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Sample records for lacz reporter gene

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Karttunen, Jaana; Shastri, Nilabh

    1991-05-01

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

  3. Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene.

    PubMed Central

    Karttunen, J; Shastri, N

    1991-01-01

    We have used the bacterial beta-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic beta-galactosidase substrate fluorescein di-beta-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/Ab complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands. PMID:1902576

  4. A lacZ Reporter-Based Strategy for Rapid Expression Analysis and Target Validation of Mycobacterium tuberculosis Latent Infection Genes.

    PubMed

    Sood, Shivani; Kaur, Satinder; Shrivastava, Rahul

    2016-02-01

    We report a novel lacZ fusion vector and demonstrate its utility for expression analysis of genes associated with Mycobacterium tuberculosis latent infection. The vector contains E. coli (oriE) and mycobacterial (oriM) origins of replication, a kanamycin resistance gene (Km(r)) as selection marker, and a lacZ reporter gene in fusion with MCS for cloning of upstream regulatory sequence of the desired genes. ?-galactosidase activity of the vector was standardized for expression analysis under latent mycobacterial conditions using Phsp60, a constitutive mycobacterial promoter, utilizing Mycobacterium smegmatis as model organism. Validation of the vector was done by cloning and expression analysis of PhspX (alpha crystalline) and Picl (isocitrate lyase), promoters from two of the genes shown to be involved in M. tuberculosis persistence. Both genes showed appreciable levels of ?-galactosidase expression under hypoxia-induced persistent conditions in comparison to their actively replicating state. Expression analysis of a set of hypothetical genes was also done, of which Rv0628c showed increased expression under persistent conditions. The reported fusion vector and the strategy can be effectively used for short listing and validation of drug targets deduced from various non-conclusive approaches such as bioinformatics and microarray analysis against latent/persistent form of mycobacterial infection. PMID:26597215

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

    PubMed

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

    1993-01-01

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

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

    PubMed

    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; Ramrez-Solis, Ramiro; Steel, Karen P; Galli, Antonella; White, Jacqueline K

    2015-11-01

    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

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

    PubMed Central

    Armit, Chris

    2015-01-01

    ABSTRACT 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. PMID:26512121

  10. Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.

    PubMed Central

    Reddy, S; Rayburn, H; von Melchner, H; Ruley, H E

    1992-01-01

    Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice. Images PMID:1495960

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

    PubMed

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

    2002-11-01

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

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

    PubMed Central

    Pestova, Ekaterina V.; Morrison, Donald A.

    1998-01-01

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

  13. An rne-1 pnp-7 double mutation suppresses the temperature-sensitive defect of lacZ gene expression in a divE mutant.

    PubMed

    Aiso, T; Ohki, R

    1998-03-01

    A divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, exhibits differential loss of the synthesis of certain proteins, such as beta-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In Escherichia coli, the UCA codon is recognized only by tRNA1Ser. Several genes containing UCA codons are normally expressed after a temperature shift to 42 degrees C in the divE mutant. Therefore, it is unlikely that the defect in protein synthesis at 42 degrees C is simply caused by a defect in the decoding function of the mutant tRNA1Ser. In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant. It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations. We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant. Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA1Ser, at 44 degrees C. We present a mechanism that may explain these results. PMID:9515904

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

    PubMed

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

    1999-01-15

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

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

    PubMed

    Jurez-Rodrguez, Mara Dolores; Torres-Escobar, Ascencin; 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 (?qseB) mutant and lsrRK double deletion mutants (?lsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria. PMID:23353051

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Differential expression of lacZ in the liver and kidney of transgenic mice carrying chimeric lacZ-erythropoietin gene constructs with or without its 1.2 kb 3'-flanking sequence.

    PubMed Central

    Haidar, M A; Loya, F; Yang, Y; Lin, H; Glassman, A; Keating, M J; Goldwasser, E; Albitar, M

    1996-01-01

    Erythropoietin (EPO) plays a key role in erythropoiesis and is expressed predominantly in the fetal liver and in the adult kidney. The EPO gene is up-regulated at the transcriptional level under hypoxic/anemic conditions. We studied the role of the 5'- and 3'-flanking sequences of the mouse EPO gene in its tissue-specific and hypoxia-induced expression by developing transgenic mouse lines carrying chimeric EPO-lacZ gene constructs. Transgenic mice carrying a 6.5 kb segment of the 5'-sequence and most of the EPO gene in which lacZ was substituted for exon 1 (5'-lacZ-EPO) demonstrated induction of lacZ expression following hypoxia/ anemia induction in both the liver and kidney of adult mice. However, transgenic mice carrying the above construct along with the 1.2 kb 3'-flanking sequence (5'-lacZ-EPO-3') showed a high level of lacZ expression following hypoxia/anemia induction in adult kidney but not in adult liver. With the aim of further understanding the role of the 3'-flanking sequence in tissue-specific expression of the EPO gene, we studied the interactions of protein factors with this 1.2 kb 3' region and demonstrated that multiple sets of protein factors interact tissue specifically with a 10 bp sequence, TCAAAGATGG, located downstream of the previously characterized 3' hypoxia-responsive enhancer element. PMID:8836192

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

    PubMed

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

    2006-04-01

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

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

    PubMed Central

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

    1994-01-01

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

  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

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

    2015-06-01

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

  2. Elimination of UL56 gene by insertion of LacZ cassette between nucleotide position 116030 to 121753 of the herpes simplex virus type 1 genome abrogates intraperitoneal pathogenicity in tree shrews and mice.

    PubMed

    Rsen-Wolff, A; Lamad, W; Berkowitz, C; Becker, Y; Darai, G

    1991-08-01

    In order to investigate whether or not the UL56 gene is involved in those processes determining the viral pathogenicity and latency, a recombinant virus HSV-1-M-LacZ was constructed in which the DNA sequences between nucleotide position (np) 116030 and 121753 were replaced by the E. coli beta-galactosidase (LacZ) gene. This deletion spans from the carboxyterminus of UL55 (np 116030) to the second exon of IE110 (np 121753) eliminating UL56 and the variable region of the BamHI DNA fragment B which were implicated in intraperitoneal pathogenicity and latency. The host range and growth kinetics of the recombinant virus HSV-1 M-LacZ were comparable to the parental strain HSV-1 F. As expected it was found that HSV-1-M-LacZ lost its virulent phenotype and was not able to develop acute infection in animals. The state of the UL56 gene was investigated by determining the cDNA sequence of the UL56 gene transcript of HSV-1 F using PCR products obtained after amplification of the cDNA with oligonucleotide primers corresponding to the translational start and stop codons of this gene. This analysis revealed that the DNA sequence of the UL56 gene of HSV-1 F differed from those DNA sequences determined for the genomic DNA of HSV-1 strain 17. Between nucleotide position 116343 and 116344 two nucleotides -AG- are inserted which prolong the ORF of the UL56 gene to 233 amino acids with a predicted molecular weight of 30 kDa. PMID:1662844

  3. 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.

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

    PubMed

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

    2007-11-30

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

  5. Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

    PubMed Central

    Feustel, Lothar; Nakotte, Stephan; Drre, Peter

    2004-01-01

    The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding ?-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in ?-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression. adc (encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower). The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data). lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter. PMID:14766557

  6. The design of a new mutation model for active genes: expression of the Escherichia coli lac operon in mammalian cells.

    PubMed

    van Sloun, P P; Lohman, P H; Vrieling, H

    1997-09-01

    The design of a novel transgenic mouse model is described that should allow analysis of mutations at a single cell level in all tissues of a model animal. The model is based on the correct regulation of the Escherichia coli lac operon in mammalian cells. Induction of a mutation in the lacI gene will result in the loss of transcriptional repression of the lacZ gene in mutated cells. Expression of beta-galactosidase can subsequently be detected at the single cell level. The model was first tested in vitro using transfection of mouse LTK- cells. LacZ expression was very heterogeneous in most of the stable transfectants and seemed to be subject to epigenetic inactivation. One clone (IIB1) was isolated that stably expressed lacZ in more than 99% of its cells. Subsequent introduction of the lacI gene into IIB1 cells resulted in correct transcriptional repression of the lacZ gene that could be alleviated by IPTG, an allosteric inducer of lacI repression. However, in time the extent of beta-galactosidase induction gradually declined suggesting that the prolonged repressed transcriptional state triggers epigenetic inactivation. Variegated expression of the lacZ gene was not confined to cultured cells since several transgenic lines also did not express the lacZ transgene. This study shows that while the susceptibility of the lacZ gene to inactivation processes poses a fundamental problem, correct regulation of the expression of a reporter gene by the lacI repressor protein is feasible in mammalian cells when assayed at the single cell level. Thus, the model can in principle be used for the detection of mutagenic events at the lacI locus. Targeting of the lacZ gene to an endogenous housekeeping gene might prevent epigenetic inactivation. Alternatively, with the use of another reporter gene in the mutation detection system the proposed transgenic mouse model could be realized. PMID:9360635

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

    PubMed Central

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

    2001-01-01

    A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 ?M sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 ?M. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves. PMID:11229926

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

    PubMed

    Wei, Jessica; Chamberlain, Joel R

    2014-01-01

    RNAi has potential for therapeutically downregulating the expression of dominantly inherited genes in a variety of human genetic disorders. Here we used the ROSA26 mouse, which constitutively expresses the bacterial lacZ gene in tissues body wide, as a model to test the ability to downregulate gene expression in striated muscles. Recombinant adeno-associated viral vectors (rAAVs) were generated that express short hairpin RNAs (shRNAs) able to target the lacZ mRNA. Systemic delivery of these rAAV6 vectors led to a decrease of ?-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

  9. 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. PMID:26723900

  10. Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.

    PubMed Central

    Sepp, K J; Auld, V J

    1999-01-01

    Since the development of the enhancer trap technique, many large libraries of nuclear localized lacZ P-element stocks have been generated. These lines can lend themselves to the molecular and biological characterization of new genes. However they are not as useful for the study of development of cellular morphologies. With the advent of the GAL4 expression system, enhancer traps have a far greater potential for utility in biological studies. Yet generation of GAL4 lines by standard random mobilization has been reported to have a low efficiency. To avoid this problem we have employed targeted transposition to generate glial-specific GAL4 lines for the study of glial cellular development. Targeted transposition is the precise exchange of one P element for another. We report the successful and complete replacement of two glial enhancer trap P[lacZ, ry+] elements with the P[GAL4, w+] element. The frequencies of transposition to the target loci were 1.3% and 0.4%. We have thus found it more efficient to generate GAL4 lines from preexisting P-element lines than to obtain tissue-specific expression of GAL4 by random P-element mobilization. It is likely that similar screens can be performed to convert many other P-element lines to the GAL4 system. PMID:10049925

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

    SciTech Connect

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

    1994-09-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Gould, B R; Zingg, H H

    2003-01-01

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

  16. Magnetic Resonance Reporter Gene Imaging

    PubMed Central

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

    2012-01-01

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

  17. Expression of a reporter gene interrupted by the Candida albicans group I intron is inhibited by base analogs.

    PubMed

    Mercure, S; Cousineau, L; Montplaisir, S; Belhumeur, P; Lemay, G

    1997-01-15

    We previously reported the identification of an intron (CaLSU) in the 25S ribosomal RNA of some Candida albicans yeast strains. CaLSU was shown to self-splice and has the potential to adopt a secondary structure typical of group I introns. The presence of CaLSU inC. albicans strains correlates with a high degree of susceptibility to base analog antifungal agents, 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). Cell death, resulting from addition of base analogs to growing cultures, precluded demonstration of a causal relationship between CaLSU presence and susceptibility to base analogs. In the present study, CaLSU was inserted in a non-essential lacZ reporter gene and expression was examined in Saccharomyces cerevisiae. Different mutations affecting in vitro self-splicing also had similar effects on reporter gene expression in vivo. This indicates that in vivo removal of CaLSU from the reporter gene occurs through the typical self-splicing mechanism of group I introns. Base analogs inhibited expression of the reporter gene product in a concentration-dependent manner upon their addition to the cultures. This supports a model in which disruption of intron secondary structure, consecutive to the incorporation of nucleotide analogs, is a major factor determining the susceptibility of C.albicans cells to base analogs. PMID:9016575

  18. Expression of a reporter gene interrupted by the Candida albicans group I intron is inhibited by base analogs.

    PubMed Central

    Mercure, S; Cousineau, L; Montplaisir, S; Belhumeur, P; Lemay, G

    1997-01-01

    We previously reported the identification of an intron (CaLSU) in the 25S ribosomal RNA of some Candida albicans yeast strains. CaLSU was shown to self-splice and has the potential to adopt a secondary structure typical of group I introns. The presence of CaLSU inC. albicans strains correlates with a high degree of susceptibility to base analog antifungal agents, 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). Cell death, resulting from addition of base analogs to growing cultures, precluded demonstration of a causal relationship between CaLSU presence and susceptibility to base analogs. In the present study, CaLSU was inserted in a non-essential lacZ reporter gene and expression was examined in Saccharomyces cerevisiae. Different mutations affecting in vitro self-splicing also had similar effects on reporter gene expression in vivo. This indicates that in vivo removal of CaLSU from the reporter gene occurs through the typical self-splicing mechanism of group I introns. Base analogs inhibited expression of the reporter gene product in a concentration-dependent manner upon their addition to the cultures. This supports a model in which disruption of intron secondary structure, consecutive to the incorporation of nucleotide analogs, is a major factor determining the susceptibility of C.albicans cells to base analogs. PMID:9016575

  19. An Integrated Approach to Functional Genomics: Construction of a Novel Reporter Gene Fusion Library for Sinorhizobium meliloti?

    PubMed Central

    Cowie, Alison; Cheng, Jiujun; Sibley, Christopher D.; Fong, Ying; Zaheer, Rahat; Patten, Cheryl L.; Morton, Richard M.; Golding, G. Brian; Finan, Turlough M.

    2006-01-01

    As a means of investigating gene function, we developed a robust transcription fusion reporter vector to measure gene expression in bacteria. The vector, pTH1522, was used to construct a random insert library for the Sinorhizobium meliloti genome. pTH1522 replicates in Escherichia coli and can be transferred to, but cannot replicate in, S. meliloti. Homologous recombination of the DNA fragments cloned in pTH1522 into the S. meliloti genome generates transcriptional fusions to either the reporter genes gfp+ and lacZ or gusA and rfp, depending on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298 clones were identified by DNA sequence analysis, and the plasmid clones were recombined into S. meliloti. Reporter enzyme activities following growth of these recombinants in complex medium (LBmc) and in minimal medium with glucose or succinate as the sole carbon source allowed the identification of genes highly expressed under one or more growth condition and those expressed at very low to background levels. In addition to generating reporter gene fusions, the vector allows Flp recombinase-directed deletion formation and gene disruption, depending on the nature of the cloned fragment. We report the identification of genes essential for growth on complex medium as deduced from an inability to recover recombinants from pTH1522 clones that carried fragments internal to gene or operon transcripts. A database containing all the gene expression activities together with a web interface showing the precise locations of reporter fusion junctions has been constructed (www.sinorhizobium.org). PMID:16963549

  20. 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-2? in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock-in allele. Inka1-LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1?/? mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP-2? is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not. PMID:20175189

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    2015-12-01

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

  4. Aequorin as a reporter gene.

    PubMed

    Plieth, Christoph

    2006-01-01

    Reporter proteins allow one to monitor cellular parameters that are involved in signal transduction, development, metabolic processes, and transport. There are targeting strategies available to direct the indicator protein exactly to the locale inside the organism from which information is desired. This circumvents experimental reductionism and allows experimentation with whole intact and undisturbed organisms. The outstanding advantages of self-reporting organisms make it worth to shoulder cost- and time-consuming molecular work. Here, the luminescent Ca2+ indicator aequorin is introduced and a rough guideline is given from early planning the molecular work and assembling an experimental setup to experimentation with luminescent Arabidopsis, data processing, and control experiments. PMID:16739587

  5. Reporter gene imaging: potential impact on therapy.

    PubMed

    Serganova, Inna; Blasberg, Ronald

    2005-10-01

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

  6. Patterns of gene expression in Bacillus subtilis colonies.

    PubMed Central

    Salhi, B; Mendelson, N H

    1993-01-01

    Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate. Images PMID:8349543

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

    PubMed

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

    1999-02-01

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

  8. Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging.

    PubMed Central

    Yuan, D S

    2000-01-01

    The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0. 1 vs. 10 microm) zinc media were isolated and the gene fusions were sequenced. Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells. All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1. ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells. ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake. These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast. PMID:10978274

  9. Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging.

    PubMed

    Yuan, D S

    2000-09-01

    The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0. 1 vs. 10 microm) zinc media were isolated and the gene fusions were sequenced. Ribonuclease protection assays demonstrated four- to eightfold increases for the RNAs of the ZAP1, ZRG17 (YNR039c), DPP1, ADH4, MCD4, and YEF3B genes in zinc-deficient cells. All but YEF3B were shown through reporter gene assays to be controlled by a master regulator of zinc homeostasis now known to be encoded by ZAP1. ZAP1 mutants lacked the flocculence and distended vacuoles characteristic of zinc-deficient cells, suggesting that flocculation and vacuolation serve homeostatic functions in zinc-deficient cells. ZRG17 mutants required extra zinc supplementation to repress these phenotypes, suggesting that ZRG17 functions in zinc uptake. These findings illustrate the utility of transposon tagging as an approach for studying regulated gene expression in yeast. PMID:10978274

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

    PubMed Central

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

    1997-01-01

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

  11. Identification of the ?4406 Regulatory Region, a Developmental Promoter of Myxococcus xanthus, and a DNA Segment Responsible for Chromosomal Position-Dependent Inhibition of Gene Expression

    PubMed Central

    Loconto, Jennifer; Viswanathan, Poorna; Nowak, Scott J.; Gloudemans, Monica; Kroos, Lee

    2005-01-01

    When starved, Myxococcus xanthus cells send signals to each other that coordinate their movements, gene expression, and differentiation. C-signaling requires cell-cell contact, and increasing contact brought about by cell alignment in aggregates is thought to increase C-signaling, which induces expression of many genes, causing rod-shaped cells to differentiate into spherical spores. C-signaling involves the product of the csgA gene. A csgA mutant fails to express many genes that are normally induced after about 6 h into the developmental process. One such gene was identified by insertion of Tn5 lac at site ?4406 in the M. xanthus chromosome. Tn5 lac fused transcription of lacZ to the upstream ?4406 promoter. In this study, the ?4406 promoter region was identified by analyzing mRNA and by testing different upstream DNA segments for the ability to drive developmental lacZ expression in M. xanthus. The 5? end of ?4406 mRNA mapped to approximately 1.3 kb upstream of the Tn5 lac insertion. A 1.0-kb DNA segment from 0.8 to 1.8 kb upstream of the Tn5 lac insertion, when fused to lacZ and integrated at a phage attachment site in the M. xanthus chromosome, showed a similar pattern of developmental expression as Tn5 lac ?4406. The DNA sequence upstream of the putative transcriptional start site was strikingly similar to promoter regions of other C-signal-dependent genes. Developmental lacZ expression from the 1.0-kb segment was abolished in a csgA mutant but was restored upon codevelopment of the csgA mutant with wild-type cells, which supply C-signal, demonstrating that the ?4406 promoter responds to extracellular C-signaling. Interestingly, the 0.8-kb DNA segment immediately upstream of Tn5 lac ?4406 inhibited expression of a downstream lacZ reporter in transcriptional fusions integrated at a phage attachment site in the chromosome but not at the normal ?4406 location. To our knowledge, this is the first example in M. xanthus of a chromosomal position-dependent effect on gene expression attributable to a DNA segment outside the promoter region. PMID:15937177

  12. Effect of dietary supplementation on the frequency of spontaneous lacZ mutations in the developing colon.

    PubMed

    Trentin, G A; Moody, J; Shima, N; Thompson, L U; Heddle, J A

    2004-07-13

    Epidemiological studies have demonstrated that dietary modifications can reduce the incidence of cancer. Specifically, diets high in vegetables and fruits are associated with lower rates of cancer at many sites. Somatic mutations have a critical role in carcinogenesis suggesting the use of in vivo mutation assays as an alternative approach to studying the relationship between diet and cancer. Since the rate of accumulation of spontaneous mutations is highest during growth and development early in life, we tested whether certain foods as dietary supplements could reduce the rate of mutation during this period using lacZ transgenic mice. Pregnant female mice were placed on a control diet or a diet supplemented to 20% final dry weight with broccoli, cabbage, carrots, flaxseed, green peas, green peppers, oranges or strawberries for the entire duration of their pregnancy and lactation. Mutation frequencies were subsequently measured at the lacZ transgene in colonic epithelial cells of the offspring at 3 weeks of age. A small number of measurements were also made on siblings at 8 weeks of age. While the control AIN-96G diet on its own resulted in lower mutant frequencies than had been observed in earlier experiments with lab chow, no significant reduction in mutant frequencies was detected for any of the foods tested as compared to the AIN-93G diet alone. Significantly more mutations were found at 3 weeks of age in mice fed diets supplemented with broccoli or oranges, but the result with oranges may be the result of jackpot mutations. PMID:15225595

  13. 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

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

    PubMed

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

    2009-05-01

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

  15. The application of reporter gene assays for the determination of the toxic potency of diffuse air pollution.

    PubMed

    Hamers, T; van Schaardenburg; Felzel, E C; Murk, A J; Koeman, J H

    2000-10-30

    Diffuse air pollution consists of a mixture of numerous compounds. It is emitted by many distributed sources and is omnipresent due to atmospheric transport. Risk assessment of the complex mixture of air pollutants on the basis of the toxicity of the individual compounds is not yet possible because the chemical identity and/or toxicity of the constituencies of a substantial fraction is unknown. In addition, no adequate procedures are available to integrate toxicity data of such complex mixtures, so that an individual risk assessment of the constituents of air pollution disregards possible combination effects. In the present study, an approach has been developed to assess the toxic potency by using in vitro bio-assay techniques. Genotoxicity was assessed in the umu-assay, a reporter gene assay using a strain of Salmonella typhimurium stably transfected with a plasmid (pSK1002) carrying the SOS-gene umuC fused to the reporter gene lacZ. Arylhydrocarbon-receptor activation was assessed in the DR-CALUX-assay, using a stably transfected H4IIE hepatoma cell line containing a plasmid for the luciferase gene under transcriptional control of dioxin-responsive elements. Samples of airborne particulate matter (APM) were collected with a high volume sampler next to a highway and in a natural conservation area. Both assays proved to be applicable to quantify genotoxicity and the presence of polycyclic aromatic hydrocarbons (PAHs) in small extracts from air-filter samples. Results indicate that PAHs from traffic exhausts seem to be largely responsible for an increased genotoxic activity of APM collected down-wind from the highway (western wind). APM collected at eastern wind directions seems to have a different composition of compounds, with a higher genotoxic activity that is less related to highway-emitted PAH-like compounds. At northern wind directions, APM is relatively less genotoxic and contains less PAHs than at other wind directions. Dioxin-like compounds contribute negligibly to the Ah-receptor agonistic potency of APM. Airborne pollutants with genotoxic and/or PAH-like characteristics form an undesired mutagenic risk, which will be evaluated in further in vivo studies. PMID:11059851

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

    PubMed Central

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

    2014-01-01

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

  17. Transfection efficiency of pORF lacZ plasmid lipopolyplex to hepatocytes and hepatoma cells

    PubMed Central

    Sun, Xun; Zhang, Hong-Wei; Zhang, Zhi-Rong

    2004-01-01

    AIM: To develop a novel non-viral gene delivery system, which has a small particle size and a high transfection efficiency to hepatocyte and hepatoma cells. METHODS: Lipid-polycation-DNA lipopolyplex (LPD) was prepared by mixing plasmid DNA and polylysine. The resulted polyplex was incubated for 10 min at room temperature, following the addition of preformed cationic liposomes. The morphology of LPD was observed by transmission electron microscopy. The diameter and surface charge of LPD were measured by photon correlation spectroscopy (PCS). The nuclease protection ability of LPD was evaluated by agarose gel electrophoresis. Estimation of the transfection efficiency was performed by galactosidase assay in Chang cells and SMMC-7721 cells. RESULTS: LPD had a regular spherical surface. The average diameter and the zeta potential of LPD were 132.1 nm and 26.8 mV respectively. LPD could protect plasmid DNA from nuclease degradation after 2 hours incubation at 37 C while the naked DNA degraded rapidly. The average transfection efficiencies were 86.2% 8.9% and 72.4% 6.5% in Chang cells and SMMC-7721 cells respectively. CONCLUSION: LPD has a rather small particle size and a high transfection activity. LPD may be a good non-viral vector for application in some gene delivery. PMID:14966911

  18. Simultaneous gene inactivation and promoter reporting in cyanobacteria.

    PubMed

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

    2015-02-01

    Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0% of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72h, decreased 49.6% compared to wild-type. Thus, we discovered that All2508may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms. PMID:25434810

  19. Characterization of a gene trap insertion into a novel gene, cordon-bleu, expressed in axial structures of the gastrulating mouse embryo.

    PubMed

    Gasca, S; Hill, D P; Klingensmith, J; Rossant, J

    1995-01-01

    We have used a gene trap (GT) vector and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. GT vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. In this paper we describe a GT insertion which displays a very restricted pattern of expression in the gastrulating embryo. beta-Galactosidase activity was first detected at 7.5 days post-coitum (E7.5) in the node region of the embryo and extended to the midline structures at E8.0. At E9.5 expression was restricted to the floor plate, the notochord, the roof of the gut, and the liver anlage. Expression appeared in the somites at E10.0 and later became more widespread. We used rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to clone a partial 360 base pair (bp) cDNA representing an endogenous sequence and containing an open reading frame (ORF) fused in frame to the lacZ reporter gene. The sequence showed no homology to any known protein or protein domain. An overlapping 1,200 bp fragment from a wild-type cDNA library was cloned and it detected the same pattern of expression as the reporter gene in E7.5, E8.5, and E9.5 wild-type embryos. It hybridized to a 5.4 kb lacZ fusion transcript and to an endogenous transcript of 6.5 kb. The gene was mapped to chromosome 11 and was named cordon-bleu (cobl). No phenotype was detected in mice homozygous for the insertion. However, the insertion may not cause a complete disruption of the gene function. The pattern of expression of cobl is very similar to that of hepatic nuclear factor 3 beta (HNF3 beta) and sonic hedgehog (Shh), both of which are involved in axial patterning. Therefore, the product of the cobl gene may also prove to be an important component of the genetic pathway regulating vertebrate axis formation. PMID:7586755

  20. Luciferase as a reporter of gene activity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2007-07-01

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

  2. Cloning-free regulated monitoring of reporter and gene expression

    PubMed Central

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

    2009-01-01

    Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile. PMID:19267938

  3. 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.

  4. 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.

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

    PubMed

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

    2013-06-15

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

  6. Long-Term Expression of the Human CFTR Gene in Mouse Airway via Helper-Dependent Adenoviral Vector Delivery and Transient Immunosuppression.

    PubMed

    Cao, Huibi; Wu, Jing; Duan, Cathleen; Du, Kai; Lee, Chan Mi; Yeger, Herman; Hu, Jim

    2016-01-01

    Sustained expression of the CFTR gene is a major challenge to gene therapy with either viral or nonviral vectors with immune response to vector and transgene products. One strategy to achieve sustained CFTR expression is to modulate the host immune system through transient immunosuppression. In this study, we examined cyclophosphamide (cytoxan), dexamethasone (Dex), and a combination of cyclosporin, methylprednisolone, and azathioprine (combination) for their effects on long-term expression of the human CFTR delivered with helper-dependent adenoviral vectors in mouse airways. We found that cyclophosphamide significantly enhanced long-term expression of the transgenic human CFTR and the reporter gene LacZ by reducing host immune responses. Dex administration greatly reduced neutralizing antibody production but had no effect on transgene expression. Treatment with a combination of cyclosporin A, azathioprine, and methylprednisolone affected neither CFTR gene expression nor inflammation. Our data suggest that transient immunosuppression might be a strategy to improve sustained expression in gene therapy. PMID:26710934

  7. Genetics in methylotrophic bacteria: Appendix. Final report

    SciTech Connect

    Lidstrom, M.E.

    1998-09-01

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

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

    PubMed

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

    2013-02-01

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2004-02-01

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

  11. Dual-modality gene reporter for in vivo imaging

    PubMed Central

    Patrick, P. Stephen; Hammersley, Jayne; Loizou, Louiza; Kettunen, Mikko I.; Rodrigues, Tiago B.; Hu, De-En; Tee, Sui-Seng; Hesketh, Robin; Lyons, Scott K.; Soloviev, Dmitry; Lewis, David Y.; Aime, Silvio; Fulton, Sandra M.; Brindle, Kevin M.

