisms and self-reported aggressive behavior and anger: Interactions with alcohol consumption.

    PubMed

    Johansson, Ada; Westberg, Lars; Sandnabba, Kenneth; Jern, Patrick; Salo, Benny; Santtila, Pekka

    2012-09-01

    Oxytocin has been implicated in the regulation of social as well as aggressive behaviors, and in a recent study we found that the effect of alcohol on aggressive behavior was moderated by the individual's genotype on an oxytocin receptor gene (OXTR) polymorphism (Johansson et al., 2012). In this study we wanted to deepen and expand the analysis by exploring associations between three (rs1488467, rs4564970, rs1042778) OXTR polymorphisms and aggressive behavior, trait anger as well as anger control in a population-based sample of Finnish men and women (N=3577) aged between 18 and 49 years (M=26.45 years, SD=5.02). A specific aim was to investigate if the polymorphisms would show interactive effects with alcohol consumption on aggressive behavior and trait anger, as well as to explore whether these polymorphisms affect differences in anger control between self-reported sober and intoxicated states. The results showed no main effects of the polymorphisms, however, three interactions between the polymorphisms and alcohol consumption were found. The effect of alcohol consumption on aggressive behavior was moderated by the genotype of the individual on the rs4564970 polymorphism, in line with previous results (Johansson et al., 2012). For trait anger, both the rs1488467 and the rs4564970 polymorphisms interacted with alcohol consumption. It appears that the region of the OXTR gene including both the rs4564970 and the rs1488467 polymorphisms may be involved in the regulation of the relationship between alcohol and aggressive behavior as well as between alcohol and the propensity to react to situations with elevated levels of anger.

  15. Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo.

    PubMed

    Park, F; Kay, M A

    2001-09-01

    Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

  16. Disruption of the pdhB pyruvate dehydrogenase [corrected] gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

    PubMed

    Hegde, Shivanand; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2015-01-01

    The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

  17. Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa.

    PubMed

    Aoshima, Ryota; Hiraoka, Rieko; Shimada, Nao; Kawata, Takefumi

    2006-01-01

    A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT (signal transducers and activators of transcription) proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. We fused promoter fragments of the cognate gene, ahhA (adducin head homologue A), to a lacZ reporter and determined their expression pattern. The proximal promoter region is necessary for the expression of ahhA at an early (pre-aggregative) stage of development and this expression is Dd-STATa independent. The distal promoter region is necessary for expression at later stages of development in pstA cells, of the slug and in upper cup and pstAB cells during culmination. The distal region is partly Dd-STATa-dependent. The ahhA-null mutant develops almost normally until culmination, but it forms slanting culminants that tend to collapse on to the substratum. The mutant also occasionally forms fruiting bodies with swollen papillae and with constrictions in the prestalk region. The AhhA protein localizes to the stalk tube entrance and also to the upper cup cells and in cells at or near to the constricted region where an F-actin ring is localized. These findings suggest that Dd-STATa regulates culmination and may be necessary for straight downward elongation of the stalk, via the putative actin-binding protein AhhA.

  18. An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.

    PubMed

    Seo, Jeong-Woo; Hong, Won-kyung; Rairakhwada, Dina; Seo, Pil-Soo; Choi, Min Ho; Song, Ki-Bang; Rhee, Sang-Ki; Kim, Chul Ho

    2009-06-01

    The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli.

  19. Expression of the Nitroarene Dioxygenase Genes in Comamonas sp. Strain JS765 and Acidovorax sp. Strain JS42 Is Induced by Multiple Aromatic Compounds

    PubMed Central

    Lessner, Daniel J.; Parales, Rebecca E.; Narayan, Shakti; Gibson, David T.

    2003-01-01

    This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator. PMID:12813084

  20. Evolution of echinoderms may not have required modification of the ancestral deuterostome HOX gene cluster: first report of PG4 and PG5 Hox orthologues in echinoderms.

    PubMed

    Long, Suzanne; Martinez, Pedro; Chen, Wei-Chung; Thorndyke, Michael; Byrne, Maria

    2003-11-01

    Is the extreme derivation of the echinoderm body plan reflected in a derived echinoderm Hox genotype? Building on previous work, we exploited the sequence conservation of the homeobox to isolate putative orthologues of several Hox genes from two asteroid echinoderms. The 5-peptide motif (LPNTK) diagnostic of PG4 Hox genes was identified immediately downstream of one of the partial homeodomains from Patiriella exigua. This constitutes the first unequivocal report of a PG4 Hox gene orthologue from an echinoderm. Subsequent screenings identified genes of both PG4 and PG4/5 in Asterias rubens. Although in echinoids only a single gene (PG4/5) occupies these two contiguous cluster positions, we conclude that the ancestral echinoderm must have had the complete deuterostome suite of medial Hox genes, including orthologues of both PG4 and PG4/5 (=PG5). The reported absence of PG4 in the HOX cluster of echinoids is therefore a derived state, and the ancestral echinoderm probably had a HOX cluster not dissimilar to that of other deuterostomes. Modification of the ancestral deuterostome Hox genotype may not have been required for evolution of the highly derived echinoderm body plan.