    2014-01-01

    The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd3+-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd3+ ion for the radionuclide, 111In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging. PMID:24347640

  12. Electrotransformation and Expression of Bacterial Genes Encoding Hygromycin Phosphotransferase and ?-Galactosidase in the Pathogenic Fungus Histoplasma capsulatum

    PubMed Central

    Woods, Jon P.; Heinecke, Elizabeth L.; Goldman, William E.

    1998-01-01

    We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional ?-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus. PMID:9529100

  13. K137R mutation on adeno-associated viral capsids had minimal effect on enhancing gene delivery in vivo.

    PubMed

    Qiao, Chunping; Li, Chengwen; Zhao, Chunxia; Li, Jianbin; Bian, Tao; Grieger, Joshua; Li, Juan; Samulski, R Jude; Xiao, Xiao

    2014-02-01

    The adeno-associated viral (AAV) vector has emerged as an attractive vector for gene therapy applications. Development of AAV vectors with enhanced gene transduction efficiency is important to ease the burden of AAV production and minimize potential immune responses. Rational mutations on AAV capsids have gained attention as a simple method of enhancing AAV transduction efficiency. A single-amino acid mutation, K137R, on AAV1 and AAV8 was recently reported to increase liver transgene expression by 5-10-fold. To determine whether the same mutation on other AAV serotypes would result in similar gene enhancement effects, K137R mutants were generated on AAV7, AAV8, and AAV9, and their effects were evaluated in vivo. Two reporter genes were utilized: the nuclear LacZ gene driven by the cytomegalovirus promoter and the luciferase gene driven by the CB promoter. Surprisingly, we found no difference in luciferase gene expression in the liver or other tissues using either the wild-type AAV8 capsid or AAV8-K137R. LacZ gene expression in the liver by AAV8-K137R was about onefold higher than that of wild-type AAV8. However, no difference was found in other tissues, such as skeletal muscle and cardiac muscle. In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo. PMID:24116972

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  15. Evaluating Reported Candidate Gene Associations with Polycystic Ovary Syndrome

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

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

    2009-10-23

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

  17. A reporter gene construct for studying the regulation of manganese peroxidase gene expression.

    PubMed Central

    Godfrey, B J; Akileswaran, L; Gold, M H

    1994-01-01

    The orotidylate decarboxylase (ODase) gene (ura1) from Schizophyllum commune was utilized as a reporter for studying Mn regulation of the manganese peroxidase (MnP) gene (mnp) from the lignin-degrading basidiomycete Phanerochaete chrysosporium. A 1,500-bp fragment of the mnp1 promoter was fused upstream of the coding region of the ODase gene in a plasmid (pAMO) containing the S. commune ade5 gene as a selectable marker. pAMO was used to transform a P. chrysosporium ade1 ura11 mutant lacking endogenous ODase activity. When the P. chrysosporium transformant was grown in nitrogen-limited, Mn(II)-sufficient cultures, ODase activity was detected only during secondary metabolic growth and the pattern of ODase expression was similar to that of endogenous MnP. When Mn was added to 6-day-old nitrogen-limited, Mn-deficient cultures, both ODase activity and MnP activity were induced synchronously with maximal activity at 30 h. Growth in high-nitrogen-concentration medium suppressed the induction of both the ODase and endogenous MnP. These results indicate that this promoter-reporter construct can be used to study the regulation of the mnp gene. PMID:8017922

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

    PubMed Central

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

    2012-01-01

    Treatment of chondrosarcoma remains a major challenge in orthopaedic oncology. Gene transfer strategies based on recombinant adenoassociated viral (rAAV) vectors may provide powerful tools to develop new, efficient therapeutic options against these tumors. In the present study, we tested the hypothesis that rAAV is adapted for a stable and safe delivery of foreign sequences in human chondrosarcoma tissue by transducing primary human chondrosarcoma cells in vitro and in situ with different reporter genes (E. coli lacZ, firefly luc, Discosoma sp. RFP). The effects of rAAV administration upon cell survival and metabolic activities were also evaluated to monitor possibly detrimental effects of the gene transfer method. Remarkably, we provide evidence that efficient and prolonged expression of transgene sequences via rAAV can be safely achieved in all the systems investigated, demonstrating the potential of the approach of direct application of therapeutic gene vectors as a means to treat chondrosarcoma. PMID:22645415

  19. 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...

  20. Inheritance of P element and reporter gene sequences in zebrafish.

    PubMed

    Gibbs, P D; Gray, A; Thorgaard, G

    1994-12-01

    We have established 15 separate lines of zebrafish transgenic for two plasmid DNAs containing Drosophila P element sequences and either the reporter genes chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), and beta-galactosidase (BGAL) or the reporter gene hygromycin phosphotransferase (HPT). Transient expression of CAT, but not NPT or BGAL, could be measured in the G0 (microinjected) generation, while expression in subsequent generations was below the limit of detection for CAT and NPT. All 15 lines contain stable low copy number integrations of between one and five transgene copies per cell with characteristic Southern blot "junction fragments" and have shown Mendelian inheritance after the G1 generation. The transgenes in 13 of 15 of the lines were originally inherited by about 10% or less of the G1 fish, suggesting a mosaic integration into the germline. Of 14 tested lines, none is associated with an embryonic recessive lethal phenotype as scored in the G3 generation. PMID:7704114

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

    PubMed Central

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

    1989-01-01

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

  2. Ferritin as a Novel Reporter Gene for Photoacoustic Molecular Imaging

    PubMed Central

    Ha, Seung Han; Carson, Andrew R.; Kim, Kang

    2013-01-01

    Reporter genes may serve as endogenous contrast agents in the field of photoacoustic (PA) molecular imaging (PMI), enabling greater characterization of detailed cellular processes and disease progression. To demonstrate the feasibility of using ferritin as a reporter gene, human melanoma SK-24 (SK-MEL-24) cells were co-transfected with plasmid expressing human heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using lipofectamine 2000. Non-transfected SK-MEL-24 cells served as a negative control. Fluorescent imaging of GFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation due to ferritin overexpression in SK-MEL-24 cells, a focused high-frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (fluence < 5 mJ/cm2) was used to scan the PA signal at a wide range NIR wavelengths (850950 nm). PA signal intensity from H-FT transfected SK-MEL-24 cells was about 59 dB higher than nontransfected SK-MEL-24 cells at 850950 nm. Immunofluorescence and RT-PCR analysis both indicate high levels of ferritin expression in H-FT transfected SK-MEL24 cells, with little ferritin expression in nontransfected SK-MEL-24 cells. In this study, the feasibility of using ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a novel concept for PMI using an endogenous contrast agent and may provide various opportunities for molecular imaging and basic science research. PMID:22949299

  3. Defining strategies to extend duration of gene expression from targeted compacted DNA vectors.

    PubMed

    Ziady, A-G; Kim, J; Colla, J; Davis, P B

    2004-09-01

    Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1 -/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally (n=10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting. PMID:15269710

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

    PubMed Central

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

    2003-01-01

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

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

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

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

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

    SciTech Connect

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

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

  7. 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.

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

    PubMed

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

    2009-01-01

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

  9. Application of the mini-mu phage for the isolation of lac transcriptional fusions in Bacillus subtilis genes.

    PubMed

    Gardiol, D; Gramajo, H C; Hirschbein, L; de Mendoza, D

    1993-01-15

    A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome. This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids. The resultant fusions can be analyzed in B. subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment. PMID:8423002

  10. Pristinamycin-inducible gene regulation in mycobacteria.

    TOXLINE Toxicology Bibliographic Information

    Forti F; Crosta A; Ghisotti D

    2009-03-25

    In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria.

  11. Pristinamycin-inducible gene regulation in mycobacteria.

    PubMed

    Forti, Francesca; Crosta, Andrea; Ghisotti, Daniela

    2009-03-25

    In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria. PMID:19428723

  12. 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

  13. Insertion of a GFP Reporter Gene in Influenza Virus

    PubMed Central

    Perez, Jasmine T.; Garca-Sastre, Adolfo; Manicassamy, Balaji

    2013-01-01

    The incorporation of a fluorescent reporter gene into a replication competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of non-structural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then cloned into the IAV rescue system backbone. Upon construction of the GFP encoding segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP IAV can subsequently be used to visualize infected cells both in vitro and in vivo. PMID:23686828

  14. 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.

  15. 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.

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

    PubMed

    Kamm, Gretel B; Lpez-Leal, Rodrigo; Lorenzo, Juan R; Franchini, Luca F

    2013-12-19

    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

  17. Photoacoustic microscopy of tyrosinase reporter gene in vivo

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

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

    PubMed

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

    1980-08-01

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

  19. In vivo retroviral gene transfer into human bronchial epithelia of xenografts.

    PubMed Central

    Engelhardt, J F; Yankaskas, J R; Wilson, J M

    1992-01-01

    Cystic fibrosis (CF) is the most common lethal inherited disease in the Caucasian population with an incidence of approximately 1 in 2,500 live births. Pulmonary complications of CF, which are the most morbid aspects of the disease, are caused by primary abnormalities in epithelial cells that lead to impaired mucociliary clearance. One potential therapeutic strategy is to reconstitute expression of the CF gene in airway epithelia by somatic gene transfer. To this end, we have developed an animal model of the human airway using bronchial xenografts and have tested the efficiency of in vivo retroviral gene transfer. Using the LacZ reporter gene, we find the efficiency of in vivo retroviral gene transfer to be dramatically dependent on the regenerative and mitotic state of the epithelium. Within an undifferentiated regenerating epithelium in which 40% of nuclei labeled with BrdU, 5-10% retroviral gene transfer was obtained. In contrast, no gene transfer was noted in a fully differentiated epithelium in which 1% of nuclei labeled with BrdU. These findings suggest that retroviral mediated gene transfer to the airway in vivo may be feasible if the proper regenerative state can be induced. Images PMID:1281842

  20. [Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

    PubMed

    Wang, Guozhong; Hu, Shenjiang; Zheng, Zhelan; Sun, Jian; Zheng, Xia; Zhu, Zhaohui; Li, Jiang; Yao, Yumei

    2006-08-01

    To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle. PMID:17002125

  1. 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.

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

    PubMed Central

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

    1987-01-01

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

  3. 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.

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

    PubMed Central

    2012-01-01

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

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

    PubMed Central

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

    1998-01-01

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

  6. Stem cell gene therapy for fanconi anemia: report from the 1st international Fanconi anemia gene therapy working group meeting.

    PubMed

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

    2011-07-01

    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

  7. 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, Julin; 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

  8. Cellular and Molecular Factors in Flexor Tendon Repair and Adhesions: A Histological and Gene Expression Analysis

    PubMed Central

    Juneja, Subhash C.; Schwarz, Edward M.; OKeefe, Regis J.; Awad, Hani A.

    2013-01-01

    Flexor tendon healing is mediated by cell proliferation, migration, and ECM synthesis that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor tendon adhesion formation has been linked to TGF-?. To elucidate the cellular and molecular events in this pathology, we implanted live FDL grafts from the reporter mouse Rosa26LacZ/+ in WT recipients, and used histological ?-galactosidase (?-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and RT-PCR to measure gene expression of TGF-? and its receptors, extracellular matrix (ECM) proteins, and MMPs and their regulators. Over the course of healing, graft cellularity and ?-gal activity progressively increased, and ?-gal-positive cells migrated out of the Rosa26LacZ/+ graft. In addition, there was evidence of influx of host cells (?-gal-negative) into the gliding space and the graft, suggesting that both graft and host cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1 expression was highest in the early phases of healing and gradually decreased thereafter, whereas Tgfb3 increased and remained upregulated later. The expression of TGF-? receptors was also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which inflammatory MMPs were highest early and matrix MMPs increased over time. These findings offer important insights into the complex cellular and molecular factors during flexor tendon healing. PMID:23586515

  9. Functional analysis of the promoter region of amphioxus ?-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 ?-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 ?-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-? 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. PMID:25078982

  10. The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

    PubMed Central

    Xiao, W; Samson, L

    1992-01-01

    We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations. PMID:1641326

  11. Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two ?70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

    PubMed Central

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

    2002-01-01

    A mycobacteriophage Ms6 strong promoter region (Plys) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem ?70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region Plys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that ?-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation. PMID:12003945

  12. 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 ?-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 ?M using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for ?-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes. PMID:23485150

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

    Chory, J.

    1993-12-31

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

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

    PubMed

    Takahashi, Junko; Iwahashi, Hitoshi

    2004-01-01

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

  17. Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

    PubMed Central

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

    2005-01-01

    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 106 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 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (?109 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 ?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. PMID:16188984

  18. 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.

  19. Inflammatory bowel disease gene discovery. CRADA final report

    SciTech Connect

    1997-09-09

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

  20. Bioluminescent reporters for catabolic gene expression and pollutant bioavailability

    SciTech Connect

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

    1991-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

    Grant, S. R.

    1999-02-05

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

  4. 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. PMID:25886903

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

    PubMed

    Wang, Baomin; Wang, Yumei; Kennedy, Christina

    2014-10-01

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

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

    SciTech Connect

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

    1993-01-01

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

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

    SciTech Connect

    Haber, J. E.

    2002-11-30

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

  8. 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

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

    Gregor, D; Pfeifer, F

    2001-07-01

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

  12. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

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

    SciTech Connect

    Jeon, C.O.; Park, M.; Ro, H.S.; Park, W.; Madsen, E.L.

    2006-02-15

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

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

    SciTech Connect

    Kuchka, M.R.

    1992-05-01

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

  15. 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.

  16. 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.

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

    PubMed

    Zi?ko, E; Kokot, T; Skubis, A; Sikora, B; Szota-Czy?, J; Kruszniewska-Rajs, C; Wierzgo?, J; Mazurek, U; Grochowska-Niedworok, E; Muc-Wierzgo?, M

    2015-01-01

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

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

    PubMed Central

    Schaefer, T S; Hays, J B

    1991-01-01

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

  19. 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.

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

    PubMed

    Clment, Thomas; Salone, Vronique; Rederstorff, Mathieu

    2015-01-01

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

  1. A DNA fragment from the cyanobacterium Synechocystis sp. PCC 6803 mediates gene expression inducible by osmotic stress in E. coli.

    PubMed

    Milkowski, C; Quinones, A; Hagemann, M

    1998-08-01

    Fragments of Synechocystis-DNA driving salt-induced gene expression in E. coli were isolated with translational fusions to a 'lacZ gene. One fragment (fragment 19) showed a NaCl-dependent activation of betaGal expression with the maximum of a ninefold increase in enzyme activity. A similar induction was triggered by the nonionic osmolyte sucrose, indicating an osmotically dependent activation. On the contrary, transcriptional activity of the DNA fragment 19 was only slightly enhanced under salt stress conditions, suggesting a posttranscriptional mechanism of induction. Primer extension assay was performed to identify the transcription initiation site. Upstream regions share weak homology to the "-10" hexamer consensus of E. coli sigma70 promoters. The most thermodynamically stable secondary structure for the nontranslated part of the mRNA indicated that potential translation initiation sites might be blocked, leading to a low basal translation, whereas osmotic stress-induced changes of mRNA structure could be involved to increase translation. In order to analyze the function of fragment 19 in Synechocystis, promoter-probe plasmids were constructed allowing the stable integration of transcriptional and translational reporter gene fusions into the cyanobacterial chromosome. Quantitative assessment of reporter gene expression revealed a weak constitutive promoter activity of fragment 19 in Synechocystis. Sequence analysis showed that fragment 19 comprises 223 bp of the ORF sll0747 of the Synechocystis genome. PMID:9662610

  2. 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

    2015-11-01

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

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

    PubMed

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

    2015-10-15

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

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

    PubMed Central

    Snchez-Sutil, Mara Celestina; Prez, Juana; Gmez-Santos, Nuria; Shimkets, Lawrence J.; Moraleda-Muoz, Aurelio; Muoz-Dorado, Jos

    2013-01-01

    Myxococcus xanthus is a soil-dwelling member of the ?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 ?corSR 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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    2003-01-01

    Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features, and, in some cases, mental retardation. NHS has been mapped to a 1.3-Mb interval on Xp22.13. We have confirmed the same localization in the original, extended Australian family with NHS and have identified protein-truncating mutations in a novel gene, which we have called NHS, in five families. The NHS gene encompasses ?650 kb of genomic DNA, coding for a 1,630amino acid putative nuclear protein. NHS orthologs were found in other vertebrates, but no sequence similarity to known genes was identified. The murine developmental expression profile of the NHS gene was studied using in situ hybridization and a mouse line containing a lacZ reporter-gene insertion in the Nhs locus. We found a complex pattern of temporally and spatially regulated expression, which, together with the pleiotropic features of NHS, suggests that this gene has key functions in the regulation of eye, tooth, brain, and craniofacial development. PMID:14564667

  7. Effect of cell-surface glycosaminoglycans on cationic carrier combined with low-MW PEI-mediated gene transfection.

    PubMed

    Lampela, Pasi; Soininen, Pivi; Puttonen, Katja A; Ruponen, Marika; Urtti, Arto; Mnnist, Pekka T; Raasmaja, Atso

    2004-10-13

    Glycosaminoglycans (GAGs) are negatively charged polysaccharides that are found, e.g. on cell surface. GAGs have been reported to influence gene transfection. We have previously reported that cationic lipid-mediated gene transfection can be improved by combining a small polyethylenimine (PEI) with cationic lipids. In the present study, we examined if GAGs have any effect on the synergism of small PEIs and other cationic carriers. We used wild-type CHO (GAG+) and pgsB-618 cells (GAG-). Transfection efficiency was studied using lacZ and GFP reporter genes. We found that GAGs decreased the overall level of transgene expression in a reagent-dependent manner, but the synergism caused by low-MW PEIs was less affected. There were no major differences between cell lines in cellular uptake or intracellular localization of plasmids when measured with flow cytometry and confocal microscopy, respectively. In conclusion, cell-surface GAGs interfere with transfection efficiency of different cationic reagents, but that is not necessarily related to the synergy of small PEIs and cationic lipids. PMID:15454295

  8. 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 by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

  9. 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.

  10. Ebs1p, a Negative Regulator of Gene Expression Controlled by the Upf Proteins in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Ford, Amanda S.; Guan, Qiaoning; Neeno-Eckwall, Eric; Culbertson, Michael R.

    2006-01-01

    Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-? mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD. PMID:16467471

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

    Boivin, C; Camut, S; Malpica, CA; Truchet, G; Rosenberg, C

    1990-01-01

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

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

    PubMed

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

    2007-02-01

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

  14. 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.

  15. 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.

  16. 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 ?-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. PMID:22127754

  17. 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.

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

    PubMed

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

    2016-01-01

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

  19. 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

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

    PubMed

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

    1995-03-01

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

  1. A Recombinant Reporter System for Monitoring Reactivation of an Endogenously DNA Hypermethylated Gene

    PubMed Central

    Cui, Ying; Hausheer, Frederick; Beaty, Robert; Zahnow, Cynthia; Issa, Jean Pierre; Bunz, Frederick; Baylin, Stephen B.

    2014-01-01

    Reversing abnormal gene silencing in cancer cells due to DNA hypermethylation of promoter CpG islands may offer new cancer prevention or therapeutic approaches. Moreover, such approaches may be broadly applicable to enhance the efficacy of radiotherapy, chemotherapy or immunotherapy. Here we demonstrate the powerful utility of a novel gene reporter system to permit studies of the dynamics, mechanisms, and translational relevance of candidate therapies of this type in human colon cancer cells. The reporter system is based on in situ modification of the endogenous locus of the tumor suppressor gene SFRP1, a pivotal regulator of the Wnt pathway that is silenced by DNA hypermethylation in many colon cancers. The modified SFRP1-GFP reporter allele employed remained basally silent, like the unaltered allele, and it was activated only by drug treatments that de-repress gene silencing by reversing DNA hypermethylation. We employed the established DNA methyltransferase inhihibitor (DNMTi) 5-aza-deoxycitidine (DAC) to show how this system can be used to address key questions in the clinical development of epigenetic cancer therapies. First, we defined conditions for which clinically relevant dosing could induce sustained induction of RNA and protein. Second, we found that, in-vivo, a more prolonged drug exposure than anticipated was essential to de-repress gene silencing in significant cell numbers and this has implications for generating effective anticancer responses in patients with hematopoietic or solid tumors. Finally, we discovered how histone deacetylase inhibitors (HDACi) alone, when administered to cells actively replicating DNA, can robustly re-express the silenced gene with no change in promoter methylation status. Taken together, our findings offer a new tool and insights for devising optimal clinical experiments to evaluate DNMTi and HDACi, alone or in combination, and with other cancer treatments, as agents for the epigenetic management and prevention of cancer. PMID:24876104

  2. Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression

    PubMed Central

    Garcia, HernanG.; Lee, HeunJin; Boedicker, JamesQ.; Phillips, Rob

    2011-01-01

    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 ?-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 ?-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. PMID:21806921

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

    PubMed Central

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

    2014-01-01

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

  4. Luciferase reporter system for studying the effect of nanoparticles on gene expression.

    PubMed

    Ding, Min; Bowman, Linda; Castranova, Vincent

    2012-01-01

    Nanotechnology exploits the fact that nanoparticles exhibit unique physicochemical properties, which are distinct from larger particles of the same composition. It follows that nanoparticles may also express distinct bioactivity and unique interactions with biological systems. Therefore, it is essential to assess the potential health risks of exposure to nanoparticles to allow development and implementation of prevention measures. One of the biggest challenges facing the field of nanotoxicology is the huge variety of different nanoparticle types possessing a variety of properties. Genetic Luciferase Reporter System or Reporter gene assay has become an invaluable tool in studies of gene expression. This is achieved by linking the firefly luciferase gene to a promoter sequence. Luciferase assays are quick, highly sensitive, have wide dynamic range, and are cheap to perform. Because of their simplicity and versatility, and because of the absence of endogenous luciferase activity in most cell types, this test can be applied for testing a large variety of nanomaterials for their pathogenic or carcinogenetic effects on a wide range of mammalian cells. This system is an ideal early-stage toxicology tool for screening nanomaterials. Here we describe the Genetic Luciferase Reporter System as the method for detecting alteration of gene expression in response to external stimuli (e.g., nanoparticles). PMID:22791452

  5. 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.

  6. Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

    PubMed Central

    Bylund, J E; Zhang, L; Haines, M A; Higgins, M L; Piggot, P J

    1994-01-01

    The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity. Images PMID:8188591

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

    PubMed Central

    2010-01-01

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

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

    PubMed

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

    1990-02-01

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

  9. A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro.

    PubMed

    Ogg, M S; Williams, J M; Tarbit, M; Goldfarb, P S; Gray, T J; Gibson, G G

    1999-03-01

    1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene. PMID:10219967

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2012-01-01

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

  12. Quantitative cell-based reporter gene assays using droplet-based microfluidics.

    PubMed

    Baret, Jean-Christophe; Beck, Yannick; Billas-Massobrio, Isabelle; Moras, Dino; Griffiths, Andrew D

    2010-05-28

    We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data. PMID:20534350

  13. Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports

    PubMed Central

    Lakraj, Amanda Amrita; Miller, Geoffrey; Vortmeyer, Alexander O.; Khokhar, Babar; Nowak, Richard J.; DiCapua, Daniel B.

    2013-01-01

    Introduction: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia. Methods: We report the clinical presentations of two individuals with Myotonia Congenita (MC). Results: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy. Conclusions: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature. PMID:23483815

  14. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodiumiodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (back-ground) was measured. Results The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2?-18F-fluoro-5-ethyl-1-?-D-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 105 T cells, respectively. 18F-FEAU was a more efficient probe (higher uptake, lower background) than 124I-1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK. Conclusion A comparison of different reporter genereporter probe systems for imaging of T cell number was performed, and the hNET/18F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 3540 103 T cells at the site of T cell injection in the animal model. PMID:26025962

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

    PubMed Central

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

    2005-01-01

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

  18. 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.

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

    PubMed Central

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

    1994-01-01

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

  20. Tyrosinase as a dual reporter gene for both photoacoustic and magnetic resonance imaging

    PubMed Central

    Paproski, Robert J.; Forbrich, Alexander E.; Wachowicz, Keith; Hitt, Mary M.; Zemp, Roger J.

    2011-01-01

    Reporter genes are useful scientific tools for analyzing promoter activity, transfection efficiency, and cell migration. The current study has validated the use of tyrosinase (involved in melanin production) as a dual reporter gene for magnetic resonance and photoacoustic imaging. MCF-7 cells expressing tyrosinase appear brown due to melanin. Magnetic resonance imaging of tyrosinase-expressing MCF-7 cells in 300 ?L plastic tubes displayed a 34 to 40% reduction in T1 compared to normal MCF-7 cells when cells were incubated with 250 ?M ferric citrate. Photoacoustic imaging of tyrosinase-expressing MCF-7 cells in 700 ?m plastic tubes displayed a 20 to 57-fold increase in photoacoustic signal compared to normal MCF-7 cells. The photoacoustic signal from tyrosinase-expressing MCF-7 cells was significantly greater than blood at 650 nm, suggesting that tyrosinase-expressing cells can be differentiated from the vasculature with in vivo photoacoustic imaging. The imaging results suggest that tyrosinase is a useful reporter gene for both magnetic resonance and photoacoustic imaging. PMID:21483602

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

    PubMed

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

    2014-01-01

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

  2. Transcription of the ehx Enterohemolysin Gene Is Positively Regulated by GrlA, a Global Regulator Encoded within the Locus of Enterocyte Effacement in Enterohemorrhagic Escherichia coli?

    PubMed Central

    Saitoh, Takehito; Iyoda, Sunao; Yamamoto, Shouji; Lu, Yan; Shimuta, Ken; Ohnishi, Makoto; Terajima, Jun; Watanabe, Haruo

    2008-01-01

    The pathogenicity island termed locus of enterocyte effacement (LEE) encodes a type 3 protein secretion system, whose function is required for full virulence of enterohemorrhagic Escherichia coli (EHEC). GrlR and GrlA are LEE-encoded negative and positive regulators, respectively, for controlling transcription of the ler gene, which encodes a central activator of LEE gene expression. We previously reported that the GrlR-GrlA regulatory system controls not only the LEE genes but also flagellar gene expression in EHEC (S. Iyoda et al., J. Bacteriol. 188:5682-5692, 2006). In order to further explore virulence-related genes under the control of the GrlR-GrlA regulatory system, we characterized a grlR-deleted EHEC O157 strain, which was found to have high and low levels of expression of LEE and flagellar genes, respectively. We report here that the grlR deletion significantly induced enterohemolysin (Ehx) activity of EHEC O157 on plates containing defibrinated sheep erythrocytes. Ehx levels were not induced in the grlR grlA double mutant strain but increased markedly by overexpression of GrlA even in the ler mutant, indicating that GrlA is responsible for this regulation. Ehx of the EHEC O157 Sakai strain is encoded by the ehxCABD genes, which are carried on the large plasmid pO157. The expression of ehxC fused with FLAG tag or a promoterless lacZ gene on pO157 was significantly induced under conditions in which GrlA was overproduced. These results together suggest that GrlA acts as a positive regulator for the ehx transcription in EHEC. PMID:18487325

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed Central

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

    2012-01-01

    Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5?-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes. PMID:22937087

  5. Identification of AAAS gene mutation in Allgrove syndrome: A report of three cases

    PubMed Central

    LI, WENJING; GONG, CHUNXIU; QI, ZHAN; WU, DI; CAO, BINGYAN

    2015-01-01

    Allgrove syndrome (AS) is an autosomal recessive congenital disease, caused by mutations in the AAAS gene, and is characterized by the triad of Addison's disease, achalasia and alacrima. The present study describes three newly diagnosed cases of AS, in which genetic analysis of the AAAS gene was used to identify AAAS gene mutations, to enhance the understanding of the pathogenesis and clinical manifestations of AS in the Chinese population. Two of the cases exhibited homozygous mutations of c.771delG (p.Arg258GlyfsX33) in exon 8 and one case exhibited a homozygous mutation of c.1366C>T (p.Q456X) in exon 15. A review of the current literature suggests that the AAAS c.771delG mutation has only been reported in the Chinese population. Genetic analysis of the AAAS gene in Chinese AS patients at a young age may facilitate an earlier diagnosis and the timely initiation of the appropriate treatment, ultimately improving the patient outcome. PMID:26622478

  6. Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay

    PubMed Central

    Jin, Yi; Chen, Zujian; Liu, Xiqiang; Zhou, Xiaofeng

    2013-01-01

    MicroRNAs are post-transcriptional regulators that control mRNA stability and the translation efficiency of their target genes. Mature microRNAs are approximately 22-nucleotide in length. They mediate post-transcriptional gene regulation by binding to the imperfect complementary sequences (a.k.a. microRNA regulatory elements, MRE) in the target mRNAs. It is estimated that more than one-third of the protein-coding genes in the human genome are regulated by microRNAs. The experimental methods to examine the interaction between the microRNA and its targeting site(s) in the mRNA are important for understanding microRNA functions. The luciferase reporter gene assay has recently been adapted to test the effect of microRNAs. In this chapter, we use a previously identified miR-138 targeting site in the 3?-untranslated region (3?-UTR) of the RhoC mRNA as an example to describe a quick method for testing the interaction of microRNA and mRNA. PMID:23007504

  7. 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. PMID:11425903

  8. Isolation, structure, and characterization of the RAD3 gene of the yeast

    SciTech Connect

    Higgins, D.R.