  1. Water quality assessment using the AREc32 reporter gene assay indicative of the oxidative stress response pathway.

    PubMed

    Escher, Beate I; Dutt, Mriga; Maylin, Erin; Tang, Janet Y M; Toze, Simon; Wolf, C Roland; Lang, Matti

    2012-11-01

    The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart

  2. The Escherichia coli rhaSR-PrhaBAD Inducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa.

    PubMed

    Meisner, Jeffrey; Goldberg, Joanna B

    2016-11-15

    The araC-ParaBAD inducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range in Escherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests that araC-ParaBAD is leaky in Pseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system in P. aeruginosa nor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of the araC-ParaBAD system in P. aeruginosa Using transcriptional fusions to a lacZ reporter gene, we determined that the noninduced expression from araC-ParaBAD is high and cannot be reduced by carbon catabolite repression as it can in E. coli Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression in P. aeruginosa significantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of the araC-ParaBAD system, we found that the lacI(q)-Ptac and rhaSR-PrhaBAD inducible promoter systems had significantly lower noninduced expression and were inducible over a broader range than araC-ParaBAD We demonstrated that noninduced expression from the araC-ParaBAD system supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis in P. aeruginosa, problems that were avoided with rhaSR-PrhaBAD. rhaSR-PrhaBAD is tightly controlled, allows gene expression over a wide range, and represents a significant improvement over araC-ParaBAD in P. aeruginosa IMPORTANCE: We report the shortcomings of the commonly used Escherichia coli araC-ParaBAD inducible promoter system in Pseudomonas aeruginosa, successfully reengineered it to improve

  3. Carbon dioxide as a regulator of gene expression in microorganisms.

    PubMed

    Stretton, S; Goodman, A E

    1998-01-01

    CO2 regulates gene expression across a diverse group of microorganisms including fungi, and both photosynthetic and non photosynthetic bacteria. The processes that CO2 regulates are diverse. Several CO2-responsive random promoter lacZ fusions of unknown function have been isolated from a marine Synechococcus and a Pseudoalteromonas sp., highlighting the wide effect of CO2 control in these organisms. Regulatory proteins have been described that mediate the CO2 response at transcription level in Bacillus anthracis, the group A streptococci and two Rhodobacter spp. These regulatory proteins include: AcpA and AtxA that are involved in CO2 control of B. anthracis capsule and toxin production; Mga that regulates surface associated virulence factors in the group A streptococci; and RegB/A, a two component signal transduction system that responds to environmental stimuli including CO2, to regulate photosynthetic apparatus and CO2 fixation enzyme synthesis in Rhodobacter spp.

  4. Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle.

    PubMed

    Wallace, Lindsay M; Moreo, Andrew; Clark, K Reed; Harper, Scott Q

    2013-04-16

    Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hr

  5. Molecular analysis of the PAX6 gene in Mexican patients with congenital aniridia: report of four novel mutations

    PubMed Central

    Villarroel, Camilo E.; Villanueva-Mendoza, Cristina; Orozco, Lorena; Alcántara-Ortigoza, Miguel Angel; Jiménez, Diana F.; Ordaz, Juan C.

    2008-01-01

    Purpose Paired box gene 6 (PAX6) heterozygous mutations are well known to cause congenital non-syndromic aniridia. These mutations produce primarily protein truncations and have been identified in approximately 40%–80% of all aniridia cases worldwide. In Mexico, there is only one previous report describing three intragenic deletions in five cases. In this study, we further analyze PAX6 variants in a group of Mexican aniridia patients and describe associated ocular findings. Methods We evaluated 30 nonrelated probands from two referral hospitals. Mutations were detected by single-strand conformation polymorphism (SSCP) and direct sequencing, and novel missense mutations and intronic changes were analyzed by in silico analysis. One intronic variation (IVS2+9G>A), which in silico analysis suggested had no pathological effects, was searched in 103 unaffected controls. Results Almost all cases exhibited phenotypes that were at the severe end of the aniridia spectrum with associated ocular alterations such as nystagmus, macular hypoplasia, and congenital cataracts. The mutation detection rate was 30%. Eight different mutations were identified: four (c.184_188dupGAGAC, c.361T>C, c.879dupC, and c.277G>A) were novel, and four (c.969C>T, IVS6+1G>C, c.853delC, and IVS7–2A>G) have been previously reported. The substitution at position 969 was observed in two patients. None of the intragenic deletions previously reported in Mexican patients were found. Most of the mutations detected predict either truncation of the PAX6 protein or conservative amino acid changes in the paired domain. We also detected two intronic non-pathogenic variations, IVS9–12C>T and IVS2+9G>A, that had been previously reported. Because the latter variation was considered potentially pathogenic, it was analyzed in 103 healthy Mexican newborns where we found an allelic frequency of 0.1116 for the A allele. Conclusions This study adds four novel mutations to the worldwide PAX6 mutational spectrum, and

  6. A novel mutation in TAP1 gene leading to MHC class I deficiency: Report of two cases and review of the literature.

    PubMed

    Hanalioglu, Damla; Ayvaz, Deniz Cagdas; Ozgur, Tuba Turul; van der Burg, Mirjam; Sanal, Ozden; Tezcan, Ilhan

    2017-02-02

    Major histocompatibility complex (MHC) class I deficiency syndrome is a rare primary immunodeficiency caused by mutations in the peptide transporter complex associated with antigen presentation (TAP) gene which plays a crucial role in intracellular peptide antigen presentation. A few cases have been reported to date. Recurrent sinopulmonary infections and skin ulcers are the main characteristics of the syndrome. Here we report two siblings diagnosed with TAP1 deficiency syndrome associated only with recurrent sinopulmonary infections with the description of a novel mutation leading to a premature stop codon in TAP1 gene and review of the relevant literature. Both of the siblings had recurrent sinopulmonary infections since childhood, responded to antibiotherapy well, neither of them had hospitalization history because of infections. One had chronic hepatitis B infection which may possibly be related to TAP1 gene defect.

  7. Thyroid vein thrombosis in a young woman with methylenetetrahydrofolate reductase gene mutation receiving cyproterone acetate/ethinylestradiol treatment for polycystic ovary syndrome: a case report.

    PubMed

    Nazli, Yunus; Colak, Necmettin; Aksoy, Omer N; Cakir, Omer

    2011-06-01

    Thyroid vein thrombosis is a rare variety of venous thrombosis. The use of oral contraceptives and heterozygous methylenetetrahydrofolate reductase (MTHFR) gene mutation could have synergistic effects on the onset of venous thromboembolism. We report a 28-year-old woman with thyroid vein thrombosis. The thrombosis was possibly provoked by a combination of heterozygous MTHFR gene mutation and the use of a combined oral contraceptive (COC) containing cyproterone acetate/ethinylestradiol (CPA/EE) for polycystic ovary syndrome (PCOS). The patient was treated successfully with low molecular weight heparin and oral anticoagulation. The thyroid vein thrombosis resolved and the patient was advised to maintain lifelong use of anticoagulation therapy. Our report emphasizes that the combination of heterozygous MTHFR gene mutation, PCOS, and the use of a COC-containing CPA/EE could increase risk of thyroid vein thrombosis in young adults.

  8. New NodW- or NifA-regulated Bradyrhizobium japonicum genes.

    PubMed

    Caldelari Baumberger, Isabelle; Fraefel, Nicole; Göttfert, Michael; Hennecke, Hauke

    2003-04-01

    A cluster of genes coding for putative plant cell-wall degrading enzymes (i.e., genes for two endoglucanases [gunA and gunA2], one pectinmethylesterase [pme], and one polygalacturonase [pgl]) was identified by sequence similarities in the symbiotic region of the Bradyrhizobium japonicum chromosome. In addition, a systematic screen of the region revealed several genes potentially transcribed by the sigma(54)-RNA polymerase and activated by the transcriptional regulator NifA (i.e., genes for proteins with similarity to outer membrane proteins [id117 and id525] and a citrate carrier [id331 or citA] and one open reading frame without similarity to known proteins [id747]). Expression studies using transcriptional lacZ fusions showed that gunA2 and pgl were strongly induced by the isoflavone genistein in a NodW-dependent manner, suggesting a role of the gene products in early events of the nodulation process; by contrast, gunA and pme expression was very weak in the conditions tested. The gunA2 gene product was purified and was shown to have cellulase activity. beta-Galactosidase activity expressed from transcriptional lacZ fusions to id117, id525, and id747 in the wild type and in nifA and rpoN mutant backgrounds confirmed that their transcription was dependent on NifA and sigma(54). Despite the presence of a -24/-12-type promoter and a NifA binding site upstream of citA, no regulation could be demonstrated in this case. Null mutations introduced in gunA, gunA2, pgl, pme, citA, id117, id525, and id747 did not impair the symbiosis with the host plants.

  9. Brief Report: High Frequency of Biochemical Markers for Mitochondrial Dysfunction in Autism: No Association with the Mitochondrial Aspartate/Glutamate Carrier "SLC25A12" Gene

    ERIC Educational Resources Information Center

    Correia, Catarina; Coutinho, Ana M.; Diogo, Luisa; Grazina, Manuela; Marques, Carla; Miguel, Teresa; Ataide, Assuncao; Almeida, Joana; Borges, Luis; Oliveira, Catarina; Oliveira, Guiomar; Vicente, Astrid M.

    2006-01-01

    In the present study we confirm the previously reported high frequency of biochemical markers of mitochondrial dysfunction, namely hyperlactacidemia and increased lactate/pyruvate ratio, in a significant fraction of 210 autistic patients. We further examine the involvement of the mitochondrial aspartate/glutamate carrier gene ("SLC25A12") in…

  10. The importance of location and orientation of male specific lethal complex binding sites of differing affinities on reporter gene dosage compensation in Drosophila.