    1987-01-01

    In Saccharomyces cerevisiae excision of UV-induced pyrimidine dimers from the DNA involves at least 10 genes. In this work, the RAD3 gene has been cloned, its nucleotide sequence determined, and some structural and functional features examined. The RAD3 gene codes for an mRNA of 2.5 kb. The RAD3 open reading frame is 2334 nucleotides long with an encoded protein of 89,779 daltons. Genomic deletions of the RAD3 gene are recessive lethal, demonstrating that it is an essential gene and suggesting that the RAD3 gene product plays a vital role in the cell in addition to its role in excision repair. To date, the RAD3 gene is the only RAD gene known to be essential for viability. It is known that the RAD1, RAD2, RAD4, RAD6, RAD7, RAD9, RAD10, RAD18, and RAD23 genes are not. A series of RAD3-lacZ fusions have been made across the N-terminal region of the RAD3 coding sequences and the subcellular localization of the fusion proteins determined. There exists an amino acid sequence within the first 32 RAD3 amino acids that allows for association of the RAD3-lacZ gene fusion with the nucleus. This region of the RAD3 protein shows no obvious similarity to any previously defined nuclear localization signal.

  9. The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient in Escherichia coli but its transcripts are poorly expressed.

    PubMed Central

    Lopez, P J; Iost, I; Dreyfus, M

    1994-01-01

    In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E. coli strain that produces saturating amounts of T7 RNA polymerase. At 37 degrees C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E. coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript. Images PMID:8165132

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

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

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

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

    PubMed

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

    2015-05-01

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

  12. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2009-01-01

    Purpose Mice lacking the Kv1.1 potassium channel ? subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1?/? (knock-out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. Methods Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1?/? animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1?/? brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E.coli lacZ reporter gene. Vector-injected mice were were examined to determine the extent of neuronal infection. Results Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1?/? mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed ectopic Kv1.1 ? channel subunit expression. Conclusions Kcna1 deletion in mice results in a seizure disorder that resembles electrographically and neuropathologically the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection PMID:17651419

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

    SciTech Connect

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

    2006-03-10

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

  17. 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.

  18. 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…

  19. 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

  20. Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis bacteriophage r1t.

    PubMed

    Nauta, A; van Sinderen, D; Karsens, H; Smit, E; Venema, G; Kok, J

    1996-03-01

    A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome has been cloned and characterized. It encompasses the two divergently oriented genes rro, encoding the phage repressor, and tec. Both genes, of which the transcription start sites have been mapped, are preceded by consensus -35 and -10 promoter sequences. The region contains three 21 bp direct repeats with internal dyad symmetry which probably act as operators. Two of these repeats partially overlap the two promoter sequences. The distant third repeat is located within the tec coding sequence. Gel mobility shift assays demonstrated that Rro specifically binds to this sequence. To study possible transcriptional regulation of the region, a lacZ translational fusion with an open reading frame following tec was constructed. Under conditions that favour the lysogenic life cycle of r1t, beta-galactosidase activity was very low. Expression of the lacZ fusion could be induced 70-fold by the addition of mitomycin C at a concentration which promotes the switch of r1t from the lysogenic to the lytic life cycle. In non-induced cells, promoter activity was repressed by Rro, as a frameshift mutation in rro resulted in constitutive expression of the lacZ gene fusion. PMID:8730874

  1. 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

  2. DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco.

    PubMed Central

    Newman, T C; Ohme-Takagi, M; Taylor, C B; Green, P J

    1993-01-01

    DST elements are highly conserved sequences located in the 3' untranslated regions (UTRs) of a set of unstable soybean transcripts known as the small auxin-up RNAs (SAURs). To test whether DST sequences could function as mRNA instability determinants in plants, a model system was developed to facilitate the direct measurement of mRNA decay rates in stably transformed cells of tobacco. Initial experiments established that the chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) transcripts degraded with similar half-lives in this system. In addition, their decay kinetics mirrored the apparent decay kinetics of the corresponding transcripts produced in transgenic plants under the control of a regulated promoter (Cab-1). The model system was then used to measure the decay rates of GUS reporter transcripts containing copies of the DST sequence inserted into the 3'UTR. An unmodified CAT gene introduced on the same vector served as the internal reference. These experiments and a parallel set utilizing a beta-globin reporter gene demonstrated that a synthetic dimer of the DST sequence was sufficient to destabilize both reporter transcripts in stably transformed tobacco cells. The decrease in transcript stability caused by the DST sequences in cultured cells was paralleled by a coordinate decrease in transcript abundance in transgenic tobacco plants. The implications of these results for the potential function of DST sequences within the SAUR transcripts are discussed. PMID:8329900

  3. Tsf1 to Tsf6, Required for Silencing the Saccharomyces Cerevisiae Gal Genes, Are Global Regulatory Genes

    PubMed Central

    Chen, S.; West-Jr, R. W.; Ma, J.; Johnson, S. L.; Gans, H.; Woldehawariat, G.

    1993-01-01

    The Saccharomyces cerevisiae GAL1 and GAL10 genes are controlled in response to the availability of galactose and glucose by multiple activating and repressing proteins bound at adjacent or overlapping sites in UAS(G). Negative control elements in UAS(G), designated GAL operators GALO(1) to GALO(6), are required to silence basal level transcription of GAL1 and GAL10 when galactose is absent. We isolated and characterized recessive mutations in six nuclear genes, TSF1 to TSF6, that impair silencing of GAL1 and GAL10 gene expression. Surprisingly, the results of several experiments suggest that the TSF genes encode global regulatory factors. tsf1 to tsf6 mutations derepressed expression from yeast CYC-GAL hybrid promoters (fused to lacZ) that harbor a variety of operator sequences, and caused pleiotropic defects in cell growth, mating, and sporulation. S1 mapping and Northern blot results for tsf3 suggest that the molecular defect is at the transcriptional level. Mutant phenotypes were additive in certain combinations of tsf double mutants, implying that more than one silencing pathway is involved in TSF1 to TSF6 function. Most significantly, mutations in all six TSF1 to TSF6 genes activated expression from GAL1 and CYC1 promoters (fused to lacZ) lacking upstream activating sequences. Combined, the simplest interpretation of these results is that TSF1 to TSF6 encode factors that control the function of the basic RNA polymerase II transcriptional machinery. PMID:8349104

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2005-01-01

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

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

    SciTech Connect

    Braam, Janet

    2008-10-28

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

  7. 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. PMID:25646605

  8. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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

  11. 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. PMID:26192461

  12. 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.

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

    PubMed Central

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

    2001-01-01

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

  14. 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)

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

    PubMed

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

    2015-07-01

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

  16. [Gene therapy].

    PubMed

    Kitajima, Isao

    2002-11-01

    Gene therapy is a method for treating hereditary disease at the gene level. In this paper, I show recent gene therapy approaches for cancer, hemophilia and arteriosclerosis obliterans, and gene medical supply including antisense and decoys are also introduced. With the rapid advances in gene therapy research, the case of dying gene therapy using the adenovirus vector was reported due to DIC. Establishment of the safety in the gene therapy and solution of the ethics problems are also important issues. PMID:12652811

  17. Allele distribution and effect on reporter gene expression of vasopressin receptor gene (AVPR1a)-linked VNTR in primates.

    PubMed

    Hong, Kyung-Won; Matsukawa, Ryota; Hirata, Yoko; Hayasaka, Ikuo; Murayama, Yuichi; Ito, Shin'ichi; Inoue-Murayama, Miho

    2009-05-01

    We surveyed repeat sequence in the 5' flanking region of primate arginine vasopressin receptor 1a gene (AVPR1a) which is important for social behavior in mammals. The amplified region was polymorphic in length in all species investigated. In addition, length variants were examined for promoter activity by in vitro expression of the luciferase gene. However, no significant difference was observed. This region could be a marker to further survey functional difference between and within species. PMID:19350216

  18. Conditional Gene Targeting in Mouse High Endothelial Venules

    PubMed Central

    Kawashima, Hiroto; Hirakawa, Jotaro; Tobisawa, Yuki; Fukuda, Minoru; Saga, Yumiko

    2009-01-01

    High endothelial venules (HEVs) are specialized blood vessels of secondary lymphoid organs composed of endothelial cells with a characteristic cuboidal morphology. Lymphocytes selectively adhere to and migrate across HEVs to initiate immune responses. In this study, we established a novel transgenic mouse line expressing Cre recombinase under the transcriptional control of the gene encoding HEV-expressed sulfotransferase, N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), using bacterial artificial chromosome recombineering. Crossing these transgenic mice with the ROSA26 reporter strain, which expresses lacZ following Cre-mediated recombination, and staining the resulting progeny with 5-bromo-4-chloro-5-indolyl-?-D-galactoside indicated that Cre recombinase was specifically expressed in mAb MECA79-reactive HEVs in secondary lymphoid organs but not in any other blood vessels of the transgenic mice. The expression of Cre recombinase correlated with a developmental switch, from immature, mAb MECA367-reactive HEVs to mature, mAb MECA79-reactive HEVs in neonatal lymph nodes. In addition to the HEVs, Cre recombinase was also strongly expressed in the colonic villi, which recapitulated the intrinsic expression of GlcNAc6ST-2 as confirmed in GlcNAc6ST-2GFP/GFP knock-in mice and by RT-PCR. Furthermore, treatment with an antimicrobial agent revealed that the colonic expression of Cre recombinase in the transgenic mice was regulated by commensal bacteria in the colon. In addition, Cre recombinase was expressed in a small subset of cells in the brain, testis, stomach, small intestine, and lung. In view of the restricted expression of Cre recombinase, this transgenic mouse line should be useful for elucidating tissue-specific gene functions using the Cre/loxP system. PMID:19380794

  19. 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

  20. A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo.

    PubMed

    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

    2014-04-25

    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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    zcab?, Bahar; Tahmiscio?lu Bucak, Feride; Ceylaner, Serdar; zcan, Rah?an; Byknal, Cenk; Ercan, Oya; Tysz, Beyhan; Evliyao?lu, 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. PMID:26831561

  3. 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.

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

    PubMed Central

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

    2015-01-01

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

  5. 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

  6. Mutations of SCN4A gene cause different diseases: 2 case reports and literature review.

    PubMed

    Liu, Xiao-li; Huang, Xiao-jun; Luan, Xing-hua; Zhou, Hai-yan; Wang, Tian; Wang, Jing-yi; Chen, Sheng-di; Tang, Hui-dong; Cao, Li

    2015-01-01

    SCN4A encodes the Nav1.4 channel and mutations in SCN4A lead to different ionic channelopathies. In this study, one sporadic individual of periodic paralysis, one paramyotonia family and 200 normal healthy controls are enrolled. Genomic DNA was extracted from peripheral blood leukocytes, followed by polymerase chain reaction and DNA sequencing of candidate genes, including SCN4A and CACNA1S. As a result, heterozygous mutations c.2024G>A (R675Q) and c.1333G>A (V445M) of gene SCN4A were identified in the hypokalemic periodic paralysis patient and the paramyotonia congenita family respectively. Both mutations were not detected in healthy controls. Compared with reported cases, patients with mutation R675Q usually do not present hypokalemic periodic paralysis but hyperkalemic or normokalemic periodic paralysis. The mutation V445M was first reported in Chinese patients with nondystrophic myotonias. In addition, we carried out literature review by summarizing clinical features of the 2 mutations and establish the genotype-phenotype correlations to provide guidance for diagnosis. PMID:25839108

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. 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 9dilution 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-?L of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted. PMID:26502231

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

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

    PubMed

    Gabriel, Gabriele V M; Viviani, Vadim R

    2014-12-01

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

  13. The ?-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 ?-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 ?-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 ?-actin gene expression across eukaryotes. Furthermore, the isolated ?-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. PMID:26204405

  14. Cloning and expression of Kluyveromyces fragilis LAC4 gene.

    PubMed

    Huo, K; Li, Y

    1995-11-01

    The genomic library of Kluyveromyces fragilis was constructed in E. coli TG1, and 5 beta-galactosidase gene (LAC4) clones have been obtained from the library by complementation of the Kluyveromyces lactis lac4-8 mutation. The studies on the structure and the function of the LAC4 gene revealed that (i) the gene can also complement E. coli lacZ mutation; (ii) the physical map of the K. fragilis LAC4 gene was very similar to that of K. lactis; (iii) the beta-galactosidase levels expressed by the clone strains were much higher than that expressed by the original strain; (iv) the variation of the beta-galactosidase level of different clone strains induced by lactose or galactose was related to the retained degree of the 5' flanking region of LAC4 gene, suggesting that there might be a lactose specific transcription activating element in the region. PMID:8745531

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2000-05-01

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

  17. Tumor-Specific Expression and Detection of a CEST Reporter Gene

    PubMed Central

    Minn, Il; Bar-Shir, Amnon; Yarlagadda, Keerthi; Bulte, Jeff W. M.; Fisher, Paul B.; Wang, Hao; Gilad, Assaf A.; Pomper, Martin G.

    2015-01-01

    Purpose To develop an imaging tool that enables the detection of malignant tissue with enhanced specificity using the exquisite spatial resolution of MRI. Methods Two mammalian gene expression vectors were created for the expression of the lysine-rich protein (LRP) under the control of the cytomegalovirus (CMV) promoter and the progression elevated gene-3 promoter (PEG-3 promoter) for constitutive and tumor-specific expression of LRP, respectively. Using those vectors, stable cell lines of rat 9L glioma, 9LCMV-LRP and 9LPEG-LRP, were established and tested for CEST contrast in vitro and in vivo. Results 9LPEG-LRP cells showed increased CEST contrast compared with 9L cells in vitro. Both 9LCMV-LRP and 9LPEG-LRP cells were capable of generating tumors in the brains of mice, with a similar growth rate to tumors derived from wild-type 9L cells. An increase in CEST contrast was clearly visible in tumors derived from both 9LCMV-LRP and 9LPEG-LRP cells at 3.4 ppm. Conclusion The PEG-3 promoter:LRP system can be used as a cancer-specific, molecular-genetic imaging reporter system in vivo. Because of the ubiquity of MR imaging in clinical practice, sensors of this class can be used to translate molecular-genetic imaging rapidly. PMID:25919119

  18. First report on interferon related developmental regulator-1 from Macrobrachium rosenbergii: bioinformatic analysis and gene expression.

    PubMed

    Arockiaraj, Jesu; Easwvaran, Sarasvathi; Vanaraja, Puganeshwaran; Singh, Arun; Othman, Rofina Yasmin; Bhassu, Subha

    2012-05-01

    This study reports the first full length gene of interferon related developmental regulator-1 (designated as MrIRDR-1), identified from the transcriptome of Macrobrachium rosenbergii. The complete gene sequence of the MrIRDR-1 is 2459 base pair long with an open reading frame of 1308 base pairs and encoding a predicted protein of 436 amino acids with a calculated molecular mass of 48 kDa. The MrIRDR-1 protein contains a long interferon related developmental regulator super family domain between 30 and 330. The mRNA expressions of MrIRDR-1 in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) infected M. rosenbergii were examined using qRT-PCR. The MrIRDR-1 is highly expressed in hepatopancreas along with all other tissues (walking leg, gills, muscle, haemocyte, pleopods, brain, stomach, intestine and eye stalk). After IHHNV infection, the expression is highly upregulated in hepatopancreas. This result indicates an important role of MrIRDR-1 in prawn defense system. PMID:22361112

  19. Use of Leishmania donovani field isolates expressing the luciferase reporter gene in in vitro drug screening.

    PubMed

    Ashutosh; Gupta, Suman; Ramesh; Sundar, Shyam; Goyal, Neena

    2005-09-01

    Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds. PMID:16131481

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

    PubMed

    Daz, Ren E; Utreras, Carlos; Ascu, Rodrigo; Hidalgo, Fernando; Vliz, Jess; Wohllk, Nelson

    2011-11-01

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

  1. 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

  2. Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila.

    PubMed Central

    Yeh, E; Gustafson, K; Boulianne, G L

    1995-01-01

    We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7624365

  3. 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. PMID:26374432

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

    PubMed

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

    2009-12-01

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

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

    PubMed Central

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

    2011-01-01

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

  6. Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

    PubMed

    Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

    2002-10-01

    Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively. PMID:12385291

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1alpha-hydroxylase promoter activity in the skin.

    PubMed

    Vanhooke, Janeen L; Prahl, Jean M; Kimmel-Jehan, Christine; Mendelsohn, Monica; Danielson, Eric W; Healy, Kevin D; DeLuca, Hector F

    2006-01-01

    The hormonally active form of vitamin D(3),1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is synthesized in the kidney through a tightly regulated reaction catalyzed by 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), the product of the CYP27B1 gene. Through gene targeting in embryonic stem cells, we engineered a mouse strain in which the coding region of the 1alpha-hydroxylase gene is replaced by the genes for beta-galactosidase (lacZ) and neomycin resistance. Null mice produced no detectable 1alpha-hydroxylase transcript. The mice grew normally when maintained on a balanced diet containing 1,25(OH)(2)D(3) but rapidly developed rickets when phosphorus and 1,25(OH)(2)D(3) were restricted. Rickets was curable through administration of 1,25(OH)(2)D(3) but not its biological precursor, 25-hydroxyvitamin D(3). Upon administration of a diet low in calcium and devoid of any form of vitamin D(3), beta-galactosidase activity was detected in the kidneys of the -/- and +/- mice and in placentas harvested from -/- females bred with -/- males. No beta-galactosidase activity was detected in skin sections or in primary keratinocyte cultures from -/- animals. Our results demonstrate we have generated 1alpha-hydroxylase null mice that display phenotypes characteristic of vitamin D-dependency rickets type I. From the histochemical analysis of reporter gene expression in these mice, we conclude that acute 1,25(OH)(2)D(3) deficiency in otherwise healthy animals does not stimulate local production of 1,25(OH)(2)D(3) in the skin. These findings stand in contrast to previously published reports of 1,25(OH)(2)D(3) production in keratinocytes. PMID:16371465

  10. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

    PubMed

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-10-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. PMID:25361954

  11. Longitudinal far red gene-reporter imaging of cancer metastasis in preclinical models: a tool for accelerating drug discovery.

    PubMed

    Zhu, Banghe; Robinson, Holly; Zhang, Songlin; Wu, Grace; Sevick-Muraca, Eva M

    2015-09-01

    In this short communication, we demonstrate for the first time, the use of far red fluorescent gene reporter, iRFP to longitudinally and non-invasively track the in vivo process of lymphatic metastases from an orthotopic site of mammary implantation through lymphatic vessels and to draining lymph nodes. Potentially useful to accelerate cancer drug discovery as an in vivo screening tool to monitor the pharmacological arrest of metastasis, we show that the custom as well as commercial small animal imaging devices have adequate performance to detect the gene reporter in stably expressing metastatic cancer cells. PMID:26417506

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

    PubMed

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

    2012-11-30

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

  13. [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. PMID:22891545

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

    PubMed

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

    2012-07-01

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

  15. Attempted replication of reported chronic obstructive pulmonary disease candidate gene associations.

    PubMed

    Hersh, Craig P; Demeo, Dawn L; Lange, Christoph; Litonjua, Augusto A; Reilly, John J; Kwiatkowski, David; Laird, Nan; Sylvia, Jody S; Sparrow, David; Speizer, Frank E; Weiss, Scott T; Silverman, Edwin K

    2005-07-01

    Case-control studies have successfully identified many significant genetic associations for complex diseases, but lack of replication has been a criticism of case-control genetic association studies in general. We selected 12 candidate genes with reported associations to chronic obstructive pulmonary disease (COPD) and genotyped 29 polymorphisms in a family-based study and in a case-control study. In the Boston Early-Onset COPD Study families, significant associations with quantitative and/or qualitative COPD-related phenotypes were found for the tumor necrosis factor (TNF)-alpha -308G>A promoter polymorphism (P < 0.02), a coding variant in surfactant protein B (SFTPB Thr131Ile) (P = 0.03), and the (GT)(31) allele of the heme oxygenase (HMOX1) promoter short tandem repeat (P = 0.02). In the case-control study, the SFTPB Thr131Ile polymorphism was associated with COPD, but only in the presence of a gene-by-environment interaction term (P = 0.01 for both main effect and interaction). The 30-repeat, but not the 31-repeat, allele of HMOX1 was associated (P = 0.04). The TNF -308G>A polymorphism was not significant. In addition, the microsomal epoxide hydrolase "fast" allele (EPHX1 His139Arg) was significantly associated in the case-control study (P = 0.03). Although some evidence for replication was found for SFTPB and HMOX1, none of the previously published COPD genetic associations was convincingly replicated across both study designs. PMID:15817713

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

    PubMed

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

    1992-10-16

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

  17. Copper homeostasis-related genes in three separate transcriptional units regulated by CsoR in Corynebacterium glutamicum.

    PubMed

    Teramoto, Haruhiko; Yukawa, Hideaki; Inui, Masayuki

    2015-04-01

    In Corynebacterium glutamicum R, CsoR acts as a transcriptional repressor not only of the cognate copA-csoR operon but also of the copZ1-copB-cgR_0126 operon. It is predicted that copA and copB encode P-type ATPases for copper efflux and copZ1 encodes a metallochaperone. Here, a CsoR-binding motif was found upstream of another copZ-like gene, copZ2, and the in vitro binding of the CsoR protein to its promoter was confirmed. The monocistronic copZ2 transcript was upregulated by excess copper in a CsoR-dependent manner. Among the extended CsoR regulon, deletion of copA, but not of copB, copZ1, or copZ2, resulted in decreased resistance to copper, indicating a major role of the CopA copper exporter in the multilayered systems for copper homeostasis. A redundant role of copZ1 and copZ2 in copper resistance was also indicated by double deletion of these genes. The copper-dependent activation of the copA, copZ1, and copZ2 promoters was confirmed by lacZ reporter assays, consistent with the coordinated derepression of the three transcriptional units. The copZ1 promoter activity showed the highest responsiveness to copper and was also induced by excess zinc and nickel. Furthermore, zinc-inducible expression observed for the CsoR-regulated genes was independent of Zur, recently found to uniquely act as a transcriptional repressor of zinc efflux genes. These results implied complicated cross talk between homeostasis of multiple transition metals. PMID:25592736

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

    PubMed

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

    2006-01-01

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

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

    PubMed

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

    2001-07-01

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

  20. Use of the pBUTR Reporter System for Scalable Analysis of 3' UTR-Mediated Gene Regulation.

    PubMed

    Chaudhury, Arindam; Neilson, Joel R

    2016-01-01

    Posttranscriptional control of mRNA subcellular localization, stability, and translation is a central aspect of gene regulation and expression. Much of this control is mediated via recognition of a given mRNA transcript's 3' untranslated region (UTR) by microRNAs and RNA-binding proteins. Here we describe how a novel, scalable piggyBac-based vector, pBUTR, can be utilized for analysis of 3' UTR-mediated posttranscriptional gene regulation (PTGR) both in vitro and in vivo. This vector is specifically designed to express a selection marker, a control reporter, and an experimental reporter from three independent transcription units. Expression of spliced reporter transcripts from medium-copy non-viral promoter elements circumvents several potential confounding factors associated with saturation and stability, while stable integration of these reporter and selection elements in the context of a DNA transposon facilitates experimental reproducibility. PMID:26463380

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

    PubMed Central

    2015-01-01

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

  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. PMID:25265085

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

    PubMed

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

    2015-07-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-12-01

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

  7. 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.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. 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.

  11. 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.

  12. Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism.

    PubMed Central

    Mosca, J D; Pitha, P M; Hayward, G S

    1992-01-01

    To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is reporter gene specific and promoter independent. Images PMID:1316484

  13. 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

  14. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.

    PubMed Central

    Ulmasov, T; Murfett, J; Hagen, G; Guilfoyle, T J

    1997-01-01

    A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression. PMID:9401121

  15. Brugada syndrome with a novel missense mutation in SCN5A gene: A case report from Bangladesh

    PubMed Central

    Sayeed, Md. Zahidus; Salam, Md. Abdus; Haque, Md. Zahirul; Islam, A.K.M. Monwarul

    2014-01-01

    Brugada syndrome is an inherited cardiac arrhythmia that follows autosomal dominant transmission and can cause sudden death. We report a case of Brugada syndrome in a 55-year-old male patient presented with recurrent palpitation, atypical chest pain and presyncope. ECG changes were consistent with type 1 Brugada. Gene analysis revealed a novel missense mutation in SCN5A gene with a genetic variation of D785N and a nucleotide change at 2353G-A. One of his children also had the same mutation. To our knowledge this is the first genetically proved case of Brugada syndrome in Bangladesh. PMID:24581105

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

    PubMed

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

    2003-05-01

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

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

    PubMed Central

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

    2003-01-01

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

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

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

    PubMed

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

    1996-07-01

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

  20. Rational design and rapid screening of antisense oligonucleotides for prokaryotic gene modulation

    PubMed Central

    Shao, Yu; Wu, Yan; Chan, Chi Yu; McDonough, Kathleen; Ding, Ye

    2006-01-01

    Antisense oligodeoxynucleotides (oligos) are widely used for functional studies of both prokaryotic and eukaryotic genes. However, the identification of effective target sites is a major issue in antisense applications. Here, we study a number of thermodynamic and structural parameters that may affect the potency of antisense inhibition. We develop a cell-free assay for rapid oligo screening. This assay is used for measuring the expression of Escherichia coli lacZ, the antisense target for experimental testing and validation. Based on a training set of 18 oligos, we found that structural accessibility predicted by local folding of the target mRNA is the most important predictor for antisense activity. This finding was further confirmed by a direct validation study. In this study, a set of 10 oligos was designed to target accessible sites, and another set of 10 oligos was selected to target inaccessible sites. Seven of the 10 oligos for accessible sites were found to be effective (>50% inhibition), but none of the oligos for inaccessible sites was effective. The difference in the antisense activity between the two sets of oligos was statistically significant. We also found that the predictability of antisense activity by target accessibility was greatly improved for oligos targeted to the regions upstream of the end of the active domain for ?-galactosidase, the protein encoded by lacZ. The combination of the structure-based antisense design and extension of the lacZ assay to include gene fusions will be applicable to high-throughput gene functional screening, and to the identification of new drug targets in pathogenic microbes. Design tools are available through the Sfold Web server at . PMID:17038332

  1. A comparative analysis of green fluorescent protein and beta-glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters.

    PubMed

    Kavita, P; Burma, Pradeep Kumar

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, beta-glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp ) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns. PMID:19005233

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

    PubMed

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

    2016-02-10

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

  3. The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection.

    PubMed

    Desai, P; Ramakrishnan, R; Lin, Z W; Osak, B; Glorioso, J C; Levine, M

    1993-10-01

    As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8396674

  4. The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection.

    PubMed Central

    Desai, P; Ramakrishnan, R; Lin, Z W; Osak, B; Glorioso, J C; Levine, M

    1993-01-01

    As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS) Images PMID:8396674

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Bogani, Debora; Morgan, Marc A J; Nelson, Andrew C; Costello, Ita; McGouran, Joanna F; Kessler, Benedikt M; Robertson, Elizabeth J; Bikoff, Elizabeth K

    2013-10-01

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

  7. 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.

  8. First Report of Carbapenem-Resistant Acinetobacter nosocomialis Isolates Harboring ISAba1-blaOXA-23 Genes in Latin America

    PubMed Central

    Teixeira, Aline Borges; Martins, Andreza Francisco; Barin, Juliana; Hermes, Djuli Milene; Pitt, Caroline Pormann

    2013-01-01

    In recent years, different resistance genes have been found in Acinetobacter spp., especially in the species A. baumannii. We describe two isolates of carbapenem-resistant A. nosocomialis harboring ISAba1-blaOXA-23 and blaOXA-51 found in patients from the city of Porto Alegre, southern Brazil. To the best of the authors' knowledge, this is the first report of carbapenem-resistant A. nosocomialis in Latin America. PMID:23740725

  9. [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.