    PubMed

    Schiemann, Anja H; Weake, Vikki M; Li, Fang; Laverty, Corey; Belikoff, Esther J; Scott, Maxwell J

    2010-11-26

    The male specific lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila. The complex binds to most actively transcribed X-linked genes in males and upregulates expression. High resolution chromatin immunoprecipitation assays have identified over one hundred high affinity binding sites on the X chromosome. One of the first high affinity sites discovered is at cytological location 18D11. The MSL complex binds weakly to a single copy of a 510bp fragment from 18D11 but strongly to a tetramer of the fragment. Here we have investigated the effect of insertion of sites of differing affinities, either upstream or within the transcribed gene, on complex binding and transcription upregulation. Insertion of four copies of the 18D11 fragment upstream or at the 3' end of a reporter gene led to strong MSL complex binding and increased expression in males. In contrast, the MSL complex did not bind consistently to autosomal transgenes that contained a single copy of the 18D11 site upstream of the gene promoter. However, MSL complex binding was observed in all lines if the single 18D11 fragment was inserted into the 3' end of the reporter gene in either orientation. This is consistent with previous studies that showed gene transcription facilitates MSL complex binding. Surprisingly, transcription elevation in males was only observed if the 18D11 fragment was in the forward orientation and only in some lines. Our results suggest that MSL complex binding to weaker sites and transcription enhancement is influenced by gene transcription, binding site orientation and the local chromatin environment. In contrast, strong binding sites do not need to be transcribed to recruit sufficient complex to cause transcription elevation of nearby genes.

  11. Understanding false positives in reporter gene assays: in silico chemogenomics approaches to prioritize cell-based HTS data.

    PubMed

    Crisman, Thomas J; Parker, Christian N; Jenkins, Jeremy L; Scheiber, Josef; Thoma, Mathis; Kang, Zhao Bin; Kim, Richard; Bender, Andreas; Nettles, James H; Davies, John W; Glick, Meir

    2007-01-01

    High throughput screening (HTS) data is often noisy, containing both false positives and negatives. Thus, careful triaging and prioritization of the primary hit list can save time and money by identifying potential false positives before incurring the expense of followup. Of particular concern are cell-based reporter gene assays (RGAs) where the number of hits may be prohibitively high to be scrutinized manually for weeding out erroneous data. Based on statistical models built from chemical structures of 650 000 compounds tested in RGAs, we created "frequent hitter" models that make it possible to prioritize potential false positives. Furthermore, we followed up the frequent hitter evaluation with chemical structure based in silico target predictions to hypothesize a mechanism for the observed "off target" response. It was observed that the predicted cellular targets for the frequent hitters were known to be associated with undesirable effects such as cytotoxicity. More specifically, the most frequently predicted targets relate to apoptosis and cell differentiation, including kinases, topoisomerases, and protein phosphatases. The mechanism-based frequent hitter hypothesis was tested using 160 additional druglike compounds predicted by the model to be nonspecific actives in RGAs. This validation was successful (showing a 50% hit rate compared to a normal hit rate as low as 2%), and it demonstrates the power of computational models toward understanding complex relations between chemical structure and biological function.

  12. Report of outbreaks of classical scrapie in Dorper sheep and associated prion protein gene polymorphisms in affected flocks.

    PubMed

    de Andrade, Caroline Pinto; de Oliveira, Eduardo Conceição; Leal, Juliano Souza; de Almeida, Laura Lopes; de Castro, Luiza Amaral; da Silva, Sergio Ceroni; Driemeier, David

    2015-08-01

    Scrapie is an infectious neurodegenerative disease affecting sheep and goats, related with conformational alteration of an isoform of the prion protein that leads to deposition and aggregation in the host's central nervous system. Occurrence of the natural disease can be influenced by host genetic factors, such as a single nucleotide polymorphism of the prion protein gene. This study reports three scrapie-affected Dorper flocks located on three different farms in Brazil. The objective of this study was to analyze these three flocks using scrapie diagnostics, combining histology, immunohistochemistry, genotyping, and western blot assays. For immunohistochemistry, 192 sheep were selected and 308 sheep blood samples were taken for genotyping. A total of 22 sheep were scrapie positive by immunohistochemistry. Of these, four presented clinical signs and had scrapie immunoreactivity at the obex in western blot assays. The sheep without clinical signs were positive in lymphoid organs, such as the third eyelid and rectal mucosa. The major genotypes found on the flocks were ARQ/ARQ, ARQ/ARR, and ARQ/VRQ for codons 136, 154, and 171. Most of the sheep were considered to be at moderate to high risk, based on risk groups for developing scrapie. Some blood samples were sequenced, and polymorphisms were identified in other codons, such as 127, 142, and 143. Our data demonstrate the importance of preclinical scrapie diagnosis in Brazilian sheep, as most of the affected sheep showed no clinical signs, and emphasize the relevance of genotyping other Dorper sheep to determine the genotypic profile of the breed.

  13. An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

    PubMed Central

    Wang, Yang; Newton, Derek C.; Miller, Tricia L.; Teichert, Anouk-Martine; Phillips, M. James; Davidoff, Michail S.; Marsden, Philip A.

    2002-01-01

    Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of β-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. β-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P450scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, β-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of β-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, β-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of

  14. [Organization and regulation of the genes for nitrogen fixation in Rhodopseudomonas capsulata]. Progress report, [June 5, 1989--June 4, 1991

    SciTech Connect

    Not Available

    1991-12-31

    In prior support periods we identified, cloned and sequenced three genes involved in the regulation of nif gene expression in Rhodobacter capsulatus. These were called nifRI, nifR2 and nifR4; they turn out to be homologue of the ntrC, ntrB and ntrA genes of enterobacteria. We subsequently found that mutations in an additional gene, nifR5. render R. capsulatus nif genes constitutive with respect to ammonia. The nifR5 gene was shown to be similar to glnB of enteric bacteria, encoding the regulatory protein PII, and furthering the intersection of the glutamine synthetase adenylylation cascade with the control of nif gene transcription. In pursuit of the mechanism of 0{sub 2} control of nif gene expression, we constructed and analyzed the topology of a small plasmid in R. capsulatus as a function of 0{sub 2} concentration. We also cloned and obtained partial sequence data for two genes encoding the B subunit of DNA gyrase. The nucleotide sequence of the rpoB gene encoding RNA polymerase was nearly completed. A method for isolation of genes expressed differentially, developed for cyanobacteria, was applied successfully to R. capsulatus. Several genes that depend on nifR4 for their transcription were isolated. A transcription start site for a nifA gene was identified and the promoter sequence was analyzed. A physical map of the R calsulatus SB1003 chromosome was prepared, based on pulsed-field electrophoresis of XbaI and AseI fragments and hybridization with a gridded cosmid library, using a device that permits 864 cosmids to be hybridized at one time with a labeled chromosomal fragment.

  15. Gene expression at the Iguazú Falls: a report from the "Gene Expression and RNA Processing" symposium; Iguazú Falls, Province of Misiones, Argentina.

    PubMed

    Rattner, Barbara P

    2012-01-01

    The fourth edition of the "Gene Expression and RNA Processing" symposium took place this year at the Iguazú Falls, one of the most renowned South American natural wonders, and brought together an outstanding array of speakers from all over the world to discuss mechanisms of transcriptional regulation and RNA processing.

  16. Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations.

    PubMed

    Das, Dhanjit Kumar; Raha, Sarbani; Sanghavi, Daksha; Maitra, Anurupa; Udani, Vrajesh

    2013-02-15

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome. A total of 62 (69%) patients remained without molecular genetics diagnosis that necessitates further search for mutations in other genes like CDKL5 and FOXG1 that are known to cause Rett phenotype. The majority of mutations are detected in exon 4 and only one mutation was present in exon 3. Therefore, our study suggests the need for screening exon 4 of MECP2 as first line of diagnosis in these patients.

  17. TUMORAL TISSUE SPECIFIC PROMOTER HYPERMETHYLATION OF DISTINCT TUMOR SUPPRESSOR GENES IN A CASE WITH NONSMALL CELL LUNG CARCINOMA: A CASE REPORT

    PubMed Central

    Arslan, Sulhattin; Dogan, Tamer; Koksal, Binnur; Yildirim, Malik Ejder; Gumus, Cesur; Elagoz, Sahenda; Akkurt, Ibrahim; Ozdemir, Oztürk

    2008-01-01

    SUMMARY Objective: Non-small cell lung carcinoma is an aggressive phenomenon and the epigenetical alterations of some tumor supressor genes have been reported for the different tumor types. Case Presentation: It is presented a case report concerning a 43 years old male with NSCLC on the lower segment of the right lung. The patient underwent a diag-nostic excisional thin-needle biopsy and after the histological confirmation. We examined the promoter methylation status of some distinct tumor supressor genes in tumoral and blood tissues of the case after sodium bisulfite conversion and DNA amplification with methylation specific multiplex PCR technique. Both tissues were also searched for G to A transitions in codons 12 and 13 of the K-ras proto-oncogene. Results: Tumor specimen showed fully methyl pattern profiles for the SFRP2, p16, DAPK1 and partially hyper-methylated profile for the p53 and MGMT genes in this case with non-small lung carci-noma. Blood speicemen showed normal hypomethylated profiles for all studied TS genes. The K-ras proto-oncogene was in normal structure both in blood and tumoral spiecemens that examined. Conclusion: Results indicate that genes exhibit tumor suppressor activi-ties in blood, but exhibit epigenetic inactivation in carcinoma cell. These findings strongly support the hypothesis that epigenetic mechanisms may play an important role in the non-small cell lung carcinogenesis in human. PMID:21264081

  18. Organ-specific gene expression in maize: The P-wr allele. Final report, August 15, 1993--August 14, 1996

    SciTech Connect

    Peterson, T.A.