  10. 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.

  11. Regulation of spo0H, an early sporulation gene in bacilli.

    PubMed Central

    Dubnau, E J; Cabane, K; Smith, I

    1987-01-01

    The construction of lacZ fusions in frame with the spo0H gene of Bacillus licheniformis enabled us to study the expression of this gene under various growth conditions and in various genetic backgrounds. spo0H was expressed during vegetative growth, but the levels increased during early stationary phase and then decreased several hours later. Expression of the gene was not repressed by glucose, but was induced by decoyinine, an inhibitor of guanine nucleotide biosynthesis, which can induce sporulation. Of those tested, the only spo0 gene required for the expression of spo0H was spo0A, and this requirement was eliminated by the abrB mutation, a partial suppressor of spo0A function. spo0H-lacZ expression was much higher in a strain with a deletion in the spo0H gene. Images PMID:3102457

  12. 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.

  13. Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity.

    PubMed

    van Gemen, B; Koets, H J; Plooy, C A; Bodlaender, J; Van Knippenberg, P H

    1987-08-01

    In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3122846

  14. cis-Acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes

    SciTech Connect

    Hofmann, A.; Garfinkel, M.D.; Meyerowitz, E.M. )

    1991-06-01

    The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between {minus}211 and {minus}43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from {minus}133 to {minus}48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements.

  15. 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.

  16. Inference of Quantitative Models of Bacterial Promoters from Time-Series Reporter Gene Data

    PubMed Central

    Stefan, Diana; Pinel, Corinne; Pinhal, Stphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for the dynamics of FliA-dependent promoters. PMID:25590141

  17. Genetic studies of a thermoregulated gene in the psychrotrophic bacterium Pseudomonas fluorescens.

    PubMed

    Regeard, C; Mérieau, A; Leriche, F; Guespin-Michel, J F

    1999-09-01

    In the psychrotrophic bacterium Pseudomonas fluorescens, some genes are thermoregulated: they are maximally expressed at a particular temperature within the broad range of temperatures that allow growth of this bacterium. To study this regulation, random transcriptional insertion fusions were obtained by means of mini-Tn5lacZ1 or mini-Tn5luxAB transposition. One fusion was studied in which beta-galactosidase production was maximal at a low-growth temperature. The mutated gene (that we call xsf) was highly homologous to xseA from Escherichia coli (and from other bacteria) which encodes the large subunit of exonuclease VII. Genetic tools were constructed in order to analyse and manipulate this fusion: a plasmid derived from R68.45 was used for chromosome transfer and a replacement vector was constructed to allow in situ marker exchange of the mini-Tn5lacZ1 by an Hg(r) interposon. This vector was used to make double mutants and hence to study the effect of the insertion in xsf on the expression of other fusions. Six genes were thereby identified with a decreased expression in an xsf- background and with different characteristics of thermoregulation. PMID:10540908

  18. 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.

  19. Gene transfer and gene therapy

    SciTech Connect

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

    1988-01-01

    This book reports the progress in gene transfer that has been made in various species, from Drosophila to higher mammals, including illustrative examples of germline gene transfer and tissue-specific somatic gene regulation in the mouse. Important new information regarding developmental control of gene transcription includes the delineation of distal elements, both cis and trans, controlling specific gene regulation. The book also offers an overview of vectors for gene transfer, including retroviral vectors and new retroviral packaging cell lines designed to minimize production of replication-competent virus.

  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. Transgene-based anthocyanin hyper-pigmentation as a visual reporter of gene silencing in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Co-suppression associated loss of flower pigmentation in transgenic petunia plants was one of the first clear indicators of the natural process of RNA-associated gene silencing in plants. We have been exploring the use of genetically engineered anthocyanin over-production in vegetative tissues as...

  2. 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.

  3. 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

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  5. A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level.

    PubMed

    Glaeser, Angela; Heermann, Ralf

    2015-08-01

    Determination of reporter gene activity at the single-cell level is a prerequisite for analyzing heterogeneous gene expression in bacteria. The insect pathogenic enteric bacterium Photorhabdus luminescens is an excellent organism in which to study heterogeneity since it exists in two phenotypically different forms, called the primary and secondary variant. A tool for generating stable genomic integrations of reporter genes has been lacking for these bacteria, and this has hampered the acquisition of reliable data sets for promoter activities at the single-cell level. We therefore generated a plasmid tool named pPINT-mCherry for the easy and stable introduction of gene fragments upstream of an mCherry reporter gene followed by stable integration of the plasmid into the P. luminescens genome at the rpmE/glmS intergenic region. We demonstrate that the genomic integration of reporter genes for single-cell analysis is necessary in P. luminescens since plasmid-borne reporter genes mimic heterogeneity and are therefore not applicable in these bacteria, in contrast to their use in single-cell analysis in other bacteria like Escherichia coli. PMID:26260085

  6. 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.

  7. Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters

    PubMed Central

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-01-01

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time. PMID:24753407

  8. Hyaline fibromatosis syndrome with mutation c.1074delT of the CMG2 gene: a case report

    PubMed Central

    2014-01-01

    Introduction Juvenile hyaline fibromatosis and infantile systemic hyalinosis are variants of the same autosomal recessive syndrome; hyaline fibromatosis syndrome, characterized by papulonodular skin lesions, gingival hypertrophy, flexion contractures of joints, osteolytic bone lesions and stunted growth. Infantile systemic hyalinosis is distinguished from juvenile hyaline fibromatosis by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections and death within the first two years of life. Hyaline fibromatosis syndrome is due to mutations of the gene-encoding capillary morphogenesis protein 2 (CMG2). Cases have been reported in different countries but to the best of our knowledge, this is the first reported Moroccan patient with hyaline fibromatosis syndrome and carrying the CMG2 mutation. Case presentation We report the case of an eight-year-old Moroccan male patient with typical features of hyaline fibromatosis syndrome: multiple recurring subcutaneous tumors, gingival hypertrophy, joint contractures and other anomalies carrying a homozygous mutation in the CMG2 gene. The identification of the mutation in our patient allowed us to do a presymptomatic diagnosis in our patients sister, a two-day-old newborn, who is carrying the familial mutation in the heterozygous state. Early recognition of this condition is important for genetic counseling and early treatment. Conclusions Hyaline fibromatosis syndrome might be underdiagnosed. Molecular diagnosis will help clinicians and geneticists, firstly to conduct genetic counseling, prenatal diagnosis and early treatment, and secondly to gain better understanding of the disease and genotype-phenotype correlations. PMID:25186005

  9. 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.

  10. Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

    PubMed Central

    2010-01-01

    Background The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1) as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi). Results Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ). This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ). Importantly, mites undergoing the immersion approach had greatly improved survival (75-80%) over 72 h, approaching that of mites not undergoing any treatment. Conclusions Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and immature stages of ticks. PMID:20712880

  11. 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.

  12. (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.

  13. [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.

  14. Phenytoin toxicity in two-month-old Thai infant with CYP2C9 gene polymorphism - A case report.

    PubMed

    Veeravigrom, Montida; Jaroonvanichkul, Vorapol; Netbaramee, Wiracha; Phaisarn, Pichaya; Uyathanarat, Thanita

    2016-01-01

    Phenytoin is one of the most well established and most effective antiepileptic medications for the treatment of focal seizures. In our clinical practice, it has proven difficult to maintain therapeutic phenytoin levels in infants less than three months of age. Incidence of phenytoin toxicity in infants is very rare. The cytochrome P450 super family plays an important role in phenytoin metabolism, especially CYP2C9 and CYP2C19. In this case report, we profiled a two-month-old Thai infant who developed phenytoin toxicity resulting from CYP2C9 gene polymorphism. PMID:25998968

  15. Analyses of both the alkB Gene Transcriptional Start Site and alkB Promoter-Inducing Properties of Rhodococcus sp. Strain BCP1 Grown on n-Alkanes?

    PubMed Central

    Cappelletti, M.; Fedi, S.; Frascari, D.; Ohtake, H.; Turner, R. J.; Zannoni, D.

    2011-01-01

    Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n-alkanes (C2 to C7) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n-alkanes (C12 to C24) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase (alkB), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (PalkB) was examined in the presence of gaseous, liquid, and solid n-alkanes along with intermediates of the putative n-alkane degradation pathway. A recombinant plasmid, pTPalkBLacZ, was constructed by inserting the lacZ gene downstream of PalkB, and it was used to transform Rhodococcus sp. strain BCP1. Measurements of ?-galactosidase activity showed that PalkB is induced by C6 to C22 n-alkanes. Conversely, C2 to C5 and >C22 n-alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on PalkB expression induced by alternative carbon sources along with putative products of n-hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n-alkanes in a Rhodococcus strain. PMID:21193665

  16. Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ER? 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 ER? 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 ER? reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17?-estradiol, estriol, and 17?-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ER? 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 ER? reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ER? reporter gene assay. PMID:25902010

  17. Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

    PubMed Central

    2011-01-01

    Background Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly. Results Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT) - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR) and gene-specific amplified fragment length polymorphism (AFLP). It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution. Conclusions RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life. PMID:21362165

  18. 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 ?-galactosidase (?-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded ?-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass. PMID:23793457

  19. oct4-EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka.

    PubMed

    Froschauer, Alexander; Khatun, Mst Muslima; Sprott, David; Franz, Alexander; Rieger, Christiane; Pfennig, Frank; Gutzeit, Herwig O

    2013-01-01

    Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4-EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4-EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4-EGFP transgenic medaka in reproductive and stem cell biology. PMID:23139203

  20. Risk assessment of gene flow from genetically engineered virus resistant cassava to wild relatives in Africa: an expert panel report.

    PubMed

    Hokanson, Karen E; Ellstrand, Norman C; Dixon, Alfred G O; Kulembeka, Heneriko P; Olsen, Kenneth M; Raybould, Alan

    2016-02-01

    The probability and consequences of gene flow to wild relatives is typically considered in the environmental risk assessment of genetically engineered crops. This is a report from a discussion by a group of experts who used a problem formulation approach to consider existing information for risk assessment of gene flow from cassava (Manihot esculenta) genetically engineered for virus resistance to the 'wild' (naturalized) relative M. glaziovii in East Africa. Two environmental harms were considered in this case: (1) loss of genetic diversity in the germplasm pool, and (2) loss of valued species, ecosystem resources, or crop yield and quality due to weediness or invasiveness of wild relatives. Based on existing information, it was concluded that gene flow will occur, but it is not likely that this will reduce the genetic diversity in the germplasm pool. There is little existing information about the impact of the virus in natural populations that could be used to inform a prediction about whether virus resistance would lead to an increase in reproduction or survival, hence abundance of M. glaziovii. However, an increase in the abundance of M. glaziovii should be manageable, and would not necessarily lead to the identified environmental harms. PMID:26667472

  1. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N.

    2013-01-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 2.1%, n = 12, vs. 44.7 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., immunization against infarction). PMID:21785893

  2. Fanconi anemia-D1 due to homozygosity for the BRCA2 gene Cypriot founder mutation: A case report

    PubMed Central

    LOIZIDOU, MARIA A.; HADJISAVVAS, ANDREAS; TANTELES, GEORGE A.; SPANOU-ARISTIDOU, ELENA; KYRIACOU, KYRIACOS; CHRISTOPHIDOU-ANASTASIADOU, VIOLETTA

    2016-01-01

    Fanconi anemia (FA) is a rare disorder characterized by multiple congenital malformations, progressive bone marrow failure and susceptibility to malignancies. Biallelic mutations in the breast cancer 2, early onset (BRCA2) gene are responsible for the FA-D1 subgroup, which accounts for ~3% of all the FA cases. Patients with biallelic BRCA2 mutations generally display a more severe phenotype, with earlier onset and increased incidence of leukaemia and other solid tumors, than other patients with FA. In the present report, the first Cypriot patient with FA-D1 is described, which is the fifth case of a homozygote for the same null allele reported thus far, and the third known case of neuroblastoma in association with FA-D1. PMID:26834852

  3. 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.

  4. 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. PMID:21409506

  5. Vitamin D-dependent rickets type II: report of a novel mutation in the vitamin D receptor gene.

    PubMed

    Shafeghati, Yousef; Momenin, Nima; Esfahani, Taher; Reyniers, Edwin; Wuyts, Wim

    2008-05-01

    Hereditary vitamin D-resistant rickets type or vitamin D-dependent rickets type II is a genetically determined and rare autosomal recessive disorder, most often caused by mutations in the vitamin D receptor gene. It usually presents with rachitic changes not responsive to vitamin D treatment and the circulating levels of 1,25 (OH)2 vitamin D-3 are elevated, differentiating it from vitamin D-dependent rickets type I. Alopecia capitis or alopecia totalis is seen in some families with vitamin D-dependent rickets type II. This is usually associated with a more severe phenotype. In this report, we present the clinical findings on a family which exhibited the typical clinical features of hereditary vitamin D-resistant rickets in two siblings. In addition, molecular analysis of the vitamin D receptor gene was performed by sequencing all coding exons. The cardinal findings in the index patient were alopecia totalis, renal tubular acidosis, mild generalized aminoaciduria, refractory rickets, high alkaline phosphatase, and hyperparathyroidism. Other routine biochemical tests were within normal limits, but 1+ glycine was detected in his urine. Skin biopsy results were compatible with alopecia areata. A previous child with similar phenotype was reported to be deceased at the age of 32 months. Mutation analysis of the vitamin D receptor gene by direct sequencing analysis of all coding exons showed a homozygous c.122GA(p.Cys41Tyr) variant in exon 2 with several arguments pointing to a pathogenic effect. We should be aware of this very rare disease whenever we see a patient with refractory rickets and alopecia. PMID:18426327

  6. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    SciTech Connect

    Zacharewski, T.

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  7. A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection.

    PubMed

    Monteiro, Freddy; Genin, Stphane; van Dijk, Irene; Valls, Marc

    2012-08-01

    Although much is known about the signals that trigger transcription of virulence genes in plant pathogens, their prevalence and timing during infection are still unknown. In this work, we address these questions by analysing expression of the main pathogenicity determinants in the bacterial pathogen Ralstonia solanacearum. We set up a quantitative, non-invasive luminescent reporter to monitor in planta transcription from single promoters in the bacterial chromosome. We show that the new reporter provides a real-time measure of promoter output in vivo - either after re-isolation of pathogens from infected plants or directly in situ - and confirm that the promoter controlling exopolysaccharide (EPS) synthesis is active in bacteria growing in the xylem. We also provide evidence that hrpB, the master regulator of type III secretion system (T3SS) genes, is transcribed in symptomatic plants. Quantitative RT-PCR assays demonstrate that hrpB and type III effector transcripts are abundant at late stages of plant infection, suggesting that their function is required throughout disease. Our results challenge the widespread view in R. solanacearum pathogenicity that the T3SS, and thus injection of effector proteins, is only active to manipulate plant defences at the first stages of infection, and that its expression is turned down when bacteria reach high cell densities and EPS synthesis starts. PMID:22609750

  8. Fusion gene vectors allowing for simultaneous drug selection, cell labeling, and reporter assay in vitro and in vivo.

    PubMed

    Zhao, Haobin; Hong, Ni; Lu, Wenqing; Zeng, Huaqiang; Song, Jianxing; Hong, Yunhan

    2012-01-17

    Vector systems allowing simultaneously for rapid drug selection, cell labeling, and reporter assay are highly desirable in biomedical research including stem cell biology. Here, we present such a vector system including pCVpf or pCVpr, plasmids that express pf or pr, a fusion protein between puromycin acetyltransferase and green or red fluorescent protein from CV, the human cytomegalovirus enhancer/promoter. Transfection with pCVpf or pCVpr produced a ?10% efficiency of gene transfer. A 2-day pulse puromycin selection resulted in ?13-fold enrichment for transgenic cells, and continuous puromycin selection produced stable transgenic stem cell clones with retained pluripotency. Furthermore, we developed a PAC assay protocol for quantification of transgene expression. To test the usefulness for cell labeling and PAC assay in vivo, we constructed pVASpf containing pf linked to the regulatory sequence of medaka germ gene vasa and generated transgenic fish with visible GFP expression in germ cells. PAC assay revealed the highest expression in the testis. Interestingly, PAC activity was also detectable in somatic organs including the eye, which was validated by fluorescence in situ hybridization. Therefore, the pf and pr vectors provide a useful system for simultaneous drug selection, live labeling, and reporter assay in vitro and in vivo. PMID:22081858

  9. RB1 gene mutations in Iranian patients with retinoblastoma: report of four novel mutations.

    PubMed

    Ahani, Ali; Behnam, Babak; Khorshid, Hamid Reza Khorram; Akbari, Mohammad Taghi

    2011-06-01

    Mutations in the RB1 gene lead to retinoblastoma, which is the most common intraocular tumor in children under the age of 6. In the present survey, the mutations of 18 unrelated Iranian retinoblastoma patients were characterized. Mutation analysis of the RB1 gene was performed in patients by sequencing all coding regions and by multiplex ligation probe-dependent amplification analysis. Clinical signs and symptoms of the retinoblastoma patients were similar to those of previously described patients with retinoblastoma. Eight known mutations and four novel mutations (c.832_833insT, c.1943delC, c.1206C>T, and c.2029delG) were determined. In silico analysis of the c.1206C>T variant showed that exon 12 contained an SC-35 consensus sequence, and this variation disrupted the splicing enhancer element and caused skipping of exon 12. Molecular genetic testing of retinoblastoma patients greatly affects the genetic counseling of the families involved, as well as the management of the disease in patients and at-risk relatives. PMID:21763628

  10. 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.

  11. Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes.

    PubMed

    Jindal, Sunita; Longchar, Bendangchuchang; Singh, Alka; Gupta, Vikrant

    2015-12-01

    Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants. PMID:26340695

  12. Radiation sensitivity of the gastrula-stage embryo: Chromosome aberrations and mutation induction in lacZ transgenic mice: The roles of DNA double-strand break repair systems.

    PubMed

    Jacquet, Paul; van Buul, Paul; van Duijn-Goedhart, Annemarie; Reynaud, Karine; Buset, Jasmine; Neefs, Mieke; Michaux, Arlette; Monsieurs, Pieter; de Boer, Peter; Baatout, Sarah

    2015-10-01

    At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/- embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including some stage-specific aspects that are not as yet understood. PMID:26433259

  13. Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe.

    PubMed

    Campbell, Dean O; Yaghoubi, Shahriar S; Su, Ying; Lee, Jason T; Auerbach, Martin S; Herschman, Harvey; Satyamurthy, Nagichettiar; Czernin, Johannes; Lavie, Arnon; Radu, Caius G

    2012-01-01

    Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-?-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies. PMID:22074768

  14. Differential gene expression in Neurospora crassa cell types: Ribosomal RNA genes. Comprehensive final terminal report of the overall activities for past 20 years

    SciTech Connect

    Dutta, S.K.

    1988-03-01

    This paper summarizes the accomplishments over the past 20 years of DOE support to the author`s research program. Highlights include molecular analysis of genome of Neurospora crassa, RNase sensitive RNA-dependent DNA polymerase, gene amplification of rRNA genes, molecular cloning of r-DNA`s, restriction fragment length polymorphism of rDNA in N. crassa., and ribosomal RNA processing genes of N. crassa.

  15. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    SciTech Connect

    Jirtle, Randy L.

    2012-10-11

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A{sup vy}) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure (<10 cGy) during early gestation. This information is particularly important to ascertain given the increased use of CT scans in disease diagnosis, increased number of people predicted to live and work in space, and the present concern about radiological terrorism. We showed for the first time that LDIR significantly increased DNA methylation at the A{sup vy} locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in animals and humans needs to be defined.

  16. RNA interference screening of interferon-stimulated genes with antiviral activities against classical swine fever virus using a reporter virus.

    PubMed

    Wang, Xiao; Li, Yongfeng; Li, Lian-Feng; Shen, Liang; Zhang, Lingkai; Yu, Jiahui; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2016-04-01

    Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is a highly contagious and often fatal disease of pigs, which leads to significant economic losses in many countries. Viral infection can induce the production of interferons (IFNs), giving rise to the transcription of hundreds of IFN-stimulated genes (ISGs) to exert antiviral effects. Although numerous ISGs have been identified to possess antiviral activities against different viruses, rare anti-CSFV ISGs have been reported to date. In this study, to screen anti-CSFV ISGs, twenty-one ISGs reported previously were individually knocked down using small interfering RNAs (siRNAs) followed by infection with a reporter CSFV expressing Renilla luciferase (Rluc). As a result, four novel anti-CSFV ISGs were identified, including natural-resistance-associated macrophage protein 1 (NRAMP1), cytosolic 5'-nucleotidase III A (NT5C3A), chemokine C-X-C motif ligand 10 (CXCL10), and 2'-5'-oligoadenylate synthetase 1 (OAS1), which were further verified to exhibit antiviral activities against wild-type CSFV. We conclude that the reporter virus is a useful tool for efficient screening anti-CSFV ISGs. PMID:26868874

  17. Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

    PubMed

    Cevenini, L; Calabretta, M M; Calabria, D; Roda, A; Michelini, E

    2016-01-01

    : The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples. PMID:25898810

  18. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    SciTech Connect

    Guldberg, P.; Henriksen, K.F.; Guettler, F.

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  19. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation

    PubMed Central

    Mendenhall, Alexander R.; Tedesco, Patricia M.; Sands, Bryan; Johnson, Thomas E.; Brent, Roger

    2015-01-01

    In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, “classical” multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms. PMID:25946008

  20. Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

    PubMed

    Kim, Young-Hwa; Youn, Hyewon; Na, Juri; Hong, Kee-Jong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene. PMID:25553100

  1. 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

  2. Sol-gel-derived materials for production of pin-printed reporter gene living-cell microarrays.

    PubMed

    Ge, Xin; Eleftheriou, Nikolas M; Dahoumane, Si Amar; Brennan, John D

    2013-12-17

    We report the fabrication of three-dimensional living-cell microarrays via pin-printing of soft sol-gel-derived silica materials containing bacterial cells. Bacterial cells entrapped in the silica-glycerol microarray spots can express reporter genes and produce strong fluorescence signals. The signals responded to the presence and concentration of inducers or repressors as expected, indicating that the entrapped cells remained metabolically active. Microscopic imaging of individual microarray spots at different culture times suggests that the entrapped cells can grow and divide, phenomena further confirmed by experiments in bulk sol-gel materials that demonstrated the increases of entrapped cell density and fluorescence during incubation in culture media. The cell microarrays can also be printed into 96-well glass bottom microtiter plates in a multiplexed manner, and the fluorescence signals generated were able to quantitatively and selectively respond to the concentration of inducers, thus demonstrating the potential for multitarget biosensing and high-throughput/high-content cell-based screening. The signal levels of bacterial cells in silica were significantly higher than those in alginate arrays, presumably due to viability of the entrapped cells in silica sol-gels. Microarray stability assays proved that the entrapped cells retained their physiological activity after storage for four weeks. Given that a large number of fluorescent and luminescent protein-based cell assays have been developed, the reporter gene living-cell microarrays demonstrated in this paper are expected to be applicable to a wide variety of research areas ranging from bioanalysis and chemical biology to drug discovery and probing of cell-material interactions. PMID:24279880

  3. Jasmonic acid stimulates the expression of nod genes in Rhizobium.

    PubMed

    Rosas, S; Soria, R; Correa, N; Abdala, G

    1998-12-01

    Jasmonates and salicylic acid are considered to be signal molecules that induce a variety of plant genes involved in wound or defence response, as well as affecting nos promoter activity. In this paper we examined whether these chemicals could also affect nod genes from isogenic rhizobia strains. Isogenic strains contain the Rhizobium leguminosarum nodA promoter fused to the lacZ gene of Escherichia coli and differ only in the source of the regulatory nodD gene. Naringenin, jasmonic acid and methyl jasmonate induced expression of nod genes in strain RBL1284 and salicylic acid showed no activity alone or when used in combination with other compounds; addition of naringenin + jasmonic acid produced a synergistic effect. Results obtained with strain RBL5284 were similar to those for RBL1284 albeit the combination of naringenin with the other compounds markedly inhibited nod gene expression. Whereas RBL5283 responded to naringenin with a strong induction, jasmonic acid, methyl jasmonate or salicylic acid showed no significant responses. The inhibitory effect of salicylic acid on nod gene expression indicates that the induction mechanism of jasmonic acid, methyl jasmonate, N-propyldihydrojasmonate and naringenin is probably different from that of salicylic acid. PMID:9869421

  4. Forebrain-specific promoter/enhancer D6 derived from the mouse Dach1 gene controls expression in neural stem cells.

    PubMed

    Machon, O; van den Bout, C J; Backman, M; Rsok, ; Caubit, X; Fromm, S H; Geronimo, B; Krauss, S

    2002-01-01

    Drosophila dachshund is involved in development of eye and limbs and in the development of mushroom bodies, a brain structure required for learning and memory in flies. Its mouse homologue mDach1 is expressed in various embryonic tissues, including limbs, the eye, the dorsal spinal cord and the forebrain. We have isolated a forebrain-specific 2.5-kb enhancer element termed D6 from the mouse mDach1 gene and created D6-LacZ and D6-green fluorescent protein (GFP) reporter gene mouse lines. In embryonic stages, the D6 enhancer activity is first detected at embryonic day 10.5 in scattered cells of the outbuldging cortical vesicles. By embryonic day 12.5, D6 activity expands throughout the developing neocortex and the hippocampus. In the adult mouse brain, D6 enhancer is active in neurons of the cortical plate, in the CA1 layer of the hippocampus and in cells of the subventricular zone and the ventricular ependymal zone. Adult mice also show D6 activity in the olfactory bulb and in the mamillary nucleus. Cultured D6-positive cells, which were derived from embryonic and postnatal brains, show characteristics of neural stem cells. They form primary and secondary neurospheres that differentiate into neurons and astrocytes as examined by cell-specific markers.Our results show that D6 enhancer exerts highly tissue-specific activity in the neurons of the neocortex and hippocampus and in neural stem cells. Moreover, the fluorescence cell sorting of D6-GFP cells from embryonic and postnatal stages allows specific selection of primary neural progenitors and their analysis. PMID:12088753

  5. Gene expression profiling and its use in adenocarcinomas of unknown primary origin: A case report

    PubMed Central

    DE MIGUEL, ANA CEBOLLERO; CID, ROBERTO PAZO; TRUFERO, JAVIER MARTINEZ; BERNAD, ISABEL PAJARES; URQUIZU, LOURDES CALERA; CUBERO, JORGE HERNANDO; TORRES, ANTONIO ANTON

    2015-01-01

    Carcinomas of unknown primary origin account for 35% of all malignancies. The current literature suggests that metastatic dissemination is able to occur in the absence of primary tumor growth. In metastatic disease that is difficult to diagnose, the origin usually remains unknown even after an exhaustive evaluation of immunohistochemistry (IHC) markers. In the current study, a 49-year-old male presented with lymph nodes metastases of unknown origin. The excisional biopsy of an inguinal node revealed an adenocarcinoma growth pattern, but the IHC could not determine the primary origin. A gene profiling test was performed to complete the diagnosis and a salivary gland adenocarcinoma was diagnosed with 90% probability. Subsequently, the patient underwent appropriate chemotherapy for salivary gland adenocarcinoma, and exhibited an improved partial response. The present case study highlights the importance of an accurate diagnosis of the primary tumor and the use of all the current tools available in order to provide patients with the best treatment possible. PMID:26622907

  6. Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model.

    PubMed

    Gorelick, Daniel A; Pinto, Caroline Lucia; Hao, Ruixin; Bondesson, Maria

    2016-01-01

    In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative dose-response information on estrogenic activity of chemical compounds. PMID:26585145

  7. Targeted Delivery of Gold Nanoparticle Contrast Agents for Reporting Gene Detection by Magnetic Resonance Imaging

    PubMed Central

    Vistain, Luke F.; Rotz, Matthew W.; Rathore, Richa; Preslar, Adam T.; Meade, Thomas J.

    2015-01-01

    Detection of protein expression by MRI requires a high payload of Gd(III) per protein binding event. Presented here is a targeted AuDNA nanoparticle capable of delivering several hundred Gd(III) chelates to the HaloTag reporter protein. Incubating this particle with HaloTag-expressing cells produced a 9.4 contrast-to-noise ratio compared to non-expressing cells. PMID:26505558

  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. Targeted delivery of gold nanoparticle contrast agents for reporting gene detection by magnetic resonance imaging.