    1997-06-01

    The ultimate aim of our work is to understand how a regulatory gene produces a specific pattern of gene expression during plant development. Our model is the P-wr gene of maize, which produces a distinctive pattern of pigmentation of maize floral organs. We are investigating this system using a combination of classical genetic and molecular approaches. Mechanisms of organ-specific gene expression are a subject of intense research interest, as it is the operation of these mechanisms during eukaryotic development which determine the characteristics of each organism Allele-specific expression has been characterized in only a few other plant genes. In maize, organ-specific pigmentation regulated by the R, B, and Pl genes is achieved by differential transcription of functionally conserved protein coding sequences. Our studies point to a strikingly different mechanism of organ-specific gene expression, involving post-transcriptional regulation of the regulatory P gene. The novel pigmentation pattern of the P-wr allele is associated with differences in the encoded protein. Furthermore, the P-wr gene itself is present as a unique tandemly amplified structure, which may affect its transcriptional regulation.

  19. Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

    PubMed

    Umemura, K; Atomi, H; Izuta, M; Kanai, T; Takeshita, S; Ueda, M; Tanaka, A

    1997-01-03

    We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was

  20. Targeted Deletion of the Antisilencer/Enhancer (ASE) Element from Intron 1 of the Myelin Proteolipid Protein Gene (Plp1) in Mouse Reveals that the Element Is Dispensable for Plp1 Expression in Brain during Development and Remyelination

    PubMed Central

    Pereira, Glauber B.; Meng, Fanxue; Kockara, Neriman T.; Yang, Baoli; Wight, Patricia A.

    2012-01-01

    Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. While removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is nonfunctional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. PMID:23157328

  1. A possible association of a human tektin-t gene mutation (A229V) with isolated non-syndromic asthenozoospermia: case report.

    PubMed

    Zuccarello, Daniela; Ferlin, Alberto; Garolla, Andrea; Pati, Mauro A; Moretti, Afra; Cazzadore, Carla; Francavilla, Sandro; Foresta, Carlo

    2008-04-01

    Asthenozoospermia (AZS), characterized by grade A + B sperm motility (as in World Health Organization Guidelines) < or =50% or A <25% in fresh ejaculate, may exist as an isolated disorder, in combination with other sperm anomalies or as part of syndromic association. The majority of syndromic patients can be ascribed to mutations in dynein genes, while, to date, no genes have been described to be associated in humans with non-syndromic, isolated AZS. An interesting family of axonemal proteins, the tektins, has been recently identified in various mammals and they are thought to play a fundamental role in ciliary movement. Recently, the human tektin-t (or h-tekB1 or Tektin-2) gene has been cloned, showing specific expression in flagella of mature sperm. We report the screening of tektin-t gene in 90 isolated non-syndromic AZS patients. We found a heterozygous mutation (A229V) in one patient. Ultrastructural analysis showed anomalies in > or =80% of examined spermatozoa involving axoneme microtubules and mitochondria. Moreover, the viability and mitochondrial function of sperm were altered in the patient with the A229V mutation. This is the first description of human pathology linked to a tektin-family gene, since only murine models are available for these genes.

  2. Gene and enhancer traps for gene discovery.

    PubMed

    Rojas-Pierce, Marcela; Springer, Patricia S

    2003-01-01

    Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as beta-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.

  3. Comparing the functions of equine and canine influenza H3N8 virus PA-X proteins: Suppression of reporter gene expression and modulation of global host gene expression.

    PubMed

    Feng, Kurtis H; Sun, Miao; Iketani, Sho; Holmes, Edward C; Parrish, Colin R

    2016-09-01

    The influenza PA-X protein is translated from the PA open reading frame from frameshifting and suppresses cellular gene expression due to its ribonuclease activity. We further defined the functional roles of PA-X by comparing PA-X proteins from two related viruses - equine influenza (EIV) and canine influenza (CIV) H3N8 - that differ in a C-terminal truncation and internal mutations. In vitro reporter gene assays revealed that both proteins were able to suppress gene expression. Interestingly, EIV PA-X demonstrated ~50% greater activity compared to CIV PA-X, and we identified the mutations that caused this difference. We used RNA-seq to evaluate the effects of PA-X on host gene expression after transfection into cultured cells. There were no significant differences in this property between EIV and CIV PA-X proteins, but expression of either resulted in the up-regulation of genes when compared to controls, most notably immunity-related proteins, trafficking proteins, and transcription factors.

  4. Regulation of the Alternative Oxidase Aox1 Gene in Chlamydomonas reinhardtii. Role of the Nitrogen Source on the Expression of a Reporter Gene under the Control of the Aox1 Promoter1

    PubMed Central

    Baurain, Denis; Dinant, Monique; Coosemans, Nadine; Matagne, René F.