    PubMed

    Vistain, Luke F; Rotz, Matthew W; Rathore, Richa; Preslar, Adam T; Meade, Thomas J

    2015-12-15

    Detection of protein expression by MRI requires a high payload of Gd(iii) per protein binding event. Presented here is a targeted AuDNA nanoparticle capable of delivering several hundred Gd(iii) chelates to the HaloTag reporter protein. Incubating this particle with HaloTag-expressing cells produced a 9.4 contrast-to-noise ratio compared to non-expressing cells. PMID:26505558

  10. New NodW- or NifA-regulated Bradyrhizobium japonicum genes.

    PubMed

    Caldelari Baumberger, Isabelle; Fraefel, Nicole; Gttfert, 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. PMID:12744463

  11. Genetic Association Studies Reporting on Variants in the C-Reactive Protein Gene and Coronary Artery Disease

    PubMed Central

    Shi, Yujie; Zhang, Jian; Tan, Chen; Xu, Wei; Sun, Qi; Li, Junxia

    2015-01-01

    Abstract C-reactive protein (CRP) is a commonly used inflammatory marker and elevated CRP levels are shown to increase the risk of coronary artery disease (CAD). Sequence variations in the CRP gene believed to influence the protein levels have been extensively investigated in CAD community. Most of the published studies, however, have reported mixed findings. The objective of the present study was to examine the associations of CRP variants (+942G>C, ?717A>G, +1444C>T) with genetic risk of CAD by use of a meta-analysis. The human casecontrol studies were identified through online search, hand search, and contacting the authors of original articles. We performed both random-effect and fixed-effect meta-analysis to estimate CAD risk (odds ratios, OR). This analysis combined 16 studies in total. We found +942G>C was not associated with CAD risk when all data were pooled together, nor did we find a significant association in subgroup analyses. Meta-analysis of +1444C>T studies showed a similar trend. However, a borderline association with CAD risk was revealed for ?717A>G (random-effect: OR?=?0.53, 95% CI?=?0.281.00 for the homozygous model; random-effect: OR?=?0.51, 95% CI?=?0.261.00 for the recessive model). These data suggest that the CRP gene variants examined may not modulate CAD risk. PMID:26266345

  12. Expression of Nodulation Genes in Rhizobium leguminosarum biovar trifolii Is Affected by Low pH and by Ca and Al Ions

    PubMed Central

    Richardson, Alan E.; Simpson, Richard J.; Djordjevic, Michael A.; Rolfe, Barry G.

    1988-01-01

    Early stages in the infection of leguminous plants by Rhizobium spp. are restricted at low pH and are further influenced by the presence of Ca and Al ions. In the experiments reported here, transcriptional and translational fusions of the Escherichia coli lacZ gene to Rhizobium leguminosarum biovar trifolii nodulation (nod) genes were used to investigate the effects of pH and of Ca and Al ions on nod gene expression. The regulatory nodD gene in R. leguminosarum biovar trifolii was constitutively expressed at a range of pH levels between 4.8 and 6.5, and expression was not affected by the addition of 22.5 ?M Al or 1,250 ?M Ca. Induction of expression of nodA, nodF, and region II nodulation genes in the presence of 5 10?7 M 7,4?-dihydroxyflavone was restricted at a pH of <5.7 and was extremely poor at pH 4.8. Induction of nodA expression was further restricted by 22.5 ?M Al over a range of pH levels but was increased in the presence of Ca. The addition of Ca, however, only slightly alleviated the Al-mediated inhibition of nodA induction. Induction of expression of nodA was equally sensitive to low pH in three strains of R. leguminosarum biovar trifolii (ANU845, ANU815, and ANU1184), which exhibited contrasting growth abilities in solution culture at a pH of <5.0. Aluminum, however, differentially affected the induction of nodA in these three strains, with the most Al-tolerant strain for growth being the most Al-sensitive strain for nod gene induction. Poor induction of expression of nodulation genes in R. leguminosarum biovar trifolii was considered to be an important factor contributing to the acid-sensitive step of legume root infection. Images PMID:16347761

  13. Mutation profile of BBS genes in Iranian patients with Bardet-Biedl syndrome: genetic characterization and report of nine novel mutations in five BBS genes.

    PubMed

    Fattahi, Zohreh; Rostami, Parvin; Najmabadi, Amin; Mohseni, Marzieh; Kahrizi, Kimia; Akbari, Mohammad Reza; Kariminejad, Ariana; Najmabadi, Hossein

    2014-07-01

    Bardet-Biedl syndrome (BBS) is a rare ciliopathy disorder that is clinically and genetically heterogeneous with 18 known genes. This study was performed to characterize responsible genes and mutation spectrum in a cohort of 14 Iranian families with BBS. Sanger sequencing of the most commonly mutated genes (BBS1, BBS2 and BBS10) accounting for ?50% of BBS patients determined mutations only in BBS2, including three novel mutations. Next, three of the remaining patients were subjected to whole exome sequencing with 96% at 20 depth of coverage that revealed novel BBS4 mutation. Observation of no mutation in the other patients represents the possible presence of novel genes. Screening of the remaining patients for six other genes (BBS3, BBS4, BBS6, BBS7, BBS9 and BBS12) revealed five novel mutations. This result represents another indication for the genetic heterogeneity of BBS and extends the mutational spectrum of the disease by introducing nine novel mutations in five BBS genes. In conclusion, although BBS1 and BBS10 are among the most commonly mutated genes in other populations like Caucasian, these two seem not to have an important role in Iranian patients. This suggests that a different strategy in molecular genetics diagnostic approaches in Middle Eastern countries such as Iran should be considered. PMID:24849935

  14. Primary renal sclerosing epithelioid fibrosarcoma: report of 2 cases with EWSR1-CREB3L1 gene fusion.

    PubMed

    Argani, Pedram; Lewin, Jack R; Edmonds, Pamela; Netto, George J; Prieto-Granada, Carlos; Zhang, Lei; Jungbluth, Achim A; Antonescu, Cristina R

    2015-03-01

    We report the first 2 genetically confirmed cases of primary renal sclerosing epithelioid fibrosarcoma (SEF), occurring in a 17-year-old boy and a 61-year-old woman. In both cases, the tumors demonstrated the typical epithelioid clear cell morphology associated with extensive hyalinizing fibrosis, raising the differential diagnosis of solitary fibrous tumor, metanephric stromal tumor, and the sclerosing variant of clear cell sarcoma of the kidney. Both neoplasms demonstrated diffuse immunoreactivity for MUC4, a highly specific marker for SEF, and both demonstrated evidence of rearrangement of both the EWSR1 and CREB3L1 genes, which have recently been shown to be fused in this entity. Both neoplasms presented with metastatic disease. Primary renal SEF represents yet another translocation-associated sarcoma now shown to arise primarily in the kidney. PMID:25353281

  15. Food flavonoid aryl hydrocarbon receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter gene assays.

    PubMed

    Van der Heiden, Edwige; Bechoux, Nathalie; Muller, Marc; Sergent, Thérèse; Schneider, Yves-Jacques; Larondelle, Yvan; Maghuin-Rogister, Guy; Scippo, Marie-Louise

    2009-04-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxin-responsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX((R)). Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cells were compared for species-dependent activities. We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24h. PMID:19286049

  16. 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

  17. Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains.

    PubMed

    Buckley, Anthony M; Jukes, Caitlin; Candlish, Denise; Irvine, June J; Spencer, Janice; Fagan, Robert P; Roe, Andrew J; Christie, John M; Fairweather, Neil F; Douce, Gillian R

    2016-01-01

    The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment. PMID:26996606

  18. Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein▿ †

    PubMed Central

    Cheng, Zhihui; Wang, Xueqi; Rikihisa, Yasuko

    2008-01-01

    The type IV secretion (T4S) system is critical for the virulence of several pathogens. In the rickettsial pathogen Ehrlichia chaffeensis, the virBD genes are split into two operons, the virB3-virB6 (preceded by sodB) and virB8-virD4 operons. Between these two operons, there are duplications of virB4, virB8, and virB9. In this study we found that transcription of all five loci was downregulated prior to the release of E. chaffeensis from host THP-1 cells and was upregulated at the initiation of exponential growth. Electrophoretic mobility shift assays revealed an E. chaffeensis-encoded protein that specifically bound to the promoter regions upstream of the virBD loci. The protein was purified from the bacterial lysate by affinity chromatography using a biotinylated promoter region upstream of sodB. Mass spectrometry identified the protein as an E. chaffeensis 12.3-kDa hypothetical protein, which was designated EcxR. Recombinant EcxR bound to the promoter regions upstream of five individual virBD loci. EcxR also activated transcription of all five virBD loci in lacZ reporter constructs. The expression of ecxR was positively autoregulated by EcxR. These results suggest that the five virBD loci are coordinately regulated by EcxR to allow developmental stage-specific expression of the T4S system in E. chaffeensis. PMID:18192398

  19. Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces.

    PubMed

    Knirschova, Renata; Novakova, Renata; Mingyar, Erik; Bekeova, Carmen; Homerova, Dagmar; Kormanec, Jan

    2015-06-01

    The integrative promoter-probe plasmid pBPSA1 was constructed using a promoterless Streptomyces aureofaciens CCM3239 bpsA gene encoding a non-ribosomal peptide synthase for the biosynthesis of a blue pigment, indigoidine. bpsA was also used to prepare pAMR4 plasmid for the deletion of genes in Streptomyces with facile identification of double crossover recombination. PMID:25801098

  20. AB130. Pseudoaldosteronism due to mutation of SCNN1A gene: a case report

    PubMed Central

    Can, Ngoc Thi Bich; Vu, Dung Chi; Bui, Thao Phuong; Nguyen, Khanh Ngoc; Zennaro, Maria-Christina; Wudy, Stefan A.

    2015-01-01

    Background Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disease characterized by resistance to the actions of aldosterone. It was first described in 1958 by Cheek and Perry, and common clinical manifestations include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in the neonatal period. Objective To describe clinical characteristics, laboratory features and management of one Vietnamese patient with pseudohypoaldosteron. Methods Clinical features, biochemical finding, mutation analysis and management in a 1 month-old-boy was studied. Based on analysis of this patient’s clinical symptoms associated with biochemical examination, the urinary steroid metabolomics analysis was performed using gas chromatography spectrometry and mutation analysis of SCNN1A was performed using PCR & direct sequencing. Results Patient is the first child normal delivery with the gestation age of 41 weeks, birth weight of 3,200 g, and onset of the disease at 7 days of age. He presented with lost weight, dehydration without vomit, diarrhea or hyperpigmentation. He was admitted with the features of cyanosis, allorhythmic, electrolyte imbalance with sodium of 119 mmoL/L, potassium of 7.4 mmoL/L. Investigation show pH 7.26, PCO2 34 mmHg, PO2 110 mmHg, HCO3- 18 mmoL/L, BE -10, plasma 17OHP level: 2.4 ng/mL, testosterone level: 1.94 nmoL/L, Cortisol 8am: 2,662.8 pmoL/L, Ure 7.4 mmoL/L, Creatinine 44.2 µmoL/L, Glucose 4.8 mmoL/L. The urine steroid metabolomics analysis showed extensive excretion of aldosterone ID-ISTD1 of 1,157.41 µg/L. Novel homozygous mutation (c.1668C > A; p.S556R) of SCNN1A gene was identified in the proband. He was treated with florinef of 0.1 mg/kg/day for electrolyte balance. He had complication of intestinal perforation and died due to infection. In conclusions, PHA1 causes severe hyponatremia, metabolic acidosis, and life-threatening hyperkalemia, with normal 17-a-hydroxyprogesterone levels and high excretion of aldosterone levels.

  1. A cross-sectional study of self-reported chemical-related sensitivity is associated with gene variants of drug-metabolizing enzymes

    PubMed Central

    Schnakenberg, Eckart; Fabig, Karl-Rainer; Stanulla, Martin; Strobl, Nils; Lustig, Michael; Fabig, Nathalie; Schloot, Werner

    2007-01-01

    Background N-acetyltransferases (NAT) and glutathione S-transferases (GST) are involved in the metabolism of several ubiquitous chemical substances leading to the activation and detoxification of carcinogenic heterocyclic and aromatic amines. Since polymorphisms within these genes are described to influence the metabolism of ubiquitous chemicals, we conducted the present study to determine if individuals with self-reported chemical-related sensitivity differed from controls without self-reported chemical-related sensitivity with regard to the distribution of genotype frequencies of NAT2, GSTM1, GSTT1, and GSTP1 polymorphisms. Methods Out of 800 subjects who answered a questionnaire of ten items with regard to their severity of chemical sensitivity 521 unrelated individuals agreed to participate in the study. Subsequently, genetic variants of the NAT2, GSTM1, GSTT1, and GSTP1 genes were analyzed. Results The results show significant differences between individuals with and without self-reported chemical-related sensitivity with regard to the distribution of NAT2, GSTM1, and GSTT1 gene variants. Cases with self-reported chemical-related sensitivity were significantly more frequently NAT2 slow acetylators (controlled OR = 1.81, 95% CI = 1.272.59, P = 0.001). GSTM1 and GSTT1 genes were significantly more often homozygously deleted in those individuals reporting sensitivity to chemicals compared to controls (GSTM1: controlled OR 2.08, 95% CI = 1.462.96, P = 0.0001; GSTT1: controlled OR = 2.80, 95% CI = 1.654.75, P = 0.0001). Effects for GSTP1 gene variants were observed in conjunction with GSTM1, GSTT1 and NAT2 gene. Conclusion The results from our study population show that individuals being slow acetylators and/or harbouring a homozygous GSTM1 and/or GSTT1 deletion reported chemical-related hypersensitivity more frequently. PMID:17291352

  2. A Novel, Photosynthesis-Associated Thioredoxin-Like Gene: Final Technical Report

    SciTech Connect

    Collier, Jackie, L

    2005-09-13

    Many aspects of the biosynthesis and physiological regulation of the photosynthetic apparatus of plants, algae and cyanobacteria remain to be understood, and are likely to involve yet-unidentified proteins that carry out oxidation/reduction (redox) reactions. TxlA from Synechococcus sp. strain PCC 7942 and its homologues from other cyanobacteria and plants, including Sll1980 from the cyanobacterium Synechocystis sp. strain PCC 6803, are likely to be among these proteins. In fact, the homologue of TxlA in the plant Arabidopsis thaliana, HCF164, may be required for synthesis of the cytochrome b6f complex that transfers electrons between the two photosynthetic reaction centers. TxlAs share an N-terminal hydrophobic domain, a central thioredoxin-like domain, and a unique C-terminal hydrophilic domain. Plant and algal TxlAs are nuclear-encoded and have an additional N-terminal domain that targets them to the chloroplast. We have found that the common N-terminal domain of TxlA anchors it to a membrane, probably the thylakoid (photosynthetic) membrane (where HCF164 is also localized, with its thioredoxin-like domain in the thylakoid lumen). We have also found that the thioredoxin-like domain is likely to assume the conformation typical of thioredoxins and possesses thioredoxin-like redox activity in vitro, and that the C-terminal domain is important to the structure and function of the thioredoxin-like domain both in vivo and in vitro. These data show that TxlAs have the cellular location and enzymatic activity expected of a protein involved in the biosynthesis of redox components or redox regulation of the photosynthetic apparatus. We were unable to inactivate the thioredoxin-like domain of TxlA in either PCC 7942 or PCC 6803, under either photosynthetic or heterotrophic growth conditions. We also found that expression of antisense txlA mRNA from an IPTG-regulated promoter in PCC 7942 was lethal, most likely because it effectively inactivated txlA by ''RNA silencing''. These results are consistent with a role for TxlA in the synthesis of the cytochrome b6f complex, which is required for both photosynthetic and respiratory electron transport in cyanobacteria. In contrast, our PCC 7942 mutants in which the C-terminal domain of TxlA was removed are viable and appear to have normal cytochrome content, but have a subtle pigmentation phenotype (increased content of phycocyanin relative to chlorophyll) that depends on both light and CO2 availability. We have also found that PCC 6803 Sll1980 inactivation mutant merodiploids have a similar pigmentation phenotype to the PCC 7942 C-terminal truncation mutants when grown photoautotrophically. In addition, when grown heterotrophically the PCC 6803 Sll1980 inactivation mutant merodiploids remain green instead of turning a golden color like the wild-type, and they are more sensitive to the b6f complex inhibitor DBMIB than is wild type PCC 6803. That the PCC 6803 Sll1980 inactivation mutant merodiploids have these phenotypes despite the fact that they still contain normal copies of the sll1980 gene suggests that the presence of truncated Sll1980 protein interferes with the function of normal Sll1980 protein. Together, these physiological data suggest that TxlA has an essential redox role in cyanobacteria, perhaps a biosynthetic one, and may also have a nonessential regulatory role reflected in the phenotypes of the PCC 7942 C-terminal truncation mutants and the PCC 6803 Sll1980 inactivation mutant merodiploids.

  3. 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.

  4. 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

  5. Mesoscopic fluorescence molecular tomography of reporter genes in bioprinted thick tissue

    PubMed Central

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

    2013-01-01

    Abstract. Three-dimensional imaging of thick tissue constructs is one of the main challenges in the field of tissue engineering and regenerative medicine. Optical methods are the most promising as they offer noninvasive, fast, and inexpensive solutions. Herein, we report the use of mesoscopic fluorescence molecular tomography (MFMT) to image function and structure of thick bioprinted tissue hosted in a 3-mm-thick bioreactor. Collagen-based tissue assembled in this study contains two vascular channels formed by green fluorescent protein- and mCherry-expressing cells. Transfected live cell imaging enables us to image function, whereas Flash Red fluorescent bead perfusion into the vascular channel allows us to image structure. The MFMT optical reconstructions are benchmarked with classical microscopy techniques. MFMT and wide-field fluorescence microscopy data match within 92% in area and 84% in location, validating the accuracy of MFMT reconstructions. Our results demonstrate that MFMT is a well-suited imaging modality for fast, longitudinal, functional imaging of thick, and turbid tissue engineering constructs. PMID:24091624

  6. 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.

  7. Direct selection of mutations reducing transcription or translation of the recA gene of Escherichia coli with a recA-lacZ protein fusion.

    PubMed

    Weisemann, J M; Weinstock, G M

    1985-08-01

    When a recA-lacZ protein fusion was cloned into phage lambda, the resulting transducing phage grew normally on wild-type Escherichia coli, but its growth was severely inhibited in lexA(Def) mutant strains that express recA constitutively at high levels. Mutants of the transducing phage that grew on the lexA(Def) strains were isolated and were found to affect production of the RecA-beta-galactosidase hybrid protein. Most mutants, including a number of nonsense mutants, were phenotypically LacZ-. LacZ+ mutants were also isolated; most of these expressed lower basal and induced levels of beta-galactosidase activity. DNA sequence analysis revealed that some of the LacZ+ mutations were in the recA promoter. One of these was found to prevent induction. Unexpectedly, three of the mutations that reduced expression were located in the recA structural gene, at codons 10, 11, and 12. Further analysis of the codon 10 mutant showed that it most likely affected translation since it had little effect on transcription as measured by beta-galactosidase synthesis from a recA-lacZ operon fusion. This expression defect was not limited to the protein fusion, since the codon 10 mutation also reduced synthesis of RecA protein when present in a complete recA gene. Analysis of the recA DNA sequence in the fusion revealed that each of the mutations at codons 10, 11, and 12 increases the homology between this region of the mRNA and a sequence found at codons 1 to 4. Thus, the secondary structure of the mutant recA mRNAs may be affecting translation. PMID:3160689

  8. A New Pyrimidine-Specific Reporter Gene: A Mutated Human Deoxycytidine Kinase Suitable for PET During Treatment with Acycloguanosine-Based Cytotoxic Drugs

    PubMed Central

    Likar, Yury; Zurita, Juan; Dobrenkov, Konstantin; Shenker, Larissa; Cai, Shangde; Neschadim, Anton; Medin, Jeffrey A.; Sadelain, Michel; Hricak, Hedvig; Ponomarev, Vladimir

    2015-01-01

    In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET. Methods Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2′-deoxy-2′-fluoroarabinofuranosylcytosine, 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs. Results Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals (18F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)–transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers (18F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine). Conclusion The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with 18F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells. PMID:20810757

  9. Genes and Gene Therapy

    MedlinePLUS

    ... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  10. Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy

    PubMed Central

    Baril, Patrick; Martin-Duque, Pilar; Vassaux, Georges

    2010-01-01

    Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer (123I-, 124I-, 99mTcO4-), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x PMID:19814733

  11. Exclusion of a schizophrenia susceptibility gene from the chromosome 5q11-q13 region: new data and a reanalysis of previous reports.

    PubMed Central

    McGuffin, P; Sargeant, M; Hetti, G; Tidmarsh, S; Whatley, S; Marchbanks, R M

    1990-01-01

    The report of a putative schizophrenia susceptibility gene linked to markers in the chromosome 5q11-q13 region and subsequent failures of replication have provoked considerable controversy. We here report six Welsh families multiply affected with schizophrenia in which there is no evidence for linkage between a dominant-like schizophrenia gene and 5q11-q13 markers. It is argued that our new results together with a combined reanalysis of previous studies suggest that a schizophrenia susceptibility gene can be excluded from the 5q11-q13 region. The apparent disparities between published results are most likely to reflect a chance finding in the one positive study and probably should not be interpreted as resulting from true linkage heterogeneity. PMID:2393025

  12. 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 from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring. PMID:23032559

  13. 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

  14. 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…

  15. Search for major genes with progeny test data to accelerate the development of genetically superior loblolly pines. Quarterly report [July - September 1999

    SciTech Connect

    Li, Bailian

    1999-08-16

    This quarterly report describes the progress made on the 3 research tasks of the project. The 3 research tasks are: (1) Develop genetic models and analytical methods; (2) Molecular confirmation of major gene segregation; and (3) Develop strategies for marker-assisted breeding.

  16. Spontaneous coronary artery dissection in a young man with a factor v leiden gene mutation: a case report and review of the literature.

    PubMed

    Khan, Tahir; Danyi, Peter; Topaz, On; Ali, Asghar; Jovin, Ion S

    2013-12-01

    Spontaneous coronary artery dissection is a rare but increasingly recognized cause of acute myocardial ischemia in young adults, especially in women. We report a case of spontaneous coronary dissection in a young healthy man who was also a carrier of the factor V Leiden gene mutation. PMID:24436622

  17. The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis.

    PubMed

    Tanabe, Noriaki; Tamoi, Masahiro; Shigeoka, Shigeru

    2015-08-10

    Sweet potato is an important crop because of its high yield and biomass production. We herein investigated the potential of the promoter activity of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) from sweet potato (Ipomoea batatas) in order to develop the high expression system of exogenous DNA in Arabidopsis. We isolated two different cDNAs (IbRbcS1 and IbRbcS2) encoding RbcS from sweet potato. Their predicted amino acid sequences were well conserved with the mature RbcS protein of other plants. The tissue-specific expression patterns of these two genes revealed that expression of IbRbcS1 was specific to green tissue, whereas that of IbRbcS2 was non-photosynthetic tissues such as roots and tubers. These results suggested that IbRbcS1 was predominantly expressed in the green tissue-specific of sweet potato over IbRbcS2. Therefore, the IbRbcS1 promoter was transformed into Arabidopsis along with ?-glucuronidase (GUS) as a reporter gene. GUS staining and semi-quantitative RT-PCR showed that the IbRbcS1 promoter conferred the expression of the GUS reporter gene in green tissue-specific and light-inducible manners. Furthermore, qPCR showed that the expression levels of GUS reporter gene in IbRbcS1 pro:GUS were same as those in CaMV 35S pro:GUS plants. These results suggest that the IbRbcS1 promoter is a potentially strong foreign gene expression system for genetic transformation in plants. PMID:25958348

  18. [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.

  19. Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).

    PubMed

    Zaniboni, Andrea; Merlo, Barbara; Zannoni, Augusta; Bernardini, Chiara; Lavitrano, Marialuisa; Forni, Monica; Mari, Gaetano; Bacci, Maria Laura

    2013-02-01

    Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene. Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein. PMID:23312469

  20. Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk.

    PubMed

    Wielogorska, E; Elliott, C T; Danaher, M; Connolly, L

    2014-07-01

    Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer. Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds. A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid-liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36 pg g(-)(1) EEQ and has been successfully applied in testing a range of milk samples. PMID:24769019

  1. Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression.

    PubMed

    Lehnherr, H; Guidolin, A; Arber, W

    1991-10-01

    Amber mutants of bacteriophage P1 were used to identify functions involved in late regulation of the P1 lytic growth cycle. A single function has been genetically identified to be involved in activation of the phage-specific late promoter sequence Ps. In vivo, P1 gene 10 amber mutants fail to trans activate a lacZ operon fusion under the transcriptional control of promoter Ps. Several P1 segments, mapping around position 95 on the P1 chromosome, were cloned into multicopy plasmid vectors. Some of the cloned DNA segments had a deleterious effect on host cells unless they were propagated in a P1 lysogenic background. By deletion and sequence analysis, the harmful effect could be delimited to a 869-bp P1 fragment, containing a 453-bp open reading frame. This open reading frame was shown to be gene 10 by sequencing the amber mutation am10.1 and by marker rescue experiments with a number of other gene 10 amber mutants. Gene 10 codes for an 18.1-kDa protein showing an unusually high density of charged amino acid residues. No significant homology to sequences present in the EMBL/GenBank data base was found, and the protein contained none of the currently known DNA-binding motifs. An in vivo trans activation assay system, consisting of gene 10 under the transcriptional control of an inducible promoter and a gene S/lacZ fusion transcribed from Ps, was used to show that gene 10 is the only phage-encoded function required for late promoter activation. PMID:1917870

  2. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection a Technical Report

    PubMed Central

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nr, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  3. Kullback-Leibler information for ordering genes using sperm typing and radiation-hybrid mapping. Technical report

    SciTech Connect

    Chernoff, H.

    1991-10-01

    Two technologies applicable to gene mapping are those of sperm typing and radiation hybrid mapping. Sperm typing makes use of the polymerase chain reaction, a biochemical technique which allows enormous amplification (production of multiple copies) of small, selected DNA fragments from a single chromosome. A sample of sperm from a single donor is analyzed to see which alleles (distinct forms of the various genes) are present in the individual sperms. The frequencies with which the various possibilities occur can be used to supply estimates of the ordering and of the recombination probabilities among the genes for which that donor is heterozygous (having different alleles of the same gene.)

  4. Inherited protein S deficiency due to a novel nonsense mutation in the PROS1 gene in the patient with recurrent vascular access thrombosis: A case report

    PubMed Central

    Cho, Eun Jin; Kim, Yong Chul; Hwang, Jin Ho; Lee, Hajung; Park, Sung Sup; Kim, So Yeon; Kim, Suhnggwon; Chin, Ho Jun

    2012-01-01

    Vascular access thrombosis is one of the major causes of morbidity in patients maintained on chronic hemodialysis. Thrombophilia has been recognized as a risk factor of vascular access thrombosis. The authors report a case of inherited protein S deficiency associated with vascular access thrombotic events. DNA sequence analysis of the PROS1 gene identified a novel heterozygous nonsense mutation in exon 10 by transition of AAG (lysine) to TAG (stop codon) at codon 473 (c.1417A>T, p.K473X). Results from the study suggest that the inherited protein S deficiency due to a PROS1 gene mutation may cause vascular access thrombosis in hemodialysis patients.

  5. Development of a new reporter gene system--dsRed/xanthine phosphoribosyltransferase-xanthine for molecular imaging of processes behind the intact blood-brain barrier.

    PubMed

    Doubrovin, Mikhail; Ponomarev, Vladimir; Serganova, Inna; Soghomonian, Suren; Myagawa, Tadashi; Beresten, Tatiana; Ageyeva, Lyudmila; Sadelain, Michel; Koutcher, Jason; Blasberg, Ronald G; Tjuvajev, Juri G Gelovani

    2003-04-01

    We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT) for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications. The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional. The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of [14C]-xanthine was in vitro (Ki = 0.124 +/- 0.008 vs. 0.00031 +/- 0.00005 mL/min/g in parental cell line), and [*]-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (logP = -0.69), meeting requirements for the blood-brain barrier (BBB) penetrating tracer. In the in vivo experiment, the concentration of [14C]-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration. The accumulation in vivo in the transfected flank tumor was to 2.4 +/- 0.3% dose/g, compared to 0.78 +/- 0.02% dose/g and 0.64 +/- 0.05% dose/g in the control flank tumors and intact muscle, respectively. [14C]-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT), which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections. The images of endogenous gene expression with the "sensory system" have to be normalized for the transfection efficiency based on the "beacon system" image data. Such an approach requires two different "reporter genes" and two different "reporter substrates." Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB. PMID:12964307

  6. A Novel Mgp-Cre Knock-In Mouse Reveals an Anticalcification/Antistiffness Candidate Gene in the Trabecular Meshwork and Peripapillary Scleral Region

    PubMed Central

    Borrs, Teresa; Smith, Matthew H.; Buie, LaKisha K.

    2015-01-01

    Purpose. Soft tissue calcification is a pathological condition. Matrix Gla (MGP) is a potent mineralization inhibitor secreted by cartilage chondrocytes and arteries' vascular smooth muscle cells. Mgp knock-out mice die at 6 weeks due to massive arterial calcification. Arterial calcification results in arterial stiffness and higher systolic blood pressure. Intriguingly, MGP was highly abundant in trabecular meshwork (TM). Because tissue stiffness is relevant to glaucoma, we investigated which additional eye tissues use Mgp's function using knock-in mice. Methods. An MgpCre-recombinase coding sequence (Cre) knock-in mouse, containing Mgp DNA plus an internal ribosomal entry site (IRES)-Cre-cassette was generated by homologous recombination. Founders were crossed with Cre-mediated reporter mouse R26R-lacZ. Their offspring expresses lacZ where Mgp is transcribed. Eyes from MgpCre/+;R26RlacZ/+ (Mgp-lacZ knock-in) and controls, 1 to 8 months were assayed for ?-gal enzyme histochemistry. Results. As expected, Mgp-lacZ knock-in's TM was intensely blue. In addition, this mouse revealed high specific expression in the sclera, particularly in the peripapillary scleral region (ppSC). Ciliary muscle and sclera above the TM were also positive. Scleral staining was located immediately underneath the choroid (chondrocyte layer), began midsclera and was remarkably high in the ppSC. Cornea, iris, lens, ciliary body, and retina were negative. All mice exhibited similar staining patterns. All controls were negative. Conclusions. Matrix Gla's restricted expression to glaucoma-associated tissues from anterior and posterior segments suggests its involvement in the development of the disease. Matrix Gla's anticalcification/antistiffness properties in the vascular tissue, together with its high TM and ppCS expression, place this gene as a strong candidate for TM's softness and sclera's stiffness regulation in glaucoma. PMID:25711639

  7. Baculovirus as an Ideal Radionuclide Reporter Gene Vector: A New Strategy for Monitoring the Fate of Human Stem Cells In Vivo

    PubMed Central

    Wu, Haifei; Lv, Jing; Xu, Xiaoqian; Zhang, Yifan

    2013-01-01

    Purpose Radionuclide reporter gene imaging holds promise for non-invasive monitoring of transplanted stem cells. Thus, the feasibility of utilizing recombinant baculoviruses carrying the sodium iodide symporter (NIS) reporter gene in monitoring stem cell therapy by radionuclide imaging was explored in this study. Methods Recombinant baculoviruses carrying NIS and green fluorescent protein (GFP) reporter genes (Bac-NIS and Bac-GFP) were constructed and used to infect human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). Infection efficiency, total fluorescence intensity and duration of transgene expression were determined by flow cytometry. Cytotoxicity/proliferative effects of baculovirus on hUCB-MSCs were assessed using CCK-8 assays. 125I uptake and perchlorate inhibition assays were performed on Bac-NIS-infected hUCB-MSCs. Radionuclide imaging of mice transplanted with Bac-NIS-infected hUCB-MSCs was performed by NanoSPECT/CT imaging. Results Infection efficiencies of recombinant baculovirus in hESCs, hiPSCs and hUCB-MSCs increased with increasing MOIs (27.3%, 35.8% and 95.6%, respectively, at MOI?=?800). Almost no cytotoxicity and only slight effects on hUCB-MSCs proliferation were observed. Obvious GFP expression (40.6%) remained at 8 days post-infection. The radioiodide was functionally accumulated by NIS gene products and specifically inhibited by perchlorate (ClO4-). Radioiodide uptake, peaking at 30 min and gradually decreasing over time, significantly correlated with hUCB-MSCs cell number (R2?=?0.994). Finally, radionuclide imaging showed Bac-NIS-infected hUCB-MSCs effectively accumulated radioiodide in vivo, which gradually weakened over time. Conclusion Baculovirus as transgenic vector of radionuclide reporter gene imaging technology is a promising strategy for monitoring stem cell transplantation therapy. PMID:23596521

  8. Focally folded myelin in Charcot-Marie-Tooth type 1B disease is associated with Asn131Lys mutation in myelin protein zero gene: short report.

    PubMed

    Kocha?ski, A; Drac, H; Jedrzejowska, H; Hausmanowa-Petrusewicz, I

    2003-09-01

    Charcot-Marie-Tooth disease type 1B (CMT1B) is a demyelinating neuropathy inherited as an autosomal dominant trait. The majority of CMT1B cases are caused by mutations in the myelin protein zero (P0) gene (MPZ). Only a few mutations in MPZ gene have been reported to be associated with focally folded myelin sheaths. We have studied five patients from one family with five generations, affected by CMT1B disease. The morphological studies of sural nerve biopsy performed in the proband revealed fibers with focally folded myelin. DNA sequencing analysis showed the Asn131Lys mutation in the MPZ gene in three members of the affected family. PMID:12940837

  9. 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.; Mgarban, 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

  10. Yeast intragenic transcriptional control: activation and repression sites within the coding region of the Saccharomyces cerevisiae LPD1 gene.

    PubMed Central

    Sinclair, D A; Kornfeld, G D; Dawes, I W

    1994-01-01

    Though widely recognized in higher eukaryotes, the regulation of Saccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPD1 gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPD1::lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYC1::lacZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate of LPD1 transcription rather than mRNA degradation or translation. Activation from the DAS1 region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3- to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements. Images PMID:8264590

  11. Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.

    PubMed Central

    Maciver, B; McHale, R H; Saul, D J; Bergquist, P L

    1994-01-01

    The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity. Images PMID:7993087

  12. Mutation analysis of the phenylalanine hydroxylase gene in Azerbaijani population, a report from West Azerbaijan province of Iran

    PubMed Central

    Bagheri, Morteza; Rad, Isa Abdi; Jazani, Nima Hosseini; Zarrin, Rasoul; Ghazavi, Ahad

    2015-01-01

    Objective(s): Phenylketonuria (PKU) is a genetic inborn error of phenylalanine (Phe) metabolism resulting from insufficiency in the hepatic enzyme, phenylalanine hydroxylase (PAH), which leads to elevated levels of Phe in the blood. The present study was carried out for mutation analysis of the PAH gene in West Azerbaijan province of Iran. Materials and Methods: A total of 218 alleles from 40 PKU families were studied using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method. Results: The frequencies of IVS10-11, S67P, R261Q, R252W, IVS11nt-1 g>c, R408Q, and Q232Q mutations were 28(35), 17(21.25), 15(18.75), 3(3.75), 3(3.75), 2(2.5), and 1(1.25), in cases group, and 51(23.4), 31(14.2), 27(12.4), 6(2.75), 6(2.75), 4(1.83), and 2(0.92) in total group, respectively. The mutations of R243Q, 364delG, L333F, 261X, I65T, and R408W were not detected in our samples. Conclusion: It can be concluded that the IVS10-11 mutation has the highest frequency in the tested population. To our knowledge, this report is the first in its own kind and provides better understanding of the genetic heterogeneity, the origin and distributions of PAH mutations in West Azerbaijan province of Iran. PMID:26351554

  13. Stimuli that induce a yeast heat shock gene fused to beta-galactosidase.

    PubMed Central

    Brazzell, C; Ingolia, T D

    1984-01-01

    Saccharomyces cerevisiae contain a multigene family related to the Drosophila heat shock gene hsp70. Two members of this family, YG100 and YG101, have been previously characterized (Ingolia et al., Mol. Cell. Biol. 2:1388-1398, 1982), and only YG100 was found to have elevated levels of transcription after heat shock. The yeast hsp70 genes contained on YG100 and YG101 were truncated and fused to the Escherichia coli lacZ gene contained on pMC1587 (Casadaban et al., Methods Enzymol. 100:283-308, 1983). The resulting plasmids directed synthesis of the beta-galactosidase gene as measured by in vitro enzyme assays and by colorimetric assays on plates. The expression level from the YG101 gene was constant under all the conditions tested, whereas expression driven by the YG100 gene could be induced over 50-fold. Other stimuli besides heat, including recovery from anoxia and high cell density, were found to strongly induce YG100 gene expression. Most physical and chemical stimuli tested, including UV irradiation, zymolyase treatment, and ethanol, did not stimulate expression of this heat shock gene. PMID:6098811

  14. Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11.

    PubMed

    Qian, Guoliang; Wang, Yansheng; Qian, Dongyu; Fan, Jiaqin; Hu, Baishi; Liu, Fengquan

    2012-02-01

    Here, three different suicide vectors were evaluated for the possibility of performing gene mutagenesis in strain OH11 using the chiA gene (accession number: DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied for disruption and in-frame deletions in the chiA gene in strain OH11, which was confirmed by PCR amplification and Southern hybridization. The chiA-deletion mutant OH11-3 did not have the ability to produce chitinase on chitine selection medium. Interestingly, the chiA-deletion mutants displayed wild-type antimicrobial activity against Saccharomyces cerevisiae, Magnaporthe grisea, Phytophthora capsici, Rhizoctonia solani, Sclerotinia sclerotiorum and Pythium ultimum. Our data suggest that chitinase might not be a unique lytic enzyme in controlling S. cerevisiae, M. grisea, P. capsici, and P. ultimum. R. solani, S. sclerotiorum. Also, suicide vector pEX18GM might be explored as a potential tool for gene deletions in L. enzymogenes, which will facilitate the molecular study of mechanisms of biological control in L. enzymogenes. PMID:22806850

  15. Fundus albipunctatus: review of the literature and report of a novel RDH5 gene mutation affecting the invariant tyrosine (p.Tyr175Phe).

    PubMed

    Skorczyk-Werner, Anna; Pawłowski, Przemysław; Michalczuk, Marta; Warowicka, Alicja; Wawrocka, Anna; Wicher, Katarzyna; Bakunowicz-Łazarczyk, Alina; Krawczyński, Maciej R

    2015-08-01

    Fundus albipunctatus (FA) is a rare, congenital form of night blindness with rod system impairment, characterised by the presence of numerous small, white-yellow retinal lesions. FA belongs to a heterogenous group of so-called flecked retina syndromes. This disorder shows autosomal recessive inheritance and is caused mostly by mutations in the RDH5 gene. This gene encodes the enzyme that is a part of the visual cycle, the 11-cis retinol dehydrogenase. This study is a brief review of the literature on FA and a report of the first molecular evidence for RDH5 gene mutation in a Polish patient with this rare disorder. We present a novel pathogenic RDH5 gene mutation in a 16-year-old female patient with symptoms of night blindness. The patient underwent ophthalmological examinations, including colour vision testing, fundus photography, automated visual field testing, full-field electroretinography (ERG) and spectral optical coherent tomography (SOCT). The patient showed typical FA ERG records, the visual field was constricted and fundus examination revealed numerous characteristic, small, white-yellowish retinal lesions. DNA sequencing of the RDH5 gene coding sequence (exons 2-5) enabled the detection of the homozygous missense substitution c.524A > T (p.Tyr175Phe) in exon 3. This is the first report of RDH5 gene mutation that affects the invariant tyrosine, one of the most conserved amino acid residues in short-chain alcohol dehydrogenases/reductases (SDRs), crucial for these enzymes' activity. The location of this substitution, together with its predicted influence on the protein function, indicate that the p.Tyr175Phe mutation is the cause of FA in our patient. PMID:25820994

  16. Differential gene expression in Neurospora crassa cell types: heterogeneity and multiple copies of rRNA genes. Annual progress report, July 1981-June 1982

    SciTech Connect

    Dutta, S.K.

    1982-01-01

    The significant results obtained were as follows: (I) Multiple copies of isolated rRNA genes from N. crassa were tested for heterogeneity by rRNA: rDNA reassociation kinetics. More than 90% of rDNA copies were identical. The possible heterogeneity of a small fraction of rDNAs could not be attributed to inclusion of any tDNA sequences. (II) Two approaches to study gross differences between rRNA genes from N. crassa cell types-conidia, germinated conidia, and mycelia were undertaken. No difference was seen in either the restriction patterns nor the autoradiographs. Either gross differences between rDNAS of N. crassa cell types were not present or they were not detected by these two approaches. (III) Using similar DNA restriction analysis procedures, differences between closely related heterothallic and homothallic species of Neurospora were detected. (IV) Successful sequencing of 317 bases of the N. crassa slime mutant pMF2 clone which includes the 5.8S rDNA and it's flanking internal spacer regions was achieved. (ERB)

  17. Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1991--August 31, 1992

    SciTech Connect

    Barkan, A.

    1992-12-31

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  18. The first report of a Pelecaniformes defensin cluster: characterization of β-defensin genes in the crested ibis based on BAC libraries.

    PubMed

    Lan, Hong; Chen, Hui; Chen, Li-Cheng; Wang, Bei-Bing; Sun, Li; Ma, Mei-Ying; Fang, Sheng-Guo; Wan, Qiu-Hong

    2014-01-01

    Defensins play a key role in the innate immunity of various organisms. Detailed genomic studies of the defensin cluster have only been reported in a limited number of birds. Herein, we present the first characterization of defensins in a Pelecaniformes species, the crested ibis (Nipponia nippon), which is one of the most endangered birds in the world. We constructed bacterial artificial chromosome libraries, including a 4D-PCR library and a reverse-4D library, which provide at least 40 equivalents of this rare bird's genome. A cluster including 14 β-defensin loci within 129 kb was assigned to chromosome 3 by FISH, and one gene duplication of AvBD1 was found. The ibis defensin genes are characterized by multiform gene organization ranging from two to four exons through extensive exon fusion. Splicing signal variations and alternative splice variants were also found. Comparative analysis of four bird species identified one common and multiple species-specific duplications, which might be associated with high GC content. Evolutionary analysis revealed birth-and-death mode and purifying selection for avian defensin evolution, resulting in different defensin gene numbers among bird species and functional conservation within orthologous genes, respectively. Additionally, we propose various directions for further research on genetic conservation in the crested ibis. PMID:25372018

  19. Microbial reporter gene assay as a diagnostic and early warning tool for the detection and characterization of toxic pollution in surface waters.

    PubMed

    Hug, Christine; Zhang, Xiaowei; Guan, Miao; Krauss, Martin; Bloch, Robert; Schulze, Tobias; Reinecke, Tim; Hollert, Henner; Brack, Werner

    2015-11-01

    Surface water samples constantly receive a vast mixture of micropollutants mainly originating from wastewater treatment plants (WWTPs). High-throughput live cell arrays provide a promising method for the characterization of the effects of chemicals and the associated molecular mechanisms. In the present study, this test system was evaluated for the first time for the characterization of a set of typical surface water extracts receiving effluent from WWTPs. The extracts containing complex mixtures of micropollutants were analyzed for the expression of 90 stress responsive genes in the Escherichia coli reporter gene assay. The most affected pathways and the genes most sensitive to surface water samples suggested prominent stress-responsive pathways for wastewater-impacted surface water, such as oxidative stress, DNA damage, and drug resistance. Samples strongly affecting particular pathways were identified by statistical analysis of gene expression. Transcription data were correlated with contamination data from chemical screening and percentages of wastewater in the samples. Samples with particular effects and outstanding chemical composition were analyzed. For these samples, hypotheses on the alteration of the transcription of genes involved in drug resistance and DNA repair attributable to the presence of pharmaceuticals were drawn. PMID:26033406

  20. Rj4, a Gene Controlling Nodulation Specificity in Soybeans, Encodes a Thaumatin-Like Protein But Not the One Previously Reported.

    PubMed

    Tang, Fang; Yang, Shengming; Liu, Jinge; Zhu, Hongyan

    2016-01-01

    Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity. PMID:26582727

  1. Patched homolog 1 gene mutation (p.G1093R) induces nevoid basal cell carcinoma syndrome and non-syndromic keratocystic odontogenic tumors: A case report

    PubMed Central

    PONTI, GIOVANNI; POLLIO, ANNAMARIA; PASTORINO, LORENZA; PELLACANI, GIOVANNI; MAGNONI, CRISTINA; NASTI, SABINA; FORTUNA, GIULIO; TOMASI, ALDO; SCARR, GIOVANNA BIANCHI; SEIDENARI, STEFANIA

    2012-01-01

    Mutations in the Patched homolog 1 (PTCH1) gene lead to an autosomal dominant disorder known as nevoid basal cell carcinoma syndrome (NBCCS) or Gorlin syndrome (GS). Several PTCH1 mutations have been observed in NBCCS associated with keratocystic odontogenic tumors (KCOTs), including non-syndromic KCOTs. The missense mutation c.3277G>C (p.G1093R) in exon 19 of the PTCH1 gene has only been reported in non-syndromic KCOTs. The present study reports for the first time a familial case (father and daughter) of NBCCS and KCOTs, carrying the same c.3277G>C (p.G1093R) germline mutation. This observation suggests that this missense mutation is involved in the pathogenesis of NBCCS as well as in a subset of non-syndromic KCOTs. The identification of a missense mutation may lead to an earlier diagnosis of NBCCS. PMID:22844361

  2. Search for genes having TNT degrading capability for bioremediation. Final report, 25 October 1993-18 September 1996

    SciTech Connect

    Dutta, S.K.

    1996-11-22

    Several achievements are made through this initial collaborative project These are: (1) creation of enormous capability at this HBcU in enhancing this research; (2) extensive studies on identification of TNT biotransformation products in several bacterial species and one fungal species; (3) identification of several genes which are capable of catalyzing degradation of metabolites of ThT in several microbial species, particularly in the flingus P. chrysosporiwn; (4) isolation and DNA sequence of these genes for probe development; and (5) PCR amplification of 2,4-DNT gene using P. chrysosporiwn genomic DNA as template; (6) DNA:DNA hybridization, (32)P labeled 2,4-DNT dioxygenase gene (used as probe) to anneal with P. chrysosporiwn genomic DNAs; and (7) selection of proper combination of mixed genotypes of bacteria for very efficient degradation of TNT.

  3. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1988--April 14, 1989

    SciTech Connect

    Okita, T.W.

    1989-12-31

    During this period researchers have been successful in determining the structure of the rice pyrophosphorylase gene. Potato tuber ADPglucose pyrophosphorylse purification and structure studies were carried out as well as recombinant DNA studies. Evidence suggests that the tuber form is made up of subunits with similar molecular weights and immunological relatedness. In contrast, the spinach leaf enzyme and presumably the maize endosperm species is composed of two dissimilar sununits encoded by different genes.

  4. (The isolation and characterization of beta-glucosidase gene and beta-glucosidase of Trichoderma viride): Progress report

    SciTech Connect

    Stafford, D.W.

    1983-01-01

    Our project was to isolate and characterize the enzyme ..beta..-glucosidase and to clone and characterize the ..beta..-glucosidase gene; our goal is to clone and characterize each of the cellulase genes from Trichoderma. The induction of the Trichoderma reesei cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. Although the induction of the cellulase complex has previously been well documented, the induction of ..beta..-glucosidase had been questioned. 49 refs., 6 figs., 2 tabs.

  5. Organization and regulation of the genes for nitrogen fixation in Rhodopseudomonas capsulata: Progress report, June 5, 1987-June 4, 1988

    SciTech Connect

    Haselkorn, R.

    1988-02-01

    We have cloned a number of fragments of DNA containing genes necessary for nitrogen fixation from the photosynthetic bacterium Rhodobacter capsulatus. The nif genes are locally clustered but the clusters are on non-neighboring DNA restriction fragments. We sought to determine the physical linkage of these fragments, to determine their relationship with the corresponding nif genes of Klebsiella, and to determine the nucleotide sequence of some of the fragments. So far we have identified six or seven regulatory genes among these, using a nifH::lac fusion. Four of the regulatory genes are required for expression of nifH. Two of these, nifR1 and nifR2, have sequences homologous to ntrC and ntrB of enteric bacteria. A third, nifR4, has sequence homology, in the C-terminal region, to the ntrA genes of Rhizobium and Klebsiella. Constitutive expression of nifR4 in R. capsulatus, from a plasmid clone, complemented a nifR4 chromosomal mutant but not a nifR1 mutant. Moreover, both oxygen and ammonia regulation of nitrogenase were maintained under these conditions. These results are consistent with a model requiring both nifR1 and nifR4 for nitrogenase gene expression; they rule out our earlier suggestion that nifR1 is needed only to turn on nifR4. Current efforts are focused on the purification of RNA polymerase and the products of nifR1, nifR2, and nifR4 to study nif gene transcription in vitro, particularly with the goal of determining the role of DNA supercoiling in transcription.

  6. First report of a clinical, multidrug-resistant Enterobacteriaceae isolate coharboring fosfomycin resistance gene fosA3 and carbapenemase gene blaKPC-2 on the same transposon, Tn1721.

    PubMed

    Li, Gang; Zhang, Ying; Bi, Dexi; Shen, Pinghua; Ai, Fuqi; Liu, Hong; Tian, Yueru; Ma, Yiming; Wang, Bei; Rajakumar, Kumar; Ou, Hong-Yu; Jiang, Xiaofei

    2015-01-01

    In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla(KPC-2) and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla(KPC-2) was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored bla(KPC-2). Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla(KPC-2) colocated in the same Tn1721-Tn3-like composite transposon on a novel IncP group plasmid. PMID:25367902

  7. First Report of a Clinical, Multidrug-Resistant Enterobacteriaceae Isolate Coharboring Fosfomycin Resistance Gene fosA3 and Carbapenemase Gene blaKPC-2 on the Same Transposon, Tn1721

    PubMed Central

    Li, Gang; Zhang, Ying; Bi, Dexi; Shen, Pinghua; Ai, Fuqi; Liu, Hong; Tian, Yueru; Ma, Yiming; Wang, Bei; Rajakumar, Kumar

    2014-01-01

    In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the blaKPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that blaKPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored blaKPC-2. Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and blaKPC-2 colocated in the same Tn1721-Tn3like composite transposon on a novel IncP group plasmid. PMID:25367902

  8. Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

    NASA Technical Reports Server (NTRS)

    Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

  9. SSTR2-based reporters for assessing gene transfer into non-small cell lung cancer: evaluation using an intrathoracic mouse model.

    PubMed

    Singh, S P; Han, L; Murali, R; Solis, L; Roth, J; Ji, L; Wistuba, I; Kundra, V

    2011-01-01

    The most common cause of cancer-related deaths in North America is lung cancer, 85% of which is non-small cell lung cancer (NSCLC). Gene therapy is a promising approach, but has been hindered by lack of methods for localizing and quantifying gene expression in vivo. Human somatostatin receptor subtype-2 (SSTR2)-based reporters can be used to follow gene expression in vivo using ligands with greater affinity for this subtype. NSCLCs can express SSTR subtypes, which may interfere with SSTR2-based reporters. We assessed whether a SSTR2-based reporter can serve as a reporter of gene transfer into NSCLCs. SSTR subtype expression was assessed in NSCLC cell lines A549, H460, and H1299 using RT-PCR. After infection with an adenovirus containing hemagglutinin-A-tagged-SSTR2 (Ad-HA-SSTR2) or control insert, expression was assessed by immunologic techniques and binding to clinically-approved (111)In-octreotide. In vivo, after magnetic resonance (MR) imaging, intrathoracic H460 tumors were injected with Ad-HA-SSTR2 or control virus (n?=?6 mice/group) under ultrasound guidance. Intravenous injection of (111)In-octreotide 2 days later was followed by planar and single-photon emission computed tomography (SPECT) imaging. Biodistribution into tumors was assessed in vivo using anatomic MR and functional gamma-camera images and ex vivo using excised organs/tumors. In human lung tumor samples (n?=?70), SSTR2 expression was assessed using immunohistochemistry. All three NSCLC cell lines expressed different SSTR subtypes, but none expressed SSTR2. Upon Ad-HA-SSTR2 infection, HA-SSTR2 expression was seen in all three cell lines using antibodies targeting the HA domain or (111)In-octreotide targeting the receptor domain (p?reporters can serve as reporters of gene transfer into NSCLCs. PMID:20653396

  10. Schizophrenia and the androgen receptor gene: Report of a sibship showing co-segregation with Reifenstein Syndrome but no evidence for linkage in 23 multiply affected families

    SciTech Connect

    Arranz, M.; Sharma, T.; Sham, P.; Kerwin, R.

    1995-10-09

    Crow et al. have reported excess sharing of alleles by male sibling pairs with schizophrenia, at a triplet repeat marker within the androgen receptor gene, indicating that mutations at or near this gene may be a risk factor for males. In this report, we describe a pair of male siblings concordant for both schizophrenia and Reifenstein syndrome, which is caused by a mutation in this gene. This provides support for the hypothesis that the androgen receptor may contribute to liability to develop schizophrenia. Because of this, we have examined a collection of 23 pedigrees multiply affected by schizophrenia for linkage to the androgen receptor. We have found no evidence for linkage by both the LOD score and affected sibling-pair methods, under a range of genetic models with a broad and narrow definition of phenotype, and when families with male-to-male transmission are excluded. However, because of the small number of informative male-male pairs in our sample, we cannot confirm or refute the excess allele sharing for males reported by Crow. 35 refs., 1 fig., 2 tabs.

  11. A New Fluorescence-Based Reporter Gene Vector as a Tool for Analyzing and Fishing Cells with Activated Wnt Signaling Pathway

    PubMed Central

    Reischmann, Patricia; Mller, Oliver

    2013-01-01

    The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or ?-galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way. PMID:24066239

  12. A new fluorescence-based reporter gene vector as a tool for analyzing and fishing cells with activated wnt signaling pathway.

    PubMed

    Apfel, Johanna; Reischmann, Patricia; Mller, Oliver

    2013-01-01

    The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or ? -galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way. PMID:24066239

  13. Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

    PubMed

    Zhang, Huimin; Zheng, Beiwen; Gao, Rongsui; Feng, Youjun

    2015-09-01

    The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other ?-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by ?-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus. PMID:26050090

  14. The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis.

    PubMed Central

    Hoffmaster, A R; Koehler, T M

    1997-01-01

    The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atx4-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes. First, total protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electrophoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing CO2-enhanced atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin coregulated) were Tox+, indicating that these genes may not be involved in anthrax toxin gene activation. Our data indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins. PMID:9234759

  15. Associations of ADH and ALDH2 gene variation with self report alcohol reactions, consumption and dependence: an integrated analysis

    PubMed Central

    Macgregor, Stuart; Lind, Penelope A.; Bucholz, Kathleen K.; Hansell, Narelle K.; Madden, Pamela A.F.; Richter, Melinda M.; Montgomery, Grant W.; Martin, Nicholas G.; Heath, Andrew C.; Whitfield, John B.

    2009-01-01

    Alcohol dependence (AD) is a complex disorder with environmental and genetic origins. The role of two genetic variants in ALDH2 and ADH1B in AD risk has been extensively investigated. This study tested for associations between nine polymorphisms in ALDH2 and 41 in the seven ADH genes, and alcohol-related flushing, alcohol use and dependence symptom scores in 4597 Australian twins. The vast majority (4296) had consumed alcohol in the previous year, with 547 meeting DSM-IIIR criteria for AD. There were study-wide significant associations (P < 2.3 10?4) between ADH1B-Arg48His (rs1229984) and flushing and consumption, but only nominally significant associations (P < 0.01) with dependence. Individuals carrying the rs1229984 G-allele (48Arg) reported a lower prevalence of flushing after alcohol (P = 8.2 10?7), consumed alcohol on more occasions (P = 2.7 10?6), had a higher maximum number of alcoholic drinks in a single day (P = 2.7 10?6) and a higher overall alcohol consumption (P = 8.9 10?8) in the previous year than those with the less common A-allele (48His). After controlling for rs1229984, an independent association was observed between rs1042026 (ADH1B) and alcohol intake (P = 4.7 10?5) and suggestive associations (P < 0.001) between alcohol consumption phenotypes and rs1693482 (ADH1C), rs1230165 (ADH5) and rs3762894 (ADH4). ALDH2 variation was not associated with flushing or alcohol consumption, but was weakly associated with AD measures. These results bridge the gap between DNA sequence variation and alcohol-related behavior, confirming that the ADH1B-Arg48His polymorphism affects both alcohol-related flushing in Europeans and alcohol intake. The absence of study-wide significant effects on AD results from the low P-value required when testing multiple single nucleotide polymorphisms and phenotypes. PMID:18996923

  16. Availability of iron to Pseudomonas fluorescens in rhizosphere and bulk soil evaluated with an ice nucleation reporter gene.

    PubMed Central

    Loper, J E; Henkels, M D

    1997-01-01

    The biological availability of iron in the rhizosphere was assessed by evaluating ice nucleation activity (INA) expressed in situ by Pseudomonas fluorescens Pf-5 containing a transcriptional fusion (pvd-inaZ) of an iron-regulated promoter to an ice nucleation reporter gene (inaZ). Pf-5 containing pvd-inaZ expresses INA that is inversely related to the iron availability of a growth medium (J. E. Loper and S. E. Lindow, Appl. Environ. Microbiol. 60:1934-1941, 1994). INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ was at a maximum within 12 to 24 h following inoculation of the bacterium onto bean roots and typically decreased gradually during the following 4 days. Iron availability in the soil, which was altered by the addition of chelators, influenced INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ. In soil adjusted to a pH of 7.0 or 8.0 by adding Ca(OH)2, rhizosphere populations of Pf-5 containing pvd-inaZ expressed greater INA, indicating lower iron availability, than they did in the nonamended soil at a pH of 5.4. Similarly, rhizosphere populations of Pf-5 containing pvd-inaZ expressed less INA in an agricultural soil of pH 5.4 than in other agricultural soils ranging in pH from 6.4 to 7.7. These results conform to the predictions of chemical models stating that pH is a major factor influencing iron availability in soil solutions. The results of this study indicate that P. fluorescens Pf-5 encountered an iron-limited environment immediately after it was inoculated onto bean roots planted in agricultural field soils. One to two days after the bacterium was inoculated onto root surfaces, however, iron became more available to rhizosphere populations of Pf-5. We speculate that iron acquisition systems of plants and other rhizosphere organisms may provide available sources of iron to established rhizosphere populations of P. fluorescens. PMID:8979343

  17. Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in Mycoplasma gallisepticum

    PubMed Central

    Indikova, Ivana; Szostak, Michael P.

    2013-01-01

    Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M.gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M.gallisepticum origin or replication (oriCMG). We failed to create a disruption vector, although by adding a further short fragment of the M.gallisepticum tufB upstream region we created a Trojan horse plasmid. This is fully integrated into the genomic DNA of M.gallisepticum, always at the same site, oriCMG, and is able to carry and express any gene of interest in the genetic background of M.gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M.gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes. PMID:24278444

  18. Differential gene expression in neurospora crassa cell types: amplification of rRNA genes. Progress report, July 1979-30 June 1980

    SciTech Connect

    Dutta, S.K.

    1980-01-01

    The significant results obtained during 1979 to 1980 of the current research program are as follows: (1) the differential rRNA gene amplification in germinated conidia of N.crassa was confirmed. N.crassa rDNAs showed differences in degrees of homology with isolated DNAs from other Neurospora species which could be due to heterogeneity in internal spacers. Studies with N.crassa rDNA clones were initiated to study their heterogeneities. The organization of the Institutional Biohazard Committee (IBC) for Recombinant DNA research was completed and necessary certifications for the laboratory and the workers were obtained in accordance with the P/sub 2/EK/sub 1/ containment regulation of N.I.H. Known 17S and 26S N.crassa rDNA probes are being used to detect differences, if any, in restriction cleavage sites in rDNAs of different cell types and developmental mutants of N.crassa. DNAs from these N.crassa cells are restricted with EcoR/sub 1/ and Hind III and cleaved fragments separated by gel electrophoresis are transferred into nitrocellulose papers. Experiments are underway now to see if there are any changes in cleavage sites by annealing with /sup 32/P or /sup 3/H-17S or 26S rDNA probes followed by autoradiography.

  19. DNA photodamage, repair, gene induction and genotoxicity following exposures to 254 nm UV and 8-methoxypsoralen plus UVA in a eukaryotic cell system.

    PubMed

    Averbeck, D; Averbeck, S

    1998-09-01

    The induction and repair of different types of photodamage and photogenotoxicity in eukaryotic cells have been the subject of many studies. Little is known about possible links between these phenomena and the induction of DNA damage-inducible genes. We explored this relationship using the yeast Saccharomyces cerevisiae, a pertinent eukaryotic model. Previous results showed that the photogenotoxic potential of 8-methoxypsoralen (8-MOP) plus UVA is higher than that of UV (254 nm). Moreover, the induction of the ribonucleotide reductase gene RNR2 by UV and 8-MOP plus UVA in an RNR2-LACZ fusion strain and the formation of DNA double-strand breaks (dsb) as repair intermediates after such treatments suggest that the latter process could involve a signal for gene induction. To further substantiate this, we measured the induction of the DNA repair gene RAD51 in RAD51-LACZ fusion strains using the dsb repair and recombination deficient mutant rad52 and the corresponding wild type, and we determined the formation of dsb by pulsed-field gel electrophoresis. After treatments, the resealing of dsb formed as repair intermediates was impaired in the rad52 mutant. At equal doses, i.e. the same number of lesions, the induction of the RAD51 gene by UV or 8-MOP plus UVA was significantly reduced in the rad52 mutant as compared with the wild type. The same was true when equitoxic doses were used. Thus, the RAD52 repair pathway appears to play an important role not only in dsb repair but also in gene induction. Furthermore, the signaling pathways initiated by DNA damage and its processing are somewhat linked to the photogenotoxic response. PMID:9747584

  20. (Nuclear genes from nicotiana encoding the small subunit of ribulose-1,5-bisphosphate carboxylase). Progress report

    SciTech Connect

    Cashmore, A.R.

    1985-01-01

    Two pea nuclear genes encoding ribulose-1,5-bisphosphate carboxylase (rbcS) were isolated and completely sequenced. These sequence studies include approximately 1 kb of 5' noncoding region and several hundred nucleotides of 3' noncoding sequences. The two genes are tightly linked being separated by 10 kb of DNA and they are oriented with their 3' ends towards one another. The two genes (ss3.6 and ss8.0) correspond to two of five EcoRI fragments of pea DNA that hybridize to a rbcS hybridization probe. The two genes ss3.6 and ss8.0 are quite divergent at their 5' and their 3' ends and in the first of the two intervening sequences. In direct contrast the second of the two intervening sequences is total conserved between the two genes. This conservation of sequence identity could result directly from evolutionary forces selecting against any sequence change. Such selection would presumably reflect a very sequence-dependent function for these introns. A role in splicing is one possibility and a transcriptional regulatory element is another possibility. 9 refs.

  1. Prolonged pancytopenia in a gene therapy patient with ADA-deficient SCID and trisomy 8 mosaicism: a case report.

    PubMed

    Engel, Barbara C; Podsakoff, Greg M; Ireland, Joanna L; Smogorzewska, E Monika; Carbonaro, Denise A; Wilson, Kathy; Shah, Ami; Kapoor, Neena; Sweeney, Mirna; Borchert, Mark; Crooks, Gay M; Weinberg, Kenneth I; Parkman, Robertson; Rosenblatt, Howard M; Wu, Shi-Qi; Hershfield, Michael S; Candotti, Fabio; Kohn, Donald B

    2007-01-15

    A patient with adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells. PMID:16973956

  2. Non-invasive imaging of firefly luciferase reporter gene expression using bioluminescence imaging in human prostate cancer models.

    PubMed

    Li, Hongwei; Li, Jin Zhong; Helm, Gregory A; Pan, Dongfeng

    2007-04-01

    Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assess the effectiveness and safety of systems of gene therapy delivery. In the present study, we employed a CCD (charge-coupled-device) imaging system to monitor how a prostate-specific adenovirus vector (AdPSA-Luc) mediated the long-term, sustained expression of firefly luciferase (Luc) in living human prostate cancer mouse models. The in vivo bioluminescence imaging revealed significantly high levels of luciferase expression up to 1 month, not only in prostate tumours, but also in lungs after intratumoural injection. Systemic tail vein injection of AdPSA-Luc revealed significant luciferase expression in lungs of both human prostate cancer mouse models and nave mice, but significantly higher in the former, while the control virus, AdCMV-Luc, containing CMV (cytomegalovirus) promoter and luciferase gene, just restricted expression in the livers. Our findings demonstrate the ability of the cooled CCD camera to sensitively and non-invasively track the location, magnitude and persistence of luciferase gene expression in human prostate cancer mouse models. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique. PMID:17073822

  3. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1987--April 14, 1988

    SciTech Connect

    Okita, T.W.

    1988-12-31

    Many agronomically important crops are viewed as significant resources of renewable energy. Overall crop productivity could be increased if the efficiency of photoassimilate conversion into dry matter such as starch were improved in storage tissues. Starch production is controlled by the catalytic activity of ADPglucose pyrophosphorylase in the first step of starch biosynthesis. This research focuses on the genetic structure and molecular mechanisms by which it is controlled during plant development and how it is affected by environmental and hormonal conditions. The current goal is to isolate the genes for this enzyme present in both cereal endosperm and potato tuber tissues, and to elucidate its structure and the controlling sequences responsible for gene expression. The long term goal is the improvement of starch production in storage organs by manipulating this gene so that it encodes an enzyme refractive to inorganic phosphate inhibition.

  4. Association of collagen type I alpha1 gene polymorphism with bone density in survivors of childhood cancer--preliminary report.

    PubMed

    Muszy?ska-Ros?an, K; Galicka, A; Sawicka, M; Krawczuk-Rybak, M; Wo?czy?ski, S

    2004-01-01

    A candidate gene, involved in the regulation of bone mass is the COLIA1 gene encoding type I collagen, the major protein of bone matrix. The disease per se, the age of its onset and treatment options might exert an impact on bone mineralization in survivors of childhood malignancy. We examined possible allelic influences of COLIA1 gene polymorphism on BMI, BMD spine and total body in 41 survivors (15 girls) of childhood cancer (the mean age 8.9 years). Genotype distribution was 33 (80.5%) SS and 8 (19.5%) Ss. There were no differences in SDS BMD and SDS BMI between patients with SS and Ss genotype. A tendency towards lower SDS values of BMD spine and BMI was observed (not significant). In conclusion, our preliminary observations suggest that COLIA1 genotype may affect bone accrual in a population treated for childhood cancer. Further investigations in a greater population are needed. PMID:15638371

  5. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase genes. Progress report, [April 15, 1990--April 14, 1991

    SciTech Connect

    Okita, T.W.

    1990-12-31

    The long term goal of this project is to assess the feasibility of increasing the conversion of photosynthate a key regulatory enzyme in starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is primarily regulated by the gene activation, expression, and allosteric regulation of ADPglucose pyrophosphorylase, as well as starch synthase, and branching enzyme. During the last year we have elucidated the structure of both subunits which compose this tetrameric enzyme and determined the temporal and spatial expression of the genes encoding each subunit as well as their correlation to starch biosynthesis. Genomic clones to both subunits have also been isolated and the gene structure of the small subunit determined. Transgenic potato plants have been produced containing deletions of the small subunit promoter. Currently, cis acting elements and their involvement in spatial and temporal expression are under investigation.

  6. Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1992--April 15, 1993

    SciTech Connect

    Barkan, A.

    1993-04-20

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that control the timing and cell-type specificity of chloroplast gene expression. Studies are being conducted with nuclear mutants of maize that are defective in the biogenesis or translation of chloroplast mRNAs. Currently studies are focused on two nuclear mutants with specific and unique lesions in chloroplast RNA processing (crp mutants). Crp1 mutants (formerly called hcf136) fail to accumulate the cytochrome f/b6 complex. The protein loss is due to a defect in the metabolism of transcripts encoding the petB and petD gene products, two subunits of the missing complex. Mutant seedlings lack the monocistronic petB and petD MRNAS, which both arise in nominal plants by endonucleolytic cleavage of the polycistronic primary transcript of the psbB gene cluster. Precursor mRNAs accumulate normally in crp1, indicating that its defect is due either to a failure to cleave the precursors, or a failure to stabilize the fully processed mRNAs. We are interested in both the biochemistry of this site-specific RNA processing and in the role of the processing in generating translatable mRNAs. To address the latter, we are quantifying the rates of synthesis of the petB and petD gene products with the goal of determining whether the missing transcripts are more efficiently translated than their precursors. To address the biochemistry of the defect in RNA metabolism, the crp1 gene is being cloned via the transposon tag. crp2 (formerly called hcf142) lacks the predominant mRNA encoding petA, but appears to be otherwise unimpaired in chloroplast RNA metabolism. The precise role of crp2 in synthesizing or stabilizing the petA mRNA is being investigated through biochemical studies.

  7. Differential gene expression in Neurospora crassa cell types: heterogeneity and amplification of rRNA genes. Progress report, July 1980-June 30, 1981

    SciTech Connect

    Dutta, S.K.

    1981-01-01

    The significant results obtained during 1980-1981 year of the current research program are as follows: I. Studies on heterogeneity of multiple copies of rDNAs from N. crassa cell types are being continued, such as: (1) Autoradiographs of Southern transfers of EcoR/sub 1/ restricted fragments of nuclear DNA from conidia, germinated conidia (sprouts) and mycelia of N. crassa were compared after hybridization with /sup 32/P-rDNA probe. The nuclear DNA of two hours sprout and of 16 hours mycelia gave similar hybridization patterns with EcoR/sub 1/ digest, but no such hybridization pattern was evident in conidial DNA digest; (2) Procedure for concentration of rDNAs from Neurospora species and cell types was standardized; restriction analysis of purified rDNAs is being done; (3) 35S total rDNA clone, 17S rDNA clone and 26S rDNA subclone are being used to see gross differences in the precursor rRNAs of different cell types; (4) Comparison of DNA:DNA homologies of rRNA genes with different Neurospora species. II. Post-mitochondrial DNAs of N. crassa are found to be rDNA-like and were further characterized by electron microscopic studies and are found to be approximately twice the size of SV-40 DNAs. These N. crassa post-mitochondrial DNAs hybridized with /sup 32/P-labeled N. crassa nuclear DNAs. III. Previous studies on differential RNase sensitive DNA polymerase activity in N. Crassa cell types and on evolution of sexual morphogenesis in the genus Neurospora are completed and published. RNase sensitive DNA polymerase activity is found to be in the post-mitochondrial fraction. Heterothallism in the genus Neurospora is evolved from homothallism.

  8. Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations.

    PubMed

    Hodgkins, M; Mead, D; Ballance, D J; Goodey, A; Sudbery, P

    1993-06-01

    The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF-alpha leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non-selective growth. The stabilized strain was grown to high cell density by fed-batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0.5 g/1 or 0.65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0.43 g/1 or 0.56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g/l) and produced 445 IU/ml(2.25 g/l or 2.2% dry weight) extracellularly and 76 IU/ml (0.38 g/l or 0.4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain. PMID:8346679

  9. Cell division gene cluster in Spiroplasma kunkelii: functional characterization of ftsZ and the first report of ftsA in mollicutes.

    PubMed

    Zhao, Yan; Hammond, Rosemarie W; Lee, Ing-Ming; Roe, Bruce A; Lin, Shaoping; Davis, Robert E

    2004-02-01

    Spiroplasma kunkelii is a helical, wall-less bacterium that causes corn stunt disease. In adaptation to its phloem-inhabiting parasitic lifestyle, the bacterium has undergone a reductive evolutionary process and, as a result, possesses a compact genome with a gene set approaching the minimal complement necessary for multiplication and pathogenesis. We cloned a much-reduced cell division gene cluster from S. kunkelii and functionally characterized the key division gene, ftsZ(sk). The 1236-bp open reading frame of ftsZ(sk) is capable of encoding a protein with a calculated molecular mass of 44.1 kDa. Protein sequence alignment revealed that FtsZ(sk) is remarkably similar to FtsZ proteins from other eubacteria, and possesses the conserved GTP-binding and hydrolyzing motifs. We demonstrated that overexpression of ftsZ(sk) in Escherichia coli causes transgression of the host cell division, resulting in a filamentous phenotype. We also report, for the first time, the presence of a ftsA gene in the cell division cluster of a mollicute species. PMID:15000753

  10. Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.

    PubMed

    Grassi, G; Maccaroni, P; Meyer, R; Kaiser, H; D'Ambrosio, E; Pascale, E; Grassi, M; Kuhn, A; Di Nardo, P; Kandolf, R; Kpper, J-H

    2003-10-01

    The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes. As a better understanding of these biological processes may contribute to the development of novel cancer treatments and to study the complex mechanisms regulating gene silencing, we established a cellular system suitable to dissect the mechanisms regulating DNA methylation and histone acetylation. For this purpose, we stably transfected the neuroblastoma cell line U87 with a cytomegalovirus promoter-driven reporter gene construct whose expression was analyzed following treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine or histone deacetylation inhibitor trichostatin A. Both substances reactivated the silenced cytomegalovirus promoter, but with different reaction kinetics. Furthermore, whereas the kinetics of reactivation by trichostatin A did not substantially change over the time range considered (5 days), reactivation induced by 5'-aza-2'-deoxycytidine showed profound differences between day 1 and longer time points. We showed that this effect is related to the down-regulation of DNA replication by 5'-aza-2'-deoxycytidine. Finally, we have shown that the simultaneous administration of trichostatin A and 5'-aza-2'-deoxycytidine results in reactivation of the CMV promoter according to a cooperative, not synergistic or additive, mechanism. In conclusion, our cellular system should represent a powerful tool to investigate the complex mechanisms regulating gene silencing and to identify new anticancer drugs. PMID:12869421

  11. Creation of immune 'stealth' genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1.

    PubMed

    Ossevoort, M; Visser, B M J; van den Wollenberg, D J M; van der Voort, E I H; Offringa, R; Melief, C J M; Toes, R E M; Hoeben, R C

    2003-11-01

    A major obstacle in gene-therapy protocols is T-cell-mediated destruction of transgene-expressing cells. Therefore new approaches are needed to prevent rapid clearance of transduced cells. We exploited the Gly-Ala repeat (GAr) domain of the Epstein-Barr virus nuclear antigen-1, since the GAr prevents cytotoxic T-lymphocyte-epitope generation. Here we show that three different enzymes (viz. the E. coli LacZ gene encoded beta-galactosidase, firefly luciferase, and HSV1 thymidine kinase) fused with the GAr retained their function. Moreover, linking GAr with beta-galactosidase successfully prevented recognition of GAr-LacZ-expressing cells by beta-galactosidase-specific CTL. Nonetheless, vaccination with a GAr-LacZ adenovirus or with an allogeneic cell line expressing GAr-LacZ resulted in the induction of beta-gal-specific CTL. This demonstrates that the GAr domain does not inhibit cross presentation of antigens, but only affects breakdown of endogenously synthesized proteins. These data demonstrate how the GAr domain can be exploited to create immuno'stealth' genes by hiding transgene products from CTL-mediated immune attack. PMID:14566361

  12. Oestrogenic activity of a textile industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN gene-reporter luciferase assay.

    PubMed

    Schilir, Tiziana; Porfido, Arianna; Spina, Federica; Varese, Giovanna Cristina; Gilli, Giorgio

    2012-08-15

    This study quantified the biological oestrogenic activity in the effluent of a textile industrial wastewater treatment plant (IWWTP) in northwestern Italy. Samples of the IWWTP effluent were collected monthly, both before and after tertiary treatment (ozonation). After solid phase extraction, all samples were subjected to two in vitro tests of total estrogenic activity, the human breast cancer cell line (MCF-7 BUS) proliferation assay, or E-screen test, and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay, to measure the 17?-oestradiol equivalent quantity (EEQ). In the E-screen test, the mean EEQ values were 2.351.68 ng/L pre-ozonation and 0.720.58 ng/L post-ozonation; in the MELN gene-reporter luciferase assay, the mean EEQ values were 4.183.54 ng/L pre-ozonation and 2.532.48 ng/L post-ozonation. These results suggest that the post-ozonation IWWTP effluent had a lower oestrogenic activity (simple paired t-tests, p<0.05). The average reduction of estrogenic activity of IWWTP effluent after ozonation was 6726% and 5227% as measured by E-screen test and MELN gene-reporter luciferase assay, respectively. There was a positive and significant correlation between the two tests (Rho S=0.650, p=0.022). This study indicates that the environmental risk is low because oestrogenic substances are deposited into the river via IWWTP at concentrations lower than those at which chronic exposure has been reported to affect the endocrine system of living organisms. PMID:22750186

  13. Brief Report: The Dopamine-3-Receptor Gene ("DRD3") Is Associated with Specific Repetitive Behavior in Autism Spectrum Disorder (ASD)

    ERIC Educational Resources Information Center

    Staal, Wouter G.; de Krom, Mariken; de Jonge, Maretha V.

    2012-01-01

    Recently the "DRD3" gene has been associated with ASD in two independent samples. Follow up analysis of the risk allele of the SNP rs167771 in 91 subjects revealed a significant association with a specific type of repetitive behavior: the factor "insistence on sameness" (IS) derived from the Autism Diagnostic Interview. This risk allele was

  14. (Analysis of cyanobacterial photosystem 2 genes by cloning and mutagenesis): Progress report, May 1986 to December 1986

    SciTech Connect

    Not Available

    1986-01-01

    We have isolated proteins that appear to be regulated by changes in environmental conditions, either iron or light. Iron-regulated proteins include: a 36 kDa protein that is involved in iron-acquisition, and a 34 kDa intrinsic thylakoid chlorophyll-binding protein associated with PS II. Utilizing a lambda gtll library we have cloned and sequenced the 36 kDa protein and are performing site-directed mutagenesis to determine the function of the protein. Insertion of a transposon into this gene and transformation of the mutated gene into a wild type host resulted in a strain which is unable to grow under low-iron conditions. We have obtained a 42 kDa carotenoid-binding protein that is induced upon growth in high light conditions, and cloned the gene for this protein. We have also isolated a carotenoid-binding protein that is localized exclusively in the plasmalemma. We have also cloned the 33 kDa extrinsic maganese-binding protein that is involved in oxygen-evolution. The gene has been cloned and sequenced and contains a 28 amino acid signal sequence. 4 figs.

  15. CELL DIVISION GENE CLUSTER IN SPIROPLASMA KUNKELII: FUNCTIONAL CHARACTERIZATION OF FTSZ AND THE FIRST REPORT OF FTSA IN MOLLICUTES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ftsZ gene, found in representatives of all bacterial groups, encodes an essential protein engaged in prokaryotic cell division. We cloned and characterized the ftsZ homologue (ftsZsk) from a pathogenic strain of the wall-less bacterium Spiroplasma kunkelii that causes corn stunt disease. The 1...

  16. Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer-plasmid complexes.

    PubMed

    Turunen, M P; Hiltunen, M O; Ruponen, M; Virkamki, L; Szoka, F C; Urtti, A; Yl-Herttuala, S

    1999-01-01

    Different lipids and cationic polymers were tested in vitro for their ability to transfect rabbit aortic smooth muscle cells and human endothelial cells with lacZ marker gene. Toxicity of the complexes was evaluated with MTT assay. Selected plasmid-polymer complexes with different charge ratios were then tested for in vivo gene transfer efficiency using adventitial gene transfer by placing a silastic gene delivery reservoir (collar) around the carotid artery. Transfection efficiency was determined by X-gal staining 3 days after the gene transfer. Based on in vitro experiments, fractured polyamidoamine dendrimers and polyethylenimines (PEI) were selected for in vivo experiments. Fractured dendrimers (generation 6, +/- charge ratio of 3) had the highest in vivo gene transfer efficiency (4.4% +/- 1.7). PEI with molecular size of 25 kDa (+/- charge ratio 4) was also effective (2.8% +/- 1.8) in this model. PEI of 800 kDa showed a constant but modest gene transfer efficiency (1.8% +/- 0.1) with all charge ratios. A low level gene transfer was also detected with naked DNA (0.5% +/- 0.3). No signs of inflammation were seen in any of the study groups. We show here that in vitro cell culture experiments can be used to identify efficient in vivo gene transfer methods for arterial gene therapy, but the charge ratios for each complex must be optimized in vivo. It is concluded that fractured dendrimer and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery route. PMID:10341870

  17. Clericuzio-type Poikiloderma with Neutropenia Syndrome in a Turkish Family: a Three Report of Siblings with Mutation in the C16orf57 gene.

    PubMed

    Patiroglu, Turkan; Akar, H Haluk

    2015-06-01

    Clericuzio-type poikiloderma with neutropenia (PN) is characterized by poikiloderma, non-cyclic neutropenia, recurrent sinopulmonary infections, pachyonychia, and palmo- plantar hyperkeratosis. Mutations in the C16orf57 gene, which is located on chromosome 16q13, have been identified as the cause of PN. PN was first described by Clericuzio in Navajo Indians. Herein, we reported the clinical presentations and laboratory investigations of PN in three siblings from Turkey.The older siblings presented with typical cutaneous poikiloderma, plantar keratoderma, pachyonychia of toenails, and recurrent upper respiratory infections. As the most affected patient, in addition to classic manifestations, the youngest sibling had recurrent pneumonia, hepatosplenomegaly, dental caries, failure to thrive, and hand malformation.Genetic study revealed a homozygous mutation (c.531delA) in the C16orf57 gene in siblings.With the presented study, we aimed to draw attention to PN which can be a predisposing factor to malignancies. PMID:26546903

  18. A Novel de novo Mutation in the G6PD Gene in a Korean Boy with Glucose-6-phosphate Dehydrogenase Deficiency: Case Report.

    PubMed

    Jang, Mi-Ae; Kim, Ji-Yoon; Lee, Ki-O; Kim, Sun-Hee; Koo, Hong Hoe; Kim, Hee-Jin

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by a mutation in the G6PD gene on Xq28. Herein, we describe a Korean boy with G6PD deficiency resulting from a novel mutation in G6PD. A 20-month-old boy with hemolytic anemia was referred for molecular diagnosis. He had no relevant family history. The G6PD activity was severely decreased at 0.2 U/g Hb (severe deficiency). Direct sequencing analyses on the G6PD gene revealed that he was hemizygous for a novel missense variant, c.1187C>G (p.Pro396Arg), in exon 10 of G6PD. Family study involving his parents revealed the de novo occurrence of the mutation. This is the first report of genetically confirmed G6PD deficiency in Korea. PMID:26275698

  19. Pediatric case report: clinical profile of a patient with PCWH with p.Q377X nonsense mutation in the SOX10 gene.

    PubMed

    Oshimo, Tomoko; Fukai, Kazuyoshi; Abe, Yuko; Hozumi, Yutaka; Yokoi, Toshiaki; Tanaka, Akemi; Yamanishi, Kiyofumi; Ishii, Masamitsu; Suzuki, Tamio

    2012-12-01

    We report the case of a Japanese patient with PCWH, a neurological variant of Waardenburg type 4. Direct sequencing of the genomic DNA obtained from peripheral leukocytes revealed the p.Q377X nonsense mutation in the SOX10 gene. The patient had mottled hypopigmented macules on the trunk since birth; such macules have not been described previously. The so-called "white forelock", a triangular or diamond shaped leukoderma on the forehead, was absent. We also reviewed and summarized the outcomes of 23 patients with Waardenburg syndrome type 4, PCWH and Yemenite deaf-blind hypopigmentation syndrome, in which SOX10 mutations were identified. Among them, 17 cases were reported to have hypopigmented skin macule(s). The five patients who had the white forelock had PCWH with severe neurological complications. Paradoxically, two cases had hyperpigmented spots. Heterochromia of the iris was reported in four patients. PMID:22963253

  20. A sugar beet chlorophyll a/b binding protein promoter void of G-box like elements confers strong and leaf specific reporter gene expression in transgenic sugar beet

    PubMed Central

    Stahl, Dietmar J; Kloos, Dorothee U; Hehl, Reinhard

    2004-01-01

    Background Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation. Results Suppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet. Conclusions This work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed. PMID:15579211

  1. Physical and functional characterization of the Bacillus subtilis spoIIM gene.

    PubMed Central

    Smith, K; Bayer, M E; Youngman, P

    1993-01-01

    The spoIIM locus of Bacillus subtilis is the most recently discovered of six genetic loci in which mutations can prevent the synthesis of a normal asymmetric septum or prevent migration of the septal structure to engulf the forespore compartment of the sporangium. Ultrastructure studies of a spoIIM mutant confirmed a block prior to the completion of engulfment. Introduction of a spoIIM mutation into a panel of strains containing lacZ fusions belonging to different regulatory classes allowed us to determine that the spoIIM gene product is required for the efficient expression of genes transcribed by sigma G-associated RNA polymerase but is not required for the expression of sigma F-controlled genes, including spoIIIG, which encodes sigma G. The results of complementation studies, gene disruption analysis, and DNA sequencing revealed that the spoIIM locus contains a single sporulation-essential gene encoding a polypeptide with a predicted molecular mass of 24,850 Da. The predicted spoIIM gene product is highly hydrophobic and very basic, and it does not exhibit significant homology to sequence files in several major data bases. Images PMID:8501064

  2. Local Gene Transfer of OPG Prevents Joint Damage and Disease Progression in Collagen-Induced Arthritis

    PubMed Central

    Zhang, Qingguo; Gong, Weiming; Ning, Bin; Nie, Lin; Wooley, Paul H.

    2013-01-01

    This study examined the influence of osteoprotegerin (OPG) gene transfer on a murine collagen-induced arthritis model. A single periarticular injection of AAV-OPG or AAV-LacZ on the arthritic paw successfully incorporated the exogenous gene to the local tissue and resulted in marked transgene expression in the joint homogenate for at least three weeks. Clinical disease scores were significantly improved in OPG treated mice starting at 28-day post-treatment (P < 0.05). Histological assessment demonstrated that OPG gene transfer dramatically protected mice from erosive joint changes compared with LacZ controls (P < 0.05), although treatment appeared less effective on the local inflammatory progress. MicroCT data suggested significant protection against subchondral bone mineral density changes in OPG treated CIA mice. Interestingly, mRNA expressions of IFN-g and MMP3 were noticeably diminished following OPG gene transfer. Overall, gene transfer of OPG effectively inhibited the arthritis-associated periarticular bone erosion and preserved the architecture of arthritic joints, and the study provides evidence that the cartilage protection of the OPG gene therapy may be associated with the down-regulation of MMP3 expression. PMID:24222748

  3. Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectors.

    PubMed

    Hibino, H; Tani, K; Sugiyama, H; Suzuki, S; Wu, M S; Izawa, K; Hase, H; Nakazaki, Y; Tanabe, T; Ooi, J; Izeki, T; Tojo, A; Saitoh, I; Tanioka, Y; Asano, S

    2001-04-01

    To establish a new non-human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti-human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid-specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c-kit mAbs could be used to purify the marmoset haematopoietic progenitor cells. Furthermore, we studied the in vitro transduction of the bacterial beta-galactosidase (LacZ) gene into CFU-GM derived from marmoset BM using retroviral and adenoviral vectors. The transduction efficiency was increased by using a mixed culture system consisting of marmoset BM stromal cells and retroviral producer cells. It was also possible to transduce LacZ gene into marmoset haematopoietic progenitor cells with adenoviral vectors as well as retroviral vectors. The percentage of adenovirally transduced LacZ-positive clusters was 15% at day 4 (multiplicity of infection=200), but only 1-2% at day 14. The differential use of viral vector systems is to be recommended in targeting different diseases. Our results suggested that marmoset BM progenitor cells were available to examine the transduction efficiency of various viral vectors in vitro. PMID:11380607

  4. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis.

    PubMed Central

    Marahiel, M A; Zuber, P; Czekay, G; Losick, R

    1987-01-01

    Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis. We identified the promoter for the B. brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B. brevis and Bacillus subtilis. Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B. brevis cells growing in sporulation medium. To study the regulation of tycA in B. subtilis, we constructed a derivative of the B. subtilis bacteriophage SP beta containing a transcriptional fusion of the tycA promoter to the lacZ gene of Escherichia coli and introduced the tycA-lacZ operon fusion by means of specialized transduction into sporulation mutants known to be blocked in sporulation-associated antibiotic production. Our principal finding was that tycA-directed lacZ expression was impaired in the stage-0 mutants with mutations spo0A, spo0B, and spo0E but not in spo0C, spo0F, spo0H, or spo+ bacteria. The dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation, which caused tycA-lacZ to be transcribed constitutively at all stages of growth. A simple model is proposed for the mechanism of tycA induction based on the Spo0A-dependent inactivation of Ab-B protein, which is proposed to be a negative regulator of tycA transcription. Images PMID:3032912

  5. Identification and sequence analysis of genes involved in late steps in cobalamin (vitamin B12) synthesis in Rhodobacter capsulatus.

    PubMed Central

    Pollich, M; Klug, G

    1995-01-01

    A 6.4-kb region of a 6.8-kb BamHI fragment carrying Rhodobacter capsulatus genes involved in late steps of cobalamin synthesis has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contains eight genes arranged in at least three operons. Five of these eight genes show homology to genes involved in the cobalamin synthesis of Pseudomonas denitrificans and Salmonella typhimurium. The arrangement of these homologous genes differs considerably in the three genera. Upstream of five overlapping genes (named bluFEDCB), a promoter activity could be detected by using lacZ fusions. This promoter shows no regulation by oxygen, vitamin B12 (cobalamin), or cobinamide. Disruption of the bluE gene by a Tn5 insertion (strain AH2) results in reduced expression of the puf and puc operons, which encode pigment-binding proteins of the photosynthetic apparatus. The mutant strain AH2 can be corrected to a wild-type-like phenotype by addition of vitamin B12 or cobinamide dicyanide. Disruption of the bluB gene by an interposon (strain BB1) also disturbs the formation of the photosynthetic apparatus. The mutation of strain BB1 can be corrected by vitamin B12 but not by cobinamide. We propose that a lack of cobalamin results in deregulation and a decreased formation of the photosynthetic apparatus. PMID:7635831

  6. Identification and sequence analysis of genes involved in late steps in cobalamin (vitamin B12) synthesis in Rhodobacter capsulatus.

    PubMed

    Pollich, M; Klug, G

    1995-08-01

    A 6.4-kb region of a 6.8-kb BamHI fragment carrying Rhodobacter capsulatus genes involved in late steps of cobalamin synthesis has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contains eight genes arranged in at least three operons. Five of these eight genes show homology to genes involved in the cobalamin synthesis of Pseudomonas denitrificans and Salmonella typhimurium. The arrangement of these homologous genes differs considerably in the three genera. Upstream of five overlapping genes (named bluFEDCB), a promoter activity could be detected by using lacZ fusions. This promoter shows no regulation by oxygen, vitamin B12 (cobalamin), or cobinamide. Disruption of the bluE gene by a Tn5 insertion (strain AH2) results in reduced expression of the puf and puc operons, which encode pigment-binding proteins of the photosynthetic apparatus. The mutant strain AH2 can be corrected to a wild-type-like phenotype by addition of vitamin B12 or cobinamide dicyanide. Disruption of the bluB gene by an interposon (strain BB1) also disturbs the formation of the photosynthetic apparatus. The mutation of strain BB1 can be corrected by vitamin B12 but not by cobinamide. We propose that a lack of cobalamin results in deregulation and a decreased formation of the photosynthetic apparatus. PMID:7635831

  7. Crosstalk between Desmoglein 2 and Patched 1 accelerates chemical-induced skin tumorigenesis

    PubMed Central

    Brennan-Crispi, Donna M.; Hossain, Claudia; Sahu, Joya; Brady, Mary; Riobo, Natalia A.; Mahoney, Mỹ G.

    2015-01-01

    Aberrant activation of Hedgehog (Hh) signaling is causative of BCCs and has been associated with a fraction of SCCs. Desmoglein 2 (Dsg2) is an adhesion protein that is upregulated in many cancers and overexpression of Dsg2 in the epidermis renders mice more susceptible to squamous-derived neoplasia. Here we examined a potential crosstalk between Dsg2 and Hh signaling in skin tumorigenesis. Our findings show that Dsg2 modulates Gli1 expression, in vitro and in vivo. Ectopic expression of Dsg2 on Ptc1+/lacZ background enhanced epidermal proliferation and interfollicular activation of the Hh pathway. Furthermore, in response to DMBA/TPA, the Dsg2/Ptc1+/lacZ mice developed squamous lessons earlier than the WT, Ptc1+/lacZ, and Inv-Dsg2 littermates. Additionally, DMBA/TPA induced BCC formation in all mice harboring the Ptc1+/lacZ gene and the presence of Dsg2 in Dsg2/Ptc1+/lacZ mice doubled the BCC tumor burden. Reporter analysis revealed activation of the Hh pathway in the BCC tumors. However, in the SCCs we observed Hh activity only in the underlying dermis of the tumors. Furthermore, Dsg2/Ptc1+/lacZ mice demonstrated enhanced MEK/Erk1/2 activation within the tumors and expression of Shh in the dermis. In summary, our results demonstrate that Dsg2 modulates Hh signaling, and this synergy may accelerate skin tumor development by different mechanisms. PMID:25871385

  8. New ex vivo reporter assay system reveals that σ factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants

    PubMed Central

    Ishii, Yoshiko; Kakizawa, Shigeyuki; Oshima, Kenro

    2013-01-01

    Abstract Analysis of the environmental regulation of bacterial gene expression is important for understanding the nature, pathogenicity, and infection route of many pathogens. “Candidatus Phytoplasma asteris”, onion yellows strain M (OY-M), is a phytopathogenic bacterium that is able to adapt to quite different host environments, including plants and insects, with a relatively small ∼850 kb genome. The OY-M genome encodes two sigma (σ) factors, RpoD and FliA, that are homologous to Escherichia coli σ70 and σ28, respectively. Previous studies show that gene expression of OY-M dramatically changes upon the response to insect and plant hosts. However, very little is known about the relationship between the two σ factors and gene regulatory systems in OY-M, because phytoplasma cannot currently be cultured in vitro. Here, we developed an Escherichia coli-based ex vivo reporter assay (EcERA) system to evaluate the transcriptional induction of phytoplasmal genes by the OY-M-derived σ factors. EcERA revealed that highly expressed genes in insect and plant hosts were regulated by RpoD and FliA, respectively. We also demonstrated that rpoD expression was significantly higher in insect than in plant hosts and fliA expression was similar between the hosts. These data indicate that phytoplasma-derived RpoD and FliA play key roles in the transcriptional switching mechanism during host switching between insects and plants. Our study will be invaluable to understand phytoplasmal transmission, virulence expression in plants, and the effect of infection on insect fitness. In addition, the novel EcERA system could be broadly applied to reveal transcriptional regulation mechanisms in other unculturable bacteria. Phytoplasma, an unculturable plant pathogen, could infect plant and insect cells, and dramatically changes their genes upon the response to these hosts. By a new system developed in this study, interactions between phytoplasma promoters and sigma factors were analyzed, and overall gene expression regulation mechanism could be revealed. This model illustrates the RpoD and FliA regulatory network in phytoplasma cells during host switching. PMID:23723081

  9. Pyrethroid and their metabolite, 3-phenoxybenzoic acid showed similar (anti)estrogenic activity in human and rat estrogen receptor α-mediated reporter gene assays.

    PubMed

    Sun, Hong; Chen, Wen; Xu, Xiaolin; Ding, Zhen; Chen, Xiaodong; Wang, Xinru

    2014-01-01

    Pyrethroids are commonly used as pesticides which are suspected as endocrine disruptors in many studies; however, still we do not know their effects on different species. To compare their effects on human estrogen receptor (hERα) and rat estrogen receptor (rERα), we developed a hERα and rERα mediated luciferase reporter assay to investigate the (anti)estrogenic activities of three frequently used pyrethroids (fenvalerate, cypermethrin, permethrin) and their metabolite 3-phenoxybenzoic acid (3-PBA). All three pyrethroids significantly induced expression of the luciferase, while none of them were antagonistic to 1 nM E₂ mediated induction. Interestingly, 3-PBA, showed antagonist activity by decreasing the effect of 1 nM E₂ to 55.12% in hERα assay and to 45.12% in rERα assay. Our results firstly demonstrated that pyrethroids and 3-PBA showed similar response in the hERα and rERα mediated reporter gene assay, which indicated that data derived from reporter gene assay or other receptor mediated assay systems with rat ER system might be used to predict to estrogenic or anti-estrogenic effects to human systems. PMID:24388911

  10. Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium

    SciTech Connect

    Wolk, C.P.; Yuping Cai; Panoff, J.M. )

    1991-06-15

    Anabaena, a filamentous cyanobacterium, is of developmental interest because, when deprived of fixed nitrogen, it shows patterned differentiation of N{sub 2}-fixing cells called heterocysts. To help elucidate its early responses to a decrease in nitrogen, the authors used a derivative of transposon Tn5 to generate transcriptional fusions of promoterless bacterial luciferase genes, luxAB, to the Anabaena genome. Genes that responded to removal of fixed nitrogen or to other environmental shifts by increased or decreased transcription were identified by monitoring the luminescence of colonies from transposon-generated libraries. The Tn5 derivative transposed in Anabaena at ca. 1-4 {times} 10{sup {minus}5} per cell and permitted high-resolution mapping of its position and orientation in the genome and facile cloning of contiguous genomic DNA.

  11. Triple-A syndrome with prominent ophthalmic features and a novel mutation in the AAAS gene: a case report

    PubMed Central

    Brooks, Brian P; Kleta, Robert; Caruso, Rafael C; Stuart, Caroline; Ludlow, Jonathan; Stratakis, Constantine A

    2004-01-01

    Background Triple-A syndrome (Allgrove syndrome) is an autosomal recessive disorder characterized by adrenal insufficiency, alacrima, achalasia, and – occasionally – autonomic instability. Mutations have been found in the AAAS gene on 12q13. Case presentation We present the case of a 12 year-old boy with classic systemic features of triple-A syndrome and several prominent ophthalmic features, including: accommodative spasm, dry eye, superficial punctate keratopathy, and pupillary hypersensitivity to dilute pilocarpine. MRI showed small lacrimal glands bilaterally. DNA sequencing of PCR-amplified fragments from the 16 exons of the AAAS gene revealed compound heterozygosity for a new, out-of-frame 5-bp deletion in exon 15, c1368-1372delGCTCA, and a previously-described nonsense mutation in exon 9, c938C>T, R286X. Conclusions In addition to known ophthalmic manifestations, triple-A syndrome can present with accommodative dysregulation and ocular signs of autonomic dysfunction. PMID:15217518

  12. Deciphering the colon cancer genes--report of the InSiGHT-Human Variome Project Workshop, UNESCO, Paris 2010.

    PubMed

    Kohonen-Corish, Maija R J; Macrae, Finlay; Genuardi, Maurizio; Aretz, Stefan; Bapat, Bharati; Bernstein, Inge T; Burn, John; Cotton, Richard G H; den Dunnen, Johan T; Frebourg, Thierry; Greenblatt, Marc S; Hofstra, Robert; Holinski-Feder, Elke; Lappalainen, Ilkka; Lindblom, Annika; Maglott, Donna; Møller, Pål; Morreau, Hans; Möslein, Gabriela; Sijmons, Rolf; Spurdle, Amanda B; Tavtigian, Sean; Tops, Carli M J; Weber, Thomas K; de Wind, Niels; Woods, Michael O

    2011-04-01

    The Human Variome Project (HVP) has established a pilot program with the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) to compile all inherited variation affecting colon cancer susceptibility genes. An HVP-InSiGHT Workshop was held on May 10, 2010, prior to the HVP Integration and Implementation Meeting at UNESCO in Paris, to review the progress of this pilot program. A wide range of topics were covered, including issues relating to genotype-phenotype data submission to the InSiGHT Colon Cancer Gene Variant Databases (chromium.liacs.nl/LOVD2/colon_cancer/home.php). The meeting also canvassed the recent exciting developments in models to evaluate the pathogenicity of unclassified variants using in silico data, tumor pathology information, and functional assays, and made further plans for the future progress and sustainability of the pilot program. PMID:21387463

  13. Chronic lymphocytic leukemia presenting with ascites diagnosed by clonality analysis via gene rearrangement assay: A case report

    PubMed Central

    HUANG, YEN-MIN; SHIH, LEE-YUNG; DUNN, PO; WANG, PO-NAN; KUO, MING-CHUNG; WU, JIN-HOU; LIN, TUNG-LIANG; TANG, TZUNG-CHIH; CHANG, HUNG; KAO, HSIAO-WEN; SHIH, HSUAN-JEN; HUNG, YU-SHIN

    2014-01-01

    The diagnosis of chronic lymphocytic leukemia (CLL) presenting with ascites is predominantly based on the morphological and immunophenotypic characteristics, which are comparable to peripheral blood and bone marrow cells. However, it is relatively difficult to diagnose CLL due to the pleomorphism of the lymphocytes in ascites. The current study presents an 80-year-old male with a prior diagnosis of CLL who developed large ascites. Predominant T lymphocytes rendered morphological and immunophenotypic diagnosis difficult. Clonality analysis of immunoglobulin (Ig) gene rearrangements was performed on the lymphocytes from the ascites to diagnose the involvement of CLL, a laparotomy and biopsy from the peritoneal node confirmed the involvement of small lymphocytic lymphoma/CLL. The clonality analysis of Ig gene rearrangements may provide a powerful and accurate method for diagnosing CLL presenting with ascites. PMID:24932257

  14. [Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, [March 15, 1989--April 14, 1990

    SciTech Connect

    Okita, T.W.

    1990-12-31

    The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of the gene that encodes for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. During the last two years we have obtained information on the structure of this enzyme from both potato tuber and rice endosperm, using a combination of biochemical and molecular biological approaches. Moreover, we present evidence that this enzyme may be localized at discrete regions of the starch grain within the amyloplast, and plays a role in controlling overall starch biosynthesis in potato tubers.

  15. Excess congenital non-synonymous variation in leukemia-associated genes in MLL? infant leukemia: a Children's Oncology Group report

    PubMed Central

    Valentine, M C; Linabery, A M; Chasnoff, S; Hughes, A E O; Mallaney, C; Sanchez, N; Giacalone, J; Heerema, N A; Hilden, J M; Spector, L G; Ross, J A; Druley, T E

    2014-01-01

    Infant leukemia (IL) is a rare sporadic cancer with a grim prognosis. Although most cases are accompanied by MLL rearrangements and harbor very few somatic mutations, less is known about the genetics of the cases without MLL translocations. We performed the largest exome-sequencing study to date on matched non-cancer DNA from pairs of mothers and IL patients to characterize congenital variation that may contribute to early leukemogenesis. Using the COSMIC database to define acute leukemia-associated candidate genes, we find a significant enrichment of rare, potentially functional congenital variation in IL patients compared with randomly selected genes within the same patients and unaffected pediatric controls. IL acute myeloid leukemia (AML) patients had more overall variation than IL acute lymphocytic leukemia (ALL) patients, but less of that variation was inherited from mothers. Of our candidate genes, we found that MLL3 was a compound heterozygote in every infant who developed AML and 50% of infants who developed ALL. These data suggest a model by which known genetic mechanisms for leukemogenesis could be disrupted without an abundance of somatic mutation or chromosomal rearrangements. This model would be consistent with existing models for the establishment of leukemia clones in utero and the high rate of IL concordance in monozygotic twins. PMID:24301523

  16. In vitro expression and mutagenesis of a gene for corticotropin releasing factor. Annual report, November 1988-October 1989

    SciTech Connect

    Vrana, K.E.

    1989-10-31

    The specific goals of this proposal are to: (1) create a recombinant gene for corticotropin releasing factor (CRF), (2) express that gene by in vitro transcription and translation, (3) test the function of this recombinant protein by receptor binding assay and agonist-induced release of ACTH from cultured pituitary cells and (4) create and test mutants of the CRF molecule (starting at the level of the DNA). The author have accomplished the first two of these goals and partially completed the third. He has have synthesized the CRF gene, expressed it and characterized the recombinant protein. This protein is active when applied to pituitary cells, but the in vitro translation extract contains substances which partially interfere with that activity. He is are presently purifying the recombinant protein from the translation extract. In a related area, he is are conducting experiments to characterize the stress non-responsive period (SNRP) in the neonatal rat. It has been found find that the spontaneously hypertensive rat (SHR) is not entirely subject to this quiescent adrenocortical period (during the first two weeks of neonatal life) when compared with the normotensive control animal. This difference is not caused by alterations in the levels of circulating (or stored) ACTH, implying that there are differences in the responsiveness of the adrenal cortex.

  17. Identification of the first intragenic deletion of the PITX2 gene causing an Axenfeld-Rieger Syndrome: case report

    PubMed Central

    de la Houssaye, Guillaume; Bieche, Ivan; Roche, Olivier; Vieira, Vronique; Laurendeau, Ingrid; Arbogast, Laurence; Zeghidi, Hatem; Rapp, Philippe; Halimi, Philippe; Vidaud, Michel; Dufier, Jean-Louis; Menasche, Maurice; Abitbol, Marc

    2006-01-01

    Background Axenfeld-Rieger syndrome (ARS) is characterized by bilateral congenital abnormalities of the anterior segment of the eye associated with abnormalities of the teeth, midface, and umbilicus. Most cases of ARS are caused by mutations in the genes encoding PITX2 or FOXC1. Here we describe a family affected by a severe form of ARS. Case presentation Two members of this family (father and daughter) presented with typical ARS and developed severe glaucoma. The ocular phenotype was much more severe in the daughter than in the father. Magnetic resonance imaging (MRI) detected an aggressive form of meningioma in the father. There was no mutation in the PITX2 gene, determined by exon screening. We identified an intragenic deletion by quantitative genomic PCR analysis and characterized this deletion in detail. Conclusion Our findings implicate the first intragenic deletion of the PITX2 gene in the pathogenesis of a severe form of ARS in an affected family. This study stresses the importance of a systematic search for intragenic deletions in families affected by ARS and in sporadic cases for which no mutations in the exons or introns of PITX2 have been found. The molecular genetics of some ARS pedigrees should be re-examined with enzymes that can amplify medium and large genomic fragments. PMID:17134502

  18. Primary mucinous adenocarcinoma of the epididymis: report of a rare case with molecular genetic characterization including mutation analysis of the TP53 gene.

    PubMed

    Gupta, Sarika; Yan, Benedict; Leow, Pay Chin; Chin, Sze Yung; Soong, Richie; Petersson, Fredrik

    2015-04-01

    We report a case of primary epididymal mucinous adenocarcinoma in a 60-year-old man who presented with a scrotal mass and subsequently developed pulmonary metastases. On immunohistochemistry the tumor was positive for villin and CK20 and negative for CK7, CDX2, and thyroid transcription factor-1. Molecular genetic analysis revealed an uncommon mutation; 249: AGG ?ATG in the TP53 gene, which has not been previously described in association with a primary epididymal adenocarcinoma. Mutational analysis showed KRAS, BRAF, and VHL to be wild-type. No microsatellite instability was found. PMID:25839701

  19. Anomalous left coronary artery from the right coronary cusp with gene positive apical hypertrophic cardiomyopathy: a case report and literature review.

    PubMed

    Georgekutty, Justin; Cross, Russell R; Rosenthal, Joanna B; Heath, Deneen M; Sinha, Pranava; John, Anitha S

    2014-06-01

    In the United States, hypertrophic cardiomyopathy and coronary artery anomalies account for the leading two causes of sudden death in athletes. We present a case of a patient with an anomalous origin of the left main from the right coronary sinus with associated gene-confirmed hypertrophic cardiomyopathy. The patient underwent surgical repair with unroofing of the intramural portion of the left main coronary artery with a good result. We also review the reported cases in the medical literature describing this uncommon association between anomalous coronary artery origin and hypertrophic cardiomyopathy. PMID:24345326

  20. 3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

    SciTech Connect

    Moorthy, Bhagavatula . E-mail: bmoorthy@bcm.tmc.edu; Muthiah, Kathirvel; Fazili, Inayat S.; Kondraganti, Sudha R.; Wang Lihua; Couroucli, Xanthi I.; Jiang Weiwu

    2007-03-23

    Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 {mu}g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 {sup o}C for 2 h, giving rise to 9 adducts, as determined by {sup 32}P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16 h, followed by MC (1 {mu}M) treatment for 24 h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

  1. Characterization of Three mnp Genes of Fomitiporia mediterranea and Report of Additional Class II Peroxidases in the Order Hymenochaetales ▿ †

    PubMed Central

    Morgenstern, Ingo; Robertson, Deborah L.; Hibbett, David S.

    2010-01-01

    We report the sequence-based characterization and expression patterns of three manganese peroxidase genes from the white rot fungus and grape vine pathogen Fomitiporia mediterranea (Agaricomycotina, Hymenochaetales), termed Fmmnp1, Fmmnp2, and Fmmnp3. The predicted open reading frames (ORFs) are 1,516-, 1,351-, and 1,345-bp long and are interrupted by seven, four, and four introns, respectively. The deduced amino acid sequences encode manganese peroxidases (EC 1.11.1.13) containing 371, 369, and 371 residues, respectively, and are similar to the manganese peroxidases of the model white rot organism Phanerochaete chrysosporium. The expression of the genes is most likely differentially regulated, as revealed by real-time PCR analysis. Phylogenetic analysis reveals that other members of the order Hymenochaetales harbor mnp genes encoding proteins that are related only distantly to those of F. mediterranea. Furthermore, multiple partial lip- and mnp-like sequences obtained for Pycnoporus cinnabarinus (Agaricomycotina, Polyporales) suggest that lignin degradation by white rot taxa relies heavily on ligninolytic peroxidases and is not efficiently achieved by laccases only. PMID:20675443

  2. Flow-type electroporation chips for gene transfection

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Jen, Chung-Min; Huang, Ming-Yuan; Lin, Xi-Zhang

    2000-08-01

    Electroporation is a technique with which DNA molecules can be delivered into cells in a chamber using high electric field pulses. The limited amount of target cells and the potential risk from the high voltage are the two drawbacks in this technique. This study aimed to fabricate an electroporation chip to manage large amount of cells continuously with a lower applied voltage. The electroporation chip, consisting of a micro-channel with thin film electrodes made of gold or platinum wire electrodes on both sides, was fabricated on PMMA material using evaporation, photolithography, wet- etching, lift-off, and fusion-bonding methods. The suspension fluid of Huh-7 cell lines (1 x 106 cells/ml) mixed with 10 micrometers plasmids equipped with lacZ genes in a volume of 500 (mu) l flowed through the channel with a variety of flow rates under a series of square pulses. The transfection rate was evaluated with blue-staining cells under X-Gal stain 24 hours later. The dimensions of the channel were 5 mm wide, 0.2 mm high, and 25 mm long. Two types of electrodes, parallel-plate type and parallel-line type electrodes, were fabricated and tested in these experiments. The fabricated microchip can deliver genes into the flowing of cells. The electric pulse frequency that determines the shock number for each cell for a fixed flow rate can be optimized for better transfection and survival rates.

  3. A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3′ untranslated region-mediated gene regulation

    PubMed Central

    Chaudhury, Arindam; Kongchan, Natee; Gengler, Jon P.; Mohanty, Vakul; Christiansen, Audrey E.; Fachini, Joseph M.; Martin, James F.; Neilson, Joel R.

    2014-01-01

    Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3′ untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3′ UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3′ UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome—a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3′ UTR of interest. The pBUTR (piggyBac-based 3′ UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting. PMID:24753411

  4. REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis

    SciTech Connect

    Larimer, F.W.; Perry, J.R.; Hardigree, A.A.

    1989-01-01

    The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents. We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant. Gene disruption was used to confirm that this DNA contained the REV1 gene. The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues. The REV1 transcript is 3.1 kilobase pairs in length. Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype. A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1. Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1. REV1 is not an essential gene. An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively. The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82. No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted. A 152-residue internal segment displayed 25% identity with UMUC protein.

  5. The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis.

    PubMed

    Larimer, F W; Perry, J R; Hardigree, A A

    1989-01-01

    The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents. We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant. Gene disruption was used to confirm that this DNA contained the REV1 gene. The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues. The REV1 transcript is 3.1 kilobase pairs in length. Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype. A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1. Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1. REV1 is not an essential gene. An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively. The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82. No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted. A 152-residue internal segment displayed 25% identity with UMUC protein. PMID:2492497

  6. Structure and expression of the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis.

    PubMed Central

    Mueller, J P; Taber, H W

    1989-01-01

    Mutations that define the ctaA gene of Bacillus subtilis block cytochrome aa3 formation and sporulation. We have recently described the isolation and initial characterization of the ctaA locus. Analysis of in vivo mRNA transcripts by RNase protection experiments located the 5' and 3' termini of the ctaA transcript, confirming a monocistronic structure. By using a nuclease protection assay, an increase in the abundance of steady-state ctaA mRNA was observed during the initiation of sporulation, followed by a decrease during subsequent stages. Transcripts originating from the ctaA gene were most abundant 2.0 h after the end of exponential growth. This pattern of ctaA mRNA accumulation was confirmed by coupling the transcription of the ctaA gene to lacZ in an integrative plasmid vector. Expression of ctaA was not repressed by glucose and was independent of the spoOA and spoOH (sigH) gene products. Postexponential expression was found to be dependent on the product of the strC gene. The expression of ctaA appears to be regulated in a growth stage-specific manner. The transcriptional start site, identified by high-resolution S1 nuclease protection experiments, was preceded by a single sigma A-dependent promoter sequence. Images PMID:2549007

  7. Replicon-Specific Regulation of Small Heat Shock Genes in Agrobacterium tumefaciens

    PubMed Central

    Balsiger, Sylvia; Ragaz, Curdin; Baron, Christian; Narberhaus, Franz

    2004-01-01

    Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (?32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5? untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive. PMID:15466035

  8. Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli.

    PubMed Central

    Khosla, C; Bailey, J E

    1989-01-01

    The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of significant homology to the catabolite activator protein-binding site near the E. coli