    2003-01-01

    In higher plants, various developmental and environmental conditions enhance expression of the alternative oxidase (AOX), whereas its induction in fungi is mainly dependent on cytochrome pathway restriction and triggering by reactive oxygen species. The AOX of the unicellular green alga Chlamydomonas reinhardtii is encoded by two different genes, the Aox1 gene being much more transcribed than Aox2. To analyze the transcriptional regulation of Aox1, we have fused its 1.4-kb promoter region to the promoterless arylsulfatase (Ars) reporter gene and measured ARS enzyme activities in transformants carrying the chimeric construct. We show that the Aox1 promoter is generally unresponsive to a number of known AOX inducers, including stress agents, respiratory inhibitors, and metabolites, possibly because the AOX activity is constitutively high in the alga. In contrast, the Aox1 expression is strongly dependent on the nitrogen source, being down-regulated by ammonium and stimulated by nitrate. Inactivation of nitrate reductase leads to a further increase of expression. The stimulation by nitrate also occurs at the AOX protein and respiratory levels. A deletion analysis of the Aox1 promoter region demonstrates that a short upstream segment (−253 to +59 with respect to the transcription start site) is sufficient to ensure gene expression and regulation, but that distal elements are required for full gene expression. The observed pattern of AOX regulation points to the possible interaction between chloroplast and mitochondria in relation to a potential increase of photogenerated ATP when nitrate is used as a nitrogen source. PMID:12644691

  5. Molecular assays in detecting EGFR gene aberrations: an updated HER2-dependent algorithm for interpreting gene signals; a short technical report.

    PubMed

    Tsiambas, Evangelos; Ragos, Vasileios; Lefas, Alicia Y; Georgiannos, Stavros N; Rigopoulos, Dimitrios N; Georgakopoulos, Georgios; Stamatelopoulos, Athanasios; Grapsa, Dimitra; Syrigos, Konstantinos

    2016-01-01

    Purpose: Among oncogenes that have already been identified and cloned, Epidermal Growth Factor Receptor (EGFR) remains one of the most significant. Understanding its deregulation mechanisms improves critically patients' selection for personalized therapies based on modern molecular biology and oncology guidelines. Anti-EGFR targeted therapeutic strategies have been developed based on specific genetic profiles and applied in subgroups of patients suffering by solid cancers of different histogenetic origin. Detection of specific EGFR somatic mutations leads to tyrosine kinase inhibitors (TKIs) application in subsets of them. Concerning EGFR gene numerical imbalances, identification of pure gene amplification is critical for targeting the molecule via monoclonal antibodies (mAbs). In the current technical paper we demonstrate the main molecular methods applied in EGFR analyses focused also on new data in interpreting numerical imbalances based on ASCO/ACAP guidelines for HER2 in situ hybridization (ISH) clarifications.

  6. [A Systematic Analysis of Oncogene and Tumor Suppressor Genes for Panitumumab-Resistant Rectal Cancer with Wild RAS Gene - A Case Report].

    PubMed

    Tajima, Yosuke; Shimada, Yoshifumi; Yagi, Ryoma; Okamura, Takuma; Nakano, Masato; Kameyama, Hitoshi; Nogami, Hitoshi; Maruyama, Satoshi; Takii, Yasumasa; Miura, Kohei; Ichikawa, Hiroshi; Nagahashi, Masayuki; Sakata, Jun; Kobayashi, Takashi; Wakai, Toshifumi

    2016-11-01

    A 58-year-old man was admitted with the complaint of bloody stools. Colonoscopy and computed tomography revealed a rectal cancer with a liver metastasis and multiple lung metastases. After administering a regimen comprising 3 courses of XELOX plus bevacizumab chemotherapy, the sizes of the primary and metastatic lesions decreased remarkably. Abdominoperineal resection was performed for local control of the cancer; the specimen from the initial tumor was found to be KRAS wild type. After 14 courses of XELOX chemotherapy, brain metastases were detected. Although 3 courses of IRIS plus panitumumab were administered, the liver, lung, and brain metastases spread rapidly. A comprehensive genomic analysis focused on cancer-related genes with CancerPlex®found a mutation of the BRAF gene(I326V). BRAF is a downstream molecule of KRAS in the RAS-RAF-MAPK pathway. Therefore, this mutation of the BRAF gene has the possibility of causing resistance against panitumumab that was found in this case. Furthermore, we expect that the systematic analysis of oncogene and suppressor oncogenes will enable us to choose the optimal regimen of chemotherapy or molecular targeting therapy for each patient with colorectal cancer.

  7. Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

    PubMed Central

    Miller, S M; Magasanik, B

    1991-01-01

    We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive. Images PMID:1682801

  8. Quantitative prediction of in vivo profiles of CYP3A4 induction in humans from in vitro results with a reporter gene assay.

    PubMed

    Kozawa, Masanari; Honma, Masashi; Suzuki, Hiroshi

    2009-06-01

    Although primary human hepatocytes are commonly used for induction studies, the evaluation method is associated with several problems. More recently, a reporter gene assay has been suggested to be an alternative, although the contribution of only transfected nuclear receptors can be evaluated. The aim of the present study was to establish a method by which the extent of in vivo CYP3A4 induction in humans can be quantitatively predicted based on in vitro results with a reporter gene assay. From previous reports, we calculated in vivo induction ratios (R(in vivo)) caused by prototypical inducers based on the alterations in the hepatic intrinsic clearance of probe drugs. Next, we derived equations by which these R(in vivo) values can be predicted from the results of a reporter gene assay. To use the data obtained from a reporter gene assay, rifampicin was used as a reference drug. The correction coefficient (CC), which is used to quantitatively correlate the activity of inducers between in vitro and in vivo situations, was calculated by comparing the predicted data with the observed R(in vivo) values for rifampicin. With the calculated CC value, good correlations were found between the predicted and observed R(in vivo) values for other inducers such as phenobarbital, phenytoin, and omeprazole. Taken together, with the equations derived in the present study, we have been able to predict the extent of in vivo induction of human CYP3A4 by inducers in a time-dependent and quantitative manner from in vitro data.

  9. Isolation of gene fusions (soi::lacZ) inducible by oxidative stress in Escherichia coli.

    PubMed Central

    Kogoma, T; Farr, S B; Joyce, K M; Natvig, D O

    1988-01-01

    Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin. The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism. Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions. On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response. Introduction of oxyR, htpR, or recA mutations did not affect the induction. Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide. The third fusion strain showed no increased sensitivity to either agent. All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome. PMID:2838846

  10. [Homozygous E387K (1159G>A) mutation of the CYP1B1 gene in a Roma boy affected with primary congenital glaucoma. Case report].

    PubMed

    Vogt, Gábor; Kádasi, Ľudevit Lajos; Czeizel, Endre

    2014-08-17

    Primary congenital glaucoma was diagnosed in a son (born in 2009) of a healthy, non-consanguineous Roma couple. This couple terminated their next two pregnancies because of the 25% recurrence risk of this autosomal recessive ophthalmological abnormality. Molecular genetic analysis showed the homozygote E387K mutation of the CYP1B1 gene in the proband and the presence of this gene mutation in heterozygous form in both parents. This gene mutation is characteristic for Slovakian Roma population. There are two objectives of this case report. On one hand this finding indicates the genetic relationship of Slovakian and Hungarian Romas. On the other hand, the couple plans to have further pregnancies, and prenatal genetic test may help to assess the possible recurrence risk of this hereditary disease.

  11. Molecular Study of Three Lebanese and Syrian Patients with Waardenburg Syndrome and Report of Novel Mutations in the EDNRB and MITF Genes

    PubMed Central

    Haddad, N.M.; Ente, D.; Chouery, E.; Jalkh, N.; Mehawej, C.; Khoueir, Z.; Pingault, V.; Mégarbané, A.

    2011-01-01

    Waardenburg syndrome (WS) is a genetic disorder characterized primarily by depigmentation of the skin and hair, heterochromia of the irides, sensorineural deafness, and sometimes by dystopia canthorum, and Hirschsprung disease. WS presents a large clinical and genetic heterogeneity. Four different types have been individualized and linked to 5 different genes. We report 2 cases of WS type II and 1 case of WS type IV from Lebanon and Syria. The genetic studies revealed 2 novel mutations in the MITF gene of the WS type II cases and 1 novel homozygous mutation in the EDNRB gene of the WS type IV case. This is the first molecular study of patients from the Arab world. Additional cases will enable a more detailed description of the clinical spectrum of Waardenburg syndrome in this region. PMID:21373256

  12. The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression

    PubMed Central

    Gasanov, N. B.; Toshchakov, S. V.; Georgiev, P. G.; Maksimenko, O. G.

    2015-01-01

    Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production. PMID:26483962

  13. Phosphatase and tensin homolog (PTEN) gene mutations and autism: literature review and a case report of a patient with Cowden syndrome, autistic disorder, and epilepsy.

    PubMed

    Conti, Sara; Condò, Maria; Posar, Annio; Mari, Francesca; Resta, Nicoletta; Renieri, Alessandra; Neri, Iria; Patrizi, Annalisa; Parmeggiani, Antonia

    2012-03-01

    Phosphatase and tensin homolog (PTEN) gene mutations are associated with a spectrum of clinical disorders characterized by skin lesions, macrocephaly, hamartomatous overgrowth of tissues, and an increased risk of cancers. Autism has rarely been described in association with these variable clinical features. At present, 24 patients with phosphatase and tensin homolog gene mutation, autism, macrocephaly, and some clinical findings described in phosphatase and tensin homolog syndromes have been reported in the literature. We describe a 14-year-old boy with autistic disorder, focal epilepsy, severe and progressive macrocephaly, and multiple papular skin lesions and palmoplantar punctate keratoses, characteristic of Cowden syndrome. The boy has a de novo phosphatase and tensin homolog gene mutation. Our patient is the first case described to present a typical Cowden syndrome and autism associated with epilepsy.

  14. Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1991--August 31, 1992

    SciTech Connect

    Barkan, A.

    1992-12-31

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene e