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1

Isolation of a novel insertion sequence from Mycobacterium fortuitum using a trap vector based on inactivation of a lacZ reporter gene  

Microsoft Academic Search

An insertion sequence of Mycobacterium fortuitum has been isolated using a trap vector following insertion in and inactivation of the lacZ reporter gene. The trap vector is a temperature-sensitive (ts) Escherichia coli-mycobacterium shuttle plasmid, pCD4, which contains ts oriM, the kanamycin-resistance gene as a selection marker and a lacZ expression cassette. The ts mutation present in pCD4 functions in mycobacteria

Morris Waskar; Deepak Kumar; Ajai Kumar; Ranjana Srivastava

2

Vertebrate caudal gene expression gradients investigated by use of chick cdx-A\\/ lacZ and mouse cdx-1\\/ lacZ reporters in transgenic mouse embryos: evidence for an intron enhancer  

Microsoft Academic Search

The vertebrate caudal proteins, being upstream regulators of the Hox genes, play a role in establishment of the body plan. We describe analysis of two orthologous caudal genes (chick cdx-A and mouse cdx-1) by use of lacZ reporters expressed in transgenic mouse embryos. The expression patterns show many similarities to the expression of endogenous mouse cdx-1. At 8.7 days, cdx\\/lacZ

Stephen J. Gaunt; Deborah Drage; Adam Cockley

2003-01-01

3

Isolation of a novel insertion sequence from Mycobacterium fortuitum using a trap vector based on inactivation of a lacZ reporter gene.  

PubMed

An insertion sequence of Mycobacterium fortuitum has been isolated using a trap vector following insertion in and inactivation of the lacZ reporter gene. The trap vector is a temperature-sensitive (ts) Escherichia coli-mycobacterium shuttle plasmid, pCD4, which contains ts oriM, the kanamycin-resistance gene as a selection marker and a lacZ expression cassette. The ts mutation present in pCD4 functions in mycobacteria and enables screening for transposable elements from the mycobacterial genome that disrupt the lacZ gene by screening for white colonies on X-Gal plates in both mycobacterial as well as E. coli hosts. The vector was used to isolate a novel 1.653 kb insertion sequence from M. fortuitum named IS219. IS219 duplicated host DNA at the target site, had inverted repeats at its ends and contained two ORFs on one strand. One of the predicted proteins showed homology to a putative transposase from Acetobacter pasteurianus. IS219 was present in two copies in the genome of M. fortuitum. The trap vector appears to be useful in trapping insertion sequences from different mycobacteria by screening for the disrupted LacZ phenotype. PMID:10832643

Waskar, M; Kumar, D; Kumar, A; Srivastava, R

2000-05-01

4

Photoacoustic imaging of lacZ gene expression  

E-print Network

for molecular imaging research. © 2007 Society of Photo-Optical Instrumentation Engineers. DOI: 10 protein enables whole body imaging of small animals, although at low spatial resolution due to multiplePhotoacoustic imaging of lacZ gene expression in vivo Li Li,a, Roger J. Zemp,a, Gina Lungu,b George

Wang, Lihong

5

Escherichia coli lacZ gene as a biochemical and histochemical marker in plant cells.  

PubMed

Several lacZ chimeric genes were constructed by fusing the truncated lacZ sequence of Escherichia coli to N-terminal sequences of few other genes. Promoters used to direct expression of the chimeric genes were the promoter for 35S RNA of cauliflower mosaic virus (P35S) as well as those of the small subunit gene of ribulose bisphosphate carboxylase and the octopine synthase gene. These constructs were introduced into tobacco cells using a Ti plasmid of Agrobacterium tumefaciens, and beta-galactosidase activity in uncloned and cloned calli derived from the crown galls were examined. The results showed that the P35S-linked lacZ chimeric gene is expressed very efficiently. When slices of the crown gall carrying this chimeric gene were placed on plates containing indicator XGal, localized areas of the outgrowth turned deep blue, whereas no such areas were found in the crown gall having promoter-less lacZ. Calli from galls containing this construct expressed beta-galactosidase activity at an eight-fold higher level (approx. 7000 units/mg protein) than the endogenous activity (approx. 900 units/mg protein). Some of the calli displayed over 20-fold higher activity. Actively growing mini calli expressing activity higher than 4000 units/mg protein dyed deep blue on XGal agar medium such that they were distinguishable from calli having no lacZ. Half of the uncloned P35S-lacZ transformant calli showed activity higher than this level. These results indicate that the lacZ gene linked to a strong promoter such as P35S is useful as a biochemical and histochemical marker gene in plant cells. PMID:3138164

Matsumoto, S; Takebe, I; Machida, Y

1988-06-15

6

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

Microsoft Academic Search

Although more than a dozen new proteins are produced when Streptococcus pneumoniae cells become com- petent 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.

EKATERINA V. PESTOVA; DONALD A. MORRISON

1998-01-01

7

[9] GENE FUSIONS IN YEAST 167 [9] Construction and Use of Gene Fusions to lacZ  

E-print Network

Z) constructed by Casada- ban and Cohen, r to investigate the expression of the sequences around the yeast URA3 in Escherichia coli. 5 These facts have led several workers to construct mutations of the lacZ gene (which

Botstein, David

8

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

PubMed

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

Fried, Luitpold; Lassak, Jürgen; Jung, Kirsten

2012-12-01

9

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

PubMed

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

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

2012-09-18

10

Spatial–temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA  

Microsoft Academic Search

Gene therapy for muscular diseases requires the efficient transfection of a large proportion of myofiber cells within a given muscle. In the present experiments, patterns of ?-galactosidase expression were examined in mouse rectus femoris muscles at various time-points after a single injection of lacZ encoded plasmid DNA. ?-Galactosidase expression was detected 3 h after injection and rose to peak levels

SG Doh; HL Vahlsing; J Hartikka; X Liang; M Manthorpe

1997-01-01

11

Chemosensitivity of micrometastases and circulating tumor cells to uracil and tegafur as evaluated using LacZ gene-tagged Lewis lung carcinoma cell  

Microsoft Academic Search

The chemosensitivity of the sequence of steps responsible for metastasis formation including circulating tumor cells and micrometastases to a 5-fluorouracil derivative (UFT) was examined with a novel micrometastasis model featuring Lewis lung carcinoma cells tagged with the bacterial LacZ gene and hygromycinR gene (hygR). Metastases in the lung could be specifically detected at the single-cell level by X-Gal staining after

Hayao Nakanishi; Kiyoshi Kobayashi; Tuyoshi Nishimura; Kenichi Inada; Tetsuya Tsukamoto; Masae Tatematsu

1999-01-01

12

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

PubMed

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

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

2014-11-01

13

Development of an integrative, lacZ transcriptional-fusion plasmid vector for Streptococcus mutans and its use to isolate expressed genes  

Microsoft Academic Search

A new integrational vector, pFP1, containing a promoterless lacZ gene has been constructed for use with Streptococcus mutans. The vector can be grown in Escherichia coli, but cannot replicate in S. mutans. pFP1 can be transformed into S. mutans with selection for kanamycin resistance. It integrates into the chromosome when homologous DNA is present in the vector. pFP1 provides a

Francesca Peruzzi; Patrick J. Piggot; Lolita Daneo-Moore

1998-01-01

14

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

Microsoft Academic Search

The mutational spectra at the lacZ gene, induced either by vacuum UV at 160 nm or UVC at 254 nm in vacuum-dried preparations of Escherichia coli plasmid pUC19 DNA, have been characterized from 72 E. coli-propagated mutants by DNA sequencing. In plasmids irradiated in vacuum, vacuum UV is five times more mutagenic than UVC. In the UV-induced mutants, base substitutions

Jörg Wehner; Gerda Horneck

1995-01-01

15

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

PubMed

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

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

2013-05-01

16

?-Galactosidase staining of lacZ fusion proteins in whole tissue preparations.  

PubMed

The lacZ gene product, ?-galactosidase, has classically been used as a reporter of gene expression. ?-Galactosidase activity can be detected using a chromogenic substrate, X-gal, which leaves an intense blue precipitate when cleaved by the enzyme. Insertion of the lacZ coding DNA targeted into a specific gene creates a ?-galactosidase-tagged fusion protein that is expressed under the endogenous promoter. Analysis of the hybrid protein takes advantage of the chromogenic detection system, as the distribution and relative abundance of the expressed protein can be efficiently visualized. PMID:23681629

Cooper, Margaret A; Zhou, Renping

2013-01-01

17

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

PubMed Central

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

Bartsch, S; Ducker, K; Wurgler, F E; Sengstag, C

1997-01-01

18

cis-acting DNA regulatory elements, including the retinoic acid response element, are required for tissue specific laminin B1 promoter \\/lacZ expression in transgenic mice  

Microsoft Academic Search

The LAMB1 gene encodes the laminin ?1 subunit of laminin, an extracellular matrix protein. Using several transgenic mouse lines containing various lengths of the LAMB1 promoter driving lacZ reporter gene expression, regions of LAMB1 promoter that contain cis-acting DNA regulatory element(s) have been identified. The 3.9LAMB1?gal transgene is expressed in various tissues during development. LAMB1 transgene expression is observed in

Karim A. Sharif; Congyi Li; Lorraine J. Gudas

2001-01-01

19

Vectors that facilitate the replacement of transcriptional lacZ fusions in Streptococcus mutans and Bacillus subtilis with fusions to gfp or gusA  

Microsoft Academic Search

Plasmid vectors have been constructed for Streptococcus mutans and Bacillus subtilis that make possible rapid replacement of the widely used reporter gene lacZ (encoding ?-galactosidase) with either gfp (encoding green fluorescent protein) or gusA (encoding ?-glucuronidase). The lacZ?gfp replacement vectors greatly facilitate the analysis of the spatial location of gene expression in biofilms of S. mutans and in sporulating B.

Vasant K. Chary; Monica Busuioc; John A. Renye; Patrick J. Piggot

2005-01-01

20

RLR1 (THO2), required for expressing lacZ fusions in yeast, is conserved from yeast to humans and is a suppressor of SIN4.  

PubMed

We isolated a mutation (rlr1-1; required for lacZ RNA) in the Saccharomyces cerevisiae (Sc) RLR1 gene as a suppressor of sin4, a component of the Mediator subcomplex of the RNA polymerase II holoenzyme and a determinant of chromatin structure. RLR1 encodes a deduced protein found also in fission yeast, nematode worms, and humans. The presence of these orthologs suggests that Rlr1 family members comprise a class of putative KEKE motif-containing proteins, characteristic of certain chaperones as well as regulators and subunits of the mammalian 20S proteasome. A role for RLR1 (THO2) in transcription appears to occur at a step subsequent to transcription initiation (see also Piruat, J.I. and Aguilera, A., 1998. EMBO J. 17, 4859-4872); Sc genes fused to the reporter gene lacZ were expressed at a very low level, while the corresponding native chromosomal genes were expressed at approximately normal levels in rlr1 mutants. Our studies show that rlr1 mutations cause a wide range of growth defects in addition to their novel affect on lacZ. PMID:10675628

West, R W; Kruger, B; Thomas, S; Ma, J; Milgrom, E

2000-02-01

21

DEAD-box RNA helicase Sub2 is required for expression of lacZ fusions in Saccharomyces cerevisiae and is a dosage-dependent suppressor of RLR1 (THO2).  

PubMed

RLR1 (THO2) encodes a novel, phylogenetically-conserved KEKE motif protein involved in transcription and transcription-associated recombination in Saccharomyces cerevisiae. One characteristic aspect of RLR1 function is its requirement for expression of the Escherichia coli lacZ reporter gene regardless of the yeast promoter to which it is fused. rlr1-1 was originally isolated (employing lacZ as a transcriptional reporter) as a suppressor of a mutation in the gene encoding Sin4, a subunit of the Mediator subcomplex of the RNA polymerase II (PolII) transcriptional machinery. To clarify the function of Rlr1, we performed a genetic screen for dosage-dependent suppressors of the cold-sensitive phenotype of rlr1-1. From this screen we isolated SUB2, encoding a conserved DEAD-box RNA helicase family member having roles in both pre-mRNA splicing and mRNA export in yeast, flies, and humans. We demonstrate that Sub2, like Rlr1, is required for lacZ to be expressed in yeast, and that sub2 mutants manifest rlr1-like growth defects. Our results are consistent with a hypothesis where expression of lacZ fusions in yeast preferentially requires a Sub2-mediated mRNP assembly/export pathway linked to transcription via Rlr1. PMID:12034490

West, Robert W; Milgrom, Elena

2002-04-17

22

Tracking Micrometastasis to Multiple Organs with lacZ -tagged CWR22R Prostate Carcinoma Cells  

Microsoft Academic Search

SUMMARY Metastasis to organs other than lung is rarely observed in animal model sys- tems of human prostate carcinoma (PCA), with the exception of already metastatic isolates of human PCA cultured for long periods of time. To analyze more directly the evolution of metastatic variants from primary PCA tumor isolates, the lacZ histochemical marker gene was transfected into the CWR22Rv1

Julianne L. Holleran; Carson J. Miller; Lloyd A. Culp

23

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

PubMed Central

The white(+) gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacW] transposant lines were collected. These represent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to cis-acting suppression of white(+) expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lacZ in P[lacW] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white(+) and lacZ may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacW] insertion site. We speculate that these putative POSE (position-specific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotor-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this ``silencer trap'' may be an efficient way of identifying genes involved in imaginal pattern formation. PMID:8582614

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

1995-01-01

24

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

Microsoft Academic Search

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

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

1989-01-01

25

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

PubMed Central

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

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

2011-01-01

26

Induction of lacZ Mutations in Muta(TM)Mouse Primary Hepatocytes  

PubMed Central

We have developed an in vitro mutation assay using primary hepatocytes from the transgenic Muta™Mouse. Primary hepatocytes were isolated using a two-step perfusion method with purification by Percoll, cultured, and treated with benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenyl- imidazo[4,5-b]pyridine (PhIP), 3-nitrobenzoanthrone (3-NBA), and cigarette smoke condensate (CSC). The mean lacZ mutant frequency (MF) for the solvent control was approximately twofold greater than the spontaneous MF observed in liver tissue. A concentration-dependent increase in MF (up to 3.7-fold above control) was observed following exposure to BaP. Fourfold and twofold increases in mutant frequency were observed for 3-NBA and PhIP exposures, respectively, without the addition of any exogenous metabolic activation. A slight but statistically significant increase in lacZ MF was observed for CSC, but only at the lowest concentration. This is the first report demonstrating that mutations can be detected in cultured primary hepatocytes from Muta™Mouse. The preliminary results presented suggest that the Muta™Mouse primary hepatocyte mutagenicity assay can be used as a cost-effective tool for screening of environmental mutagens and therapeutic products. Environ. Mol. Mutagen. 51:330–337, 2010. © 2009 Wiley-Liss, Inc. PMID:19953605

Chen, Guosheng; Gingerich, John; Soper, Lynda; Douglas, George R; White, Paul A

2010-01-01

27

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

PubMed Central

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

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

2011-01-01

28

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

Microsoft Academic Search

Using Escherichia coli plasmid pUC19 as a test system to study the effects of radiation on DNA at the molecular level, the wavelength (160–254 nm) dependence of inactivation (loss of the ability to transform E. coli), mutation induction in the target gene lacZ and induction of single-strand breaks and double-strand breaks was investigated. The same fluences were applied for all

Jörg Wehner; Gerda Horneck

1995-01-01

29

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

SciTech Connect

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

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

1997-10-01

30

Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae.  

PubMed

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability. PMID:16233719

Nakazawa, Nobushige; Iwano, Kimio

2004-01-01

31

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

PubMed

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

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

1997-01-15

32

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

PubMed Central

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

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

2012-01-01

33

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

PubMed

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

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

2014-07-01

34

Herpes simplex type 1 : lacZ recombinant viruses. II. Microtiter plate-based colorimetric assays for the discovery of new antiherpes agents and the points at which such agents disrupt the viral replication cycle  

Microsoft Academic Search

A panel of microtiter plate-based colorimetric assays for monitoring HSV-1 growth has been made. The panel consists of 4 different HSV-1 (strain KOS) lacZ recombinant viruses which express ?-galactosidase under the control of different HSV-1 promoters derived from each class of herpes simplex gene expression: immediate-early (ICP4), early (TK), delayed early (gD) and late (gC). Inhibitors of HSV-1 growth were

Ira B. Dicker; John W. Blasecki; Shobha Seetharam

1995-01-01

35

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

PubMed Central

Mutants of the mec-7 beta-tubulin gene of Caenorhabditis elegans lack the large diameter 15-protofilament microtubules normally found only in the set of six touch receptor neurons. Both a mec-7-lacZ reporter gene and affinity-purified anti-mec-7 antibodies were used to show that mec-7 is expressed primarily in the touch neurons. These data are consistent with a possible instructive role for the mec-7 tubulin in determining microtubule protofilament number. The antibodies and the mec-7-lacZ transgene were also used to examine mec-7 expression in mutants affecting the generation, differentiation or maintenance of the touch neurons. Decreased expression was observed in mutants of unc-86 and mec-3, genes that encode transcription factors essential for touch receptor neuron generation and differentiation, respectively. Images PMID:1639062

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

1992-01-01

36

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

Microsoft Academic Search

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

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

1996-01-01

37

Modified HIV1 Based Lentiviral Vectors Have an Effect on Viral Transduction Efficiency and Gene Expression in Vitro and in Vivo  

Microsoft Academic Search

Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human ?1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that

Frank Park; Mark A. Kay

2001-01-01

38

Indexing TNF-? gene expression using a gene-targeted reporter cell line  

Microsoft Academic Search

BACKGROUND: Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with

Ziying Yan; Diana Lei-Butters; John F Engelhardt; Gregory H Leno

2009-01-01

39

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

PubMed Central

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

TOTTEN, Daniel C.; VUONG, Mai; LITVINOVA, Oksana V.; JINWAL, Umesh K.; GULIA-NUSS, Monika; HARRELL, Robert A.; BENES, Helen

2014-01-01

40

A new reporter gene for transient transfection of Plasmodium falciparum.  

PubMed

Transfection of Plasmodium falciparum has remained difficult and laborious due to a lack of suitable reporter genes and low transfection efficiency. Therefore, the luciferase gene of Renilla reniformis, a sensitive mammalian reporter gene, was evaluated as a reporter gene in this system. Our studies indicate that the R. reniformis luciferase gene can be expressed in P. falciparum and is easily detected by luminometry. P. falciparum extracts do not contain endogenous R. reniformis luciferase activity, which is essential for its use as a reporter gene in this organism. Moreover, both firefly and R. reniformis luciferase genes can be co-expressed in P. falciparum and their respective enzyme activities can be measured from the same sample. Thus, the R. reniformis luciferase gene can be used as an experimental reporter gene and/or used in conjunction with the firefly luciferase gene where one gene would be used to control transfection efficiency. The R. reniformis luciferase gene thus provides a valuable tool to facilitate transient transfection analysis in P. falciparum. PMID:12489017

Militello, Kevin T; Wirth, Dyann F

2003-01-01

41

Construction of a Reporter Vector System for In Vivo Analysis of Promoter Activity in Propionibacterium freudenreichii? †  

PubMed Central

A ?-galactosidase reporter system for the analysis of promoter elements in Propionibacterium freudenreichii was designed. The pTD210 in vivo reporter vector was constructed using a promoterless lacZ gene from Bifidobacterium longum cloned into the pAMT1 plasmid. The utility of the pTD210 reporter vector was demonstrated by an investigation of six predicted promoters in P. freudenreichii. The system produced accurate and reproducible measurements that facilitated both promoter identification and the quantification of promoter activities. PMID:18424545

Faye, Therese; Ĺsebř, Anita; Salehian, Zhian; Langsrud, Thor; Nes, Ingolf F.; Brede, Dag Anders

2008-01-01

42

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

PubMed

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

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

2009-05-01

43

Disruption of murine alpha-enolase by a retroviral gene trap results in early embryonic lethality.  

PubMed

Gene trapping with the retroviral ROSA beta geo vector was used to generate lines of mice carrying disrupted genes. Both cDNA and genomic flanks have been cloned from a number of these lines. One mutation has been shown to disrupt the alpha-enolase gene by insertion of the splice-trap vector into the first intron. In adult mice, lacZ expression was detected only in testes. Embryonic expression was detected from 10.5-day postcoitum embryos and was seen as a diffuse staining pattern over much of the embryo, consistent with the housekeeping gene function of alpha-enolase. This mutation results in an early recessive embryonic lethality. Mice heterozygous for the mutation have no obvious phenotype. Mutations of this gene in humans are reported to be associated with rare autosomal-dominant, non-spherocytic haemolytic anaemia. This phenotype is not reproduced in mice heterozygous for this mutation. PMID:9626503

Couldrey, C; Carlton, M B; Ferrier, J; Colledge, W H; Evans, M J

1998-06-01

44

Reporter genes for embryogenesis research in livestock species.  

PubMed

Currently, our knowledge of early mammalian embryogenesis, stem cell differentiation and development is largely based on studies performed in mouse models. However, in important aspects, e.g. the timing of epigenetic reprogramming and embryonic genome activation, livestock species probably reflect far more closely the situation in men and other non-rodent mammals. A major challenge is the fact that in mammals, the development of individual zygotes is highly variable and vulnerable, and the outcome is uncertain. Valid indicators of the highly heterogeneous development and health status, and the actual developmental potential of individual oocytes, zygotes or embryos would be crucially important to tap the full power of holistic transcriptome and proteome analyses. Fluorescent reporter proteins opened new vistas for embryology and stem cell research: they can be used as reporters for the activity of gene promoters or tagged to functional proteins to study their intracellular localization in living cells, tissues and organisms. Fluorescent reporter genes may be used to microscopically observe key processes of early development. Thus, novel information related to developmental potential can be obtained from living embryos before processing them, e.g. for "-omic" studies. This review summarizes the main current reporter gene techniques and gene transfer approaches, which might be suitable for the investigation of early embryogenesis in livestock mammals. The potential of promoter reporter genes is exemplified by a bovine model system for quantitative monitoring of transcriptional reactivation of the so-called pluripotency gene POU5F1 in cloned bovine embryos. PMID:17583783

Habermann, F A; Wuensch, A; Sinowatz, F; Wolf, E

2007-09-01

45

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

SciTech Connect

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

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

2005-01-05

46

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

PubMed

Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation. PMID:24452856

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

2014-06-01

47

Use of Reporter Genes to Study the Activity of Promoters in Ovarian Granulosa Cells  

E-print Network

and b-galactosidase genes as reporters and a cationic liposome mediated cell transfection method genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation of reporter genes and cell-based transfection assays in endocrine research. Key words: Reporter genes

Mayo, Kelly E.

48

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

Microsoft Academic Search

A system for the in vivo analysis of fungal transcription control sequences, based on a mini-promoter, was designed. The mini-promoter, providing all sequences necessary and sufficient for transcription initiation, was derived from the Aspergillus nidulans gpdA promoter region. Transcription initiation was not affected by the introduction of transcription control sequences directly upstream from the mini-promoter. Furthermore, the expression of the

Peter J. Punt; Anneke Kuyvenhoven; Cees A. M. J. J. van den Hondel

1995-01-01

49

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects  

Microsoft Academic Search

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in

Pritha Ray; Roger Tsien; Sanjiv Sam Gambhir

2007-01-01

50

Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis.  

PubMed Central

We have used Tn917lacZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsI. An additional transposon insert that maps in the hag locus was obtained. The pattern of beta-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtilis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response. Images PMID:2174860

Zuberi, A R; Ying, C W; Parker, H M; Ordal, G W

1990-01-01

51

Dual-modality gene reporter for in vivo imaging.  

PubMed

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, Gd(3+)-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 Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging. PMID:24347640

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

52

Dual-modality gene reporter for in vivo imaging  

PubMed Central

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

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

53

Transformation of Nonselectable Reporter Genes in Marine Diatoms  

Microsoft Academic Search

:   We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the\\u000a exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations.

Angela Falciatore; Raffaella Casotti; Catherine Leblanc; Chiara Abrescia; Chris Bowler

1999-01-01

54

Photoacoustic imaging of gene expression using tyrosinase as a reporter gene  

NASA Astrophysics Data System (ADS)

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 ?m (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.

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

2011-03-01

55

Genetics in methylotrophic bacteria: Appendix. Final report  

SciTech Connect

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

Lidstrom, M.E.

1998-09-01

56

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

Microsoft Academic Search

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

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

2006-01-01

57

Application of lambda Red recombination system to Vibrio cholerae genetics: simple methods for inactivation and modification of chromosomal genes.  

PubMed

The lambda Red-based recombination system is very useful for genetic manipulation of some Gram-negative bacteria. Here we report simple procedures for the inactivation and modification of genes of interest on Vibrio cholerae chromosome using this recombination technique. For this purpose, a polymerase chain reaction (PCR) fragment carrying an antibiotic resistance cassette flanked by regions homologous to the target locus was electroporated into recipient V. cholerae strains expressing a highly proficient lambda Red recombination system. Two PCR procedures were tested to generate an amplification product carrying an antibiotic resistance cassette flanked by short (50 or 100 nt) or long (1000 nt) homologous extensions, which allowed successful disruption of four chromosomal loci (ctxB, toxT, lacZ, and recA). Our results suggest that 100-nt homology between the PCR product and the target gene is sufficient to stimulate the lambda Red-dependent recombination. To increase recombination efficiency, however, the PCR procedure should be used to generate a product with 1000-nt homologous extensions. Furthermore, we applied this gene replacement method to create lacZ reporter fusion to the target gene. Transcriptional fusion to the V. cholerae ctxA gene was constructed using a PCR product that contains the 100-nt homologous extension to ctxA on each side of the lacZ::cat cassette, and was shown to respond appropriately to a null mutation in the regulatory gene, toxT. Use of the techniques presented here should prompt rapid and efficient mutagenesis/modification of V. cholerae chromosomal genes. PMID:19268696

Yamamoto, Shouji; Izumiya, Hidemasa; Morita, Masatomo; Arakawa, Eiji; Watanabe, Haruo

2009-06-01

58

DIMACS Technical Report 200514 Inapproximability Results for the Lateral Gene Transfer  

E-print Network

losses and lateral gene transfers (a.k.a. horizontal gene transfers) that may occur during evolutionDIMACS Technical Report 2005­14 May 2005 Inapproximability Results for the Lateral Gene Transfer the most parsimonious lateral gene transfer scenario for a given pair of gene and species trees. We

59

Ferritin as a Novel Reporter Gene for Photoacoustic Molecular Imaging  

PubMed Central

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 (850–950 nm). PA signal intensity from H-FT transfected SK-MEL-24 cells was about 5–9 dB higher than nontransfected SK-MEL-24 cells at 850–950 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

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

2013-01-01

60

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

PubMed

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii . PMID:15604722

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

2004-08-01

61

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

DOEpatents

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

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

2009-04-28

62

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

DOEpatents

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.

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

2011-06-07

63

Imaging Tri-Fusion Multimodality Reporter Gene Expression in Living Subjects  

Microsoft Academic Search

Imaging reporter gene expression in living subjects with various imag- ing modalities is a rapidly accelerating area of research. Applications of these technologies to cancer research, gene therapy, and transgenic mod- els are rapidly expanding. We report construction and testing of several triple fusion reporter genes compatible with bioluminescence, fluores- cence and positron emission tomography (PET) imaging. A triple fusion

Pritha Ray; Abhijit De; Jung-Jun Min; Roger Y. Tsien; Sanjiv S. Gambhir

2004-01-01

64

BCS1L gene mutation causing GRACILE syndrome: case report.  

PubMed

GRACILE syndrome is a rare autosomal recessive disease characterized by fetal growth retardation, Fanconi type aminoaciduria, cholestasis, iron overload, profound lactic acidosis, and early death. It is caused by homozygosity for a missense mutation in the BCS1L gene. The BCS1L gene encodes a chaperone responsible for assembly of respiratory chain complex III. Here we report that a homozygous mutation c.296C > T (p.P99L), in the first exon of BCS1L gene found in an affected 2-month-old boy of asymptomatic consanguineous parents results in GRACILE syndrome. This genotype is associated with a severe clinical presentation. So far no available treatments have changed the fatal course of the disease, and the metabolic disturbance responsible is still not clearly identified. Therefore, providing prenatal diagnosis in families with previous affected infants is of major importance. Mitochondrial disorders are an extremely heterogeneous group of diseases sharing, in common, the fact that they all ultimately impair the function of the mitochondrial respiratory chain. A clinical picture with fetal growth restriction, postnatal lactacidosis, aminoaciduria, hypoglycemia, coagulopathy, elevated liver enzymes, and cholestasis should direct investigations on mitochondrial disorder. PMID:24655110

Kasapkara, Çi?dem Seher; Tümer, Leyla; Ezgü, Fatih Suheyl; Küçükçongar, Aynur; Hasano?lu, Alev

2014-07-01

65

Effect of the Steroid K-canrenoate on hsp70 Expression and Tissue Damage in Transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9  

PubMed Central

In the present study the effect of 0.1, 0.2, 0.4, 0.8, and 1.0 µL/mL of the steroid K-canrenoate was evaluated in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg9 for 6, 24, and 48 hours of duration. The treatment of 0.1, 0.2, and 0.4 µL/mL of K-canrenoate did not induce the activity of hsp70 significantly compared to the control. The treatments of 0.8 and 1.0 µL/mL of K-canrenoate not only caused tissue damage but also induced a significant increase in the expression of hsp70 for the different durations of exposure. The results of the present study suggest that the K-canrenoate at 0.8 and 1.0 µL/mL is cytotoxic and caused tissue damage in the third instar larvae of transgenic D. melanogaster (hsp70-lacZ) Bg9. PMID:23427921

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

2012-01-01

66

The Iroquois Homeobox Gene Irx2 Is Not Essential for Normal Development of the Heart and Midbrain-Hindbrain Boundary in Mice  

PubMed Central

The Iroquois homeobox (Irx) genes have been implicated in the specification and patterning of several organs in Drosophila and several vertebrate species. Misexpression studies of chick, Xenopus, and zebra fish embryos have demonstrated that Irx genes are involved in the specification of the midbrain-hindbrain boundary. All six murine Irx genes are expressed in the developing heart, suggesting that they might possess distinct functions during heart development, and a role for Irx4 in normal heart development has been recently demonstrated by gene-targeting experiments. Here we describe the generation and phenotypic analysis of an Irx2-deficient mouse strain. By targeted insertion of a lacZ reporter gene into the Irx2 locus, we show that lacZ expression reproduces most of the endogenous Irx2 expression pattern. Despite the dynamic expression of Irx2 in the developing heart, nervous system, and other organs, Irx2-deficient mice are viable, are fertile, and appear to be normal. Although chick Irx2 has been implicated in the development of the midbrain-hindbrain region, we show that Irx2-deficient mice develop a normal midbrain-hindbrain boundary. Furthermore, Irx2-deficient mice have normal cardiac morphology and function. Functional compensation by other Irx genes might account for the absence of a phenotype in Irx2-deficient mice. Further studies of mutant mice of other Irx genes as well as compound mutant mice will be necessary to uncover the functional roles of these evolutionarily conserved transcriptional regulators in development and disease. PMID:14585979

Lebel, Melanie; Agarwal, Pooja; Cheng, Chi Wa; Kabir, M. Golam; Chan, Toby Y.; Thanabalasingham, Vijitha; Zhang, Xiaoyun; Cohen, Dana R.; Husain, Mansoor; Cheng, Shuk Han; Bruneau, Benoit G.; Hui, Chi-Chung

2003-01-01

67

Role of GCR2 in transcriptional activation of yeast glycolytic genes.  

PubMed Central

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together. PMID:1508187

Uemura, H; Jigami, Y

1992-01-01

68

Direct Detection and Quantification of Horizontal Gene Transfer by Using Flow Cytometry and gfp as a Reporter Gene  

Microsoft Academic Search

A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene ( gfp) encoding green fluorescent protein. A chromosomal encoded repressor ( lacI q1)

Sřren J. Sřrensen; Anders H. Sřrensen; Lars H. Hansen; Gunnar Oregaard; Duncan Veal

2003-01-01

69

Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector.  

PubMed

A 9.0-kb fragment of the tyrosine hydroxylase (TH) promoter, previously shown to direct tissue-specific expression in transgenic mice, was fused to an Escherichia coli LacZ reporter gene in a defective herpes simplex virus type-1 (HSV-1) amplicon vector (THlac). The HSV immediate early (IE) 4/5 promoter (HSVlac) was used as a control. LacZ gene expression was visualized by X-Gal histochemical and TH immunocytochemical analysis. Two days and 10 weeks after THlac injection into rat caudate nucleus (CN), X-Gal-stained cells were observed in the substantia nigra (SN) and locus ceruleus (LC) ipsilateral to the injection site. These blue cells were TH-positive neurons as evidenced by double labeling with immunocytochemistry. Moreover, the number of X-Gal+, TH+ (double-positive) neurons in the SN increased at 10 weeks as compared to that seen 2 days after THlac injection. In marked contrast, few double-positive nigral neurons were observed either 2 days or 10 weeks after direct injection of THlac into SN. However, neither nigral nor striatal injection of HSVlac resulted in prolonged gene expression. These results suggest that a neuronal, but not a viral, promoter in an HSV vector can produce cell-type-specific, prolonged, and stable gene expression following retrograde transport. In addition, THlac produced infrequent gene expression in TH-negative cells (CN and dorsal to SN) after THlac injection into CN and SN, respectively. Overall, these results suggest that in some in vivo contexts cell-type-preferred expression can be achieved by a cellular promoter in an amplicon vector. Moreover, they underscore the need for the careful and systematic study of neuronal promoters in HSV vectors. PMID:8930662

Jin, B K; Belloni, M; Conti, B; Federoff, H J; Starr, R; Son, J H; Baker, H; Joh, T H

1996-10-20

70

Photoacoustic microscopy of tyrosinase reporter gene in vivo  

PubMed Central

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. PMID:21895303

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

2011-01-01

71

Photoacoustic microscopy of tyrosinase reporter gene in vivo  

NASA Astrophysics Data System (ADS)

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.

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

2011-08-01

72

Rapid Engineering of Bacterial Reporter Gene Fusions by Using Red Recombination? †  

PubMed Central

Reporter gene fusions are essential tools for the investigation of gene regulation. Such fusions are traditionally generated by transposon mutagenesis and identified by a suitable selection procedure. Alternatively, specific reporter fusions can be generated by cloning of DNA fragments containing promoters or other regulatory elements in reporter plasmids. Here, we describe a novel approach for the rapid generation of reporter gene fusions in single copies at defined positions in bacterial genomes. This technique utilizes the Red recombinase for the homologous recombination of PCR-generated cassettes containing various currently used reporter genes, such as those for ?-galactosidase, luciferase, and green fluorescent protein. The approach allows the generation of transcriptional or translational reporter fusions in a single step without the requirement for recombinant DNA constructs and is applicable to various enterobacterial species. Generation of reporter fusions by Red recombination is rapid, overcomes the current limitations of transposon mutagenesis or reporter plasmids, and offers new options for the study of bacterial gene regulation. PMID:17513596

Gerlach, Roman G.; Holzer, Stefanie U.; Jackel, Daniela; Hensel, Michael

2007-01-01

73

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

PubMed

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

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

2013-12-19

74

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

SciTech Connect

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.

Fields, C.A.

1994-09-01

75

A retroviral gene trap insertion into the histone 3.3A gene causes partial neonatal lethality, stunted growth, neuromuscular deficits and male sub-fertility in transgenic mice.  

PubMed

Spermatogenesis is a complex developmental pro-cess involving cell division and differentiation. Approximately half of all sterile males have defects in spermatogenesis or sperm function. An insight into the molecular control points regulating this process might help in treating male infertility. Gene trapping in embryonic stem cells and the generation of transgenic mice represents one route to identify genes expressed during spermatogenesis. The trapped gene is tagged with a lacZ reporter gene so that the expression pattern of the gene can be visualized by staining for beta-galactosidase activity. We have screened transgenic mouse lines for expression of trapped genes in the gonads. One such trap event was shown to be in the replacement histone 3.3A gene ( H3.3A ). This gene was expressed ubiquitously during embryonic development until 13.5 days post-coitum and in the adult heart, kidney, brain, testes and ovaries. This mutation resulted in postnatal death of 50% of homozygous mutants. Surviving mutants displayed reduced growth rates when competing with wild-type siblings for food. Mutant mice also had a neuro-muscular deficit and males displayed reduced copulatory activity. When copulations did occur, these resulted in very few pregnancies, suggesting that mutations in the H3.3A gene may contribute to some cases of impaired fertility in man. PMID:10556297

Couldrey, C; Carlton, M B; Nolan, P M; Colledge, W H; Evans, M J

1999-12-01

76

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

NASA Astrophysics Data System (ADS)

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.

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

2010-11-01

77

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

PubMed Central

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported here aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. We screened a collection of 97 recessive lethal P-lacZ enhancer trap lines for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, we identified six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration. PMID:18036784

McClure, Kimberly D.; Schubiger, Gerold

2008-01-01

78

An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene  

Microsoft Academic Search

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in

Danielle Janus; Birgit Hoff; Eckhard Hofmann; Ulrich Kuck

2007-01-01

79

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

PubMed Central

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 ?-galactosidase (?-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 ?-Gal expression. PMID:9566926

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

1998-01-01

80

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

PubMed Central

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

2012-01-01

81

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

Microsoft Academic Search

The expression of the ice nucleation gene inaZ from Pseudomonas syringae in Zymomonas mobilis strains under the control of three different promoters was investigated to establish the utility of the gene as a reporter and examine the possible use of the organism as a source of ice nuclei for biotechnological applications. A promoterlessversionoftheinaZgenewasplacedunderthecontrolofthreedifferentpromoters:Ppdc(pyruvate decarboxylase), a homologous strong promoter from Z.

CONSTANTIN DRAINAS; GEORGIOS VARTHOLOMATOS; ANDNICHOLAS J. PANOPOULOS

1995-01-01

82

Optical imaging of Renilla luciferase reporter gene expression in living mice  

Microsoft Academic Search

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

S. Bhaumik; S. S. Gambhir

2002-01-01

83

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

PubMed Central

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

Komorowski, Micha?; Finkenstädt, Bärbel; Rand, David

2010-01-01

84

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

PubMed Central

Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly, or through the use of targeted contrast media. However many genes and proteins, and particularly those located in intracellular compartments, are not accessible for targeted agents. The transcriptional regulation of these genes can never the less be detected, though indirectly, through the use of reporter genes encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent or bioluminescent proteins, or the reaction product of expressed enzymes and exogenous substrates, are examined using in vitro histological methods, or in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raise the possibility that MRI could become a powerful tool for concomitant high resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods is for monitoring gene expression patterns in gene therapy and for in vivo imaging of the survival, proliferation, migration, and differentiation of pluripotent or multipotent cells used in cell based regenerative therapies for cancer, myocardial infarction, and neural degeneration. In this review, we characterize the variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, cell migration, and cell differentiation. In particular, we discuss which methods are best suited for translation to clinical use in regenerative therapies. PMID:23225197

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

2013-01-01

85

Visualization of ecdysteroid activity using a reporter gene in the crustacean, Daphnia.  

PubMed

Ecdysone is a hormone known to play a pivotal role in crustaceans and insects. In order to evaluate the ecdysone activities in the environment and within the organism, we have developed a biomonitoring Daphnia strain by introducing a reporter gene. In this study, the ecdysone response element was inserted in the upstream region of a reporter gene, and the DNA construct was injected into Daphnia eggs. The expression of the reporter gene was detected during the early embryonic development stage. In addition, when the eggs expressing the reporter gene were exposed to ecdysone, there was enhanced expression of the reporter gene at detectable levels, while the presence of an antagonist led to its downregulation. These results suggested that this system could be potentially developed for monitoring ecdysone activities in media. PMID:24296240

Asada, Miki; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

2014-02-01

86

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

PubMed

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

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

2012-11-01

87

246. Efficient Gene Transfer to Peripheral Nervous System Via Systemic Delivery of AAV Vectors  

Microsoft Academic Search

Gene transfer to the peripheral nervous system has great therapeutic potential for the treatment of many neurological diseases, such as spinal muscular atrophy, amyotrophic lateral sclerosis, hereditary axonal motor neuropathies, and so on. Here we describe evidence that AAV vectors can be efficiently delivered to peripheral nervous system by a single intraperitoneal injection into neonatal mice. We delivered AAV 8-LacZ

Chunping Qiao; Jianbin Li; Tong Zhu; Bing Wang; Xiao Xiao

2006-01-01

88

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

PubMed Central

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

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

2000-01-01

89

Mapping and regulation of the cel genes in Erwinia chrysanthemi  

Microsoft Academic Search

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

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

1988-01-01

90

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

PubMed

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

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

2014-10-01

91

Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.  

PubMed Central

A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence differs from those of IFN-alpha genes A-L at approximately equal to 10% of the positions and is most similar to IFN-alpha C, -alpha F, and -alpha H. IFN-alpha WA has been found to encode amino acids that differ from those conserved at each of five positions in all previously reported IFN-alpha species. The IFN-alpha WA gene codes for an active interferon, which has been expressed in Escherichia coli using an M13-lacZ fusion as an expression vector. About 5 X 10(6) units of IFN-alpha WA were obtained per liter of bacterial culture. The described screening procedure using short probes should permit the isolation of genes for which sequence information is available from animal or plant genomic libraries. Images PMID:6387705

Torczynski, R M; Fuke, M; Bollon, A P

1984-01-01

92

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

PubMed Central

Background Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. Methods Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). Results Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. Conclusions We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer. PMID:23937994

2013-01-01

93

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

PubMed

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

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

2013-05-17

94

Simultaneous deletion of floxed genes mediated by CaMKII?-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Go?  

PubMed Central

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKII?) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKII?-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26+/stop-lacZ::CaMKII?-Cre+/Cre mice generated by crossing CaMKII?-Cre+/Cre mice with floxed ROSA26 lacZ reporter (Rosa26+/stop-lacZ) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26+/stop-lacZ::CaMKII?-Cre+/Cre mice. Similarly, when double transgenic Gnao+/f::CaMKII?-Cre+/Cre mice carrying a floxed Go-alpha gene (Gnaof/f) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao?) without inheriting the Cre transgene. The Gnao?/? mice closely resembled conventional Go-alpha knockout mice (Gnao?/?) with respect to impairment of their behavior. Thus, we conclude that CaMKII?-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKII?-Cre mice as breeding pairs. PMID:24787734

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

2014-01-01

95

Mutation in a reporter gene depends on proximity to and transcription of immunoglobulin variable transgenes.  

PubMed Central

Somatic mutation in immunoglobulin genes is localized to a 2-kilobase region of DNA surrounding and including rearranged variable (V), diversity, and joining (J) gene segments encoding heavy and light chains. To examine the structural basis for targeted mutation, we developed an assay to score mutation on plasmid substrates by using a reporter gene: a bacterial gene encoding an amber-suppressor tRNA molecule was placed 3' of a rearranged kappa VJ gene within the boundaries of mutation. The reporter gene is exquisitely suited for mutational analysis because it is only 200 base pairs (bp), which should not greatly disrupt structure of the immunoglobulin locus, and gene function depends on secondary structure, which means mutation can be scored in many different nucleotide positions. The plasmid was used to make transgenic mice, which were then immunized. The shuttle vector was retrieved by plasmid rescue into an indicator strain of Escherichia coli that contained an amber mutation in its beta-galactosidase gene. Integrity of the tRNA molecule was monitored by colony color, which permitted many transformants to be screened visually. Mutations were not seen in DNA from a transfected B-cell line grown in vitro or in DNA from nonlymphoid tissue of transgenic mice, indicating that the reporter gene was stable during cell division and DNA manipulations. However, when the transgenic mice were immunized, DNA from splenic B cells contained point mutations in the reporter gene at a frequency of 10(-3) per transformant. Sequence analysis of 17 mutated transgenes revealed that the mutations were 1- and 2-bp deletions in the tRNA gene, and one plasmid had an additional 2-bp deletion in the V gene. In contrast, previous studies have shown that mutations in endogenous VJ genes are predominantly nucleotide substitutions and have only 6% deletions. Two other plasmid constructs were analyzed in transgenic lines: no mutations were found when the tRNA gene was placed distal to the VJ gene, and no mutations were seen when the immunoglobulin promoter was deleted. Although we lack direct evidence that the deletions in the tRNA gene are caused by the same mechanism that acts on VJ genes, we have shown that mutations in this assay occur in a manner consistent with immunoglobulin-specific mutation in that they are found in splenic B cells and not in tail tissue, depend on position next to the VJ gene, and require transcription of the VJ gene. Images PMID:1905016

Umar, A; Schweitzer, P A; Levy, N S; Gearhart, J D; Gearhart, P J

1991-01-01

96

Viral gene delivery of superoxide dismutase attenuates experimental cholestasis-induced liver fibrosis in the rat  

Microsoft Academic Search

Hydrophobic bile acids lead to generation of oxygen free radicals in mitochondria. Accordingly, this study investigated if gene delivery of superoxide dismutase (SOD) would reduce hepatic injury caused by experimental cholestasis. Rats were given adenovirus (Ad; 3 × 109 p.f.u., i.v.) carrying the bacterial control gene lacZ, mitochondrial Mn-SOD or cytosolic Cu\\/Zn-SOD genes 3 days before bile duct ligation. Both

Z Zhong; M Froh; O Smutney; TG Lehmann; RG Thurman

2002-01-01

97

The Wilms’ tumor gene WT1 is a common marker of progenitor cells in fetal liver  

Microsoft Academic Search

It is well known that the Wilms’ tumor gene WT1 plays an important role in cell proliferation and differentiation, and in organ development. In this study, to examine the role of the WT1 gene in lineage determination, fetal liver cells from LacZ-transgenic mice, in which WT1 expression was marked by the expression of the LacZ gene driven by WT1 promoter,

Keisuke Kanato; Naoki Hosen; Masashi Yanagihara; Naomi Nakagata; Toshiaki Shirakata; Tsutomu Nakazawa; Sumiyuki Nishida; Akihiro Tsuboi; Manabu Kawakami; Tomoki Masuda; Yoshihiro Oka; Yusuke Oji; Annemieke IJpenberg; Nicholas D. Hastie; Haruo Sugiyama

2005-01-01

98

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

PubMed Central

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

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

2013-01-01

99

Use of URA3 as a reporter of gene expression in C. albicans  

Microsoft Academic Search

The C. albicans URA3 gene was tested as a reporter of gene expression. An integrating vector was constructed which contained ADE2 as a selectable marker together with a truncated form of URA3 lacking the first three codons. A DNA fragment containing the promoter and the first 90 codons of the C. albicans CEF3 gene was inserted into the unique XhoI

Kristi K. Myers; Paul S. Sypherd; William A. Fonzi

1995-01-01

100

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

Microsoft Academic Search

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

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

1997-01-01

101

[CANCER RESEARCH 64, 13231330, February 15, 2004] Imaging Tri-Fusion Multimodality Reporter Gene Expression in Living Subjects  

E-print Network

to cancer research, gene therapy, and transgenic mod- els are rapidly expanding. We report construction to living animals with the help of a single tri-fusion reporter gene will have the potential to accelerate events, it is desirable to image reporter gene expression in individual cells, living animals, and humans

Tsien, Roger Y.

102

Bioluminescent reporters for catabolic gene expression and pollutant bioavailability  

SciTech Connect

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.

Heitzer, A.; DiGrazia, P.M.; Sayler, G.S. (Tennessee Univ., Knoxville, TN (United States). Center for Environmental Biotechnology); Burlage, R.S. (Oak Ridge National Lab., TN (United States))

1991-01-01

103

Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression.  

PubMed

To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression. PMID:22484615

Orrů, Beatrice; Cunniffe, Ciaran; Ryan, Fergus; Schwartz, Stefan

2012-08-01

104

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

SciTech Connect

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

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

1993-01-01

105

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

SciTech Connect

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

Grant, S. R.

1999-02-05

106

MAMMALIAN CELL GENE MUTATION ASSAYS WORKING GROUP REPORT  

EPA Science Inventory

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

107

Recombination can lead to spurious results in retroviral transduction with dually fluorescent reporter genes.  

PubMed

Fluorescent proteins are routinely employed as reporters in retroviral vectors. Here, we demonstrate that transduction with retroviral vectors carrying a tandem-dimer Tomato (TdTom) reporter produces two distinct fluorescent cell populations following template jumping due to a single nucleotide polymorphism between the first and second Tomato genes. Template jumping also occurs between repeated sequences in the Tomato and green fluorescent protein (GFP) genes. Thus, proper interpretation of the fluorescence intensity of transduced cells requires caution. PMID:24067983

Salamango, Daniel J; Evans, David A; Baluyot, Mariju F; Furlong, Jackson N; Johnson, Marc C

2013-12-01

108

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

PubMed

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

Wang, Baomin; Wang, Yumei; Kennedy, Christina

2014-10-01

109

A Novel Binary T-Vector with the GFP Reporter Gene for Promoter Characterization  

PubMed Central

Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens. PMID:25197968

Jiang, Shu-Ye; Vanitha, Jeevanandam; Bai, Yanan; Ramachandran, Srinivasan

2014-01-01

110

A novel binary T-vector with the GFP reporter gene for promoter characterization.  

PubMed

Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens. PMID:25197968

Jiang, Shu-Ye; Vanitha, Jeevanandam; Bai, Yanan; Ramachandran, Srinivasan

2014-01-01

111

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

SciTech Connect

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.

Matin, A. [Stanford Univ., CA (United States). Dept. of Microbiology and Immunology; Schmidt, T. [Michigan State Univ., East Lansing, MI (United States). Dept. of Microbiology; Caldwell, D. [Univ. of Saskatchewan, Saskatoon, Saskatchewan (Canada). Dept. of Microbiology

1998-11-01

112

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

SciTech Connect

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

Kuchka, M.R.

1992-05-01

113

A 4.2 kb upstream region of the human corneodesmosin gene directs site-specific expression in hair follicles and hyperkeratotic epidermis of transgenic mice.  

PubMed

Corneodesmosin (CDSN) is a desmosomal protein expressed in the epidermis during the late stages of differentiation and in the inner root sheath of hair follicles. The homophilic adhesive properties of the protein suggest that it reinforces keratinocyte cohesion in the upper layers of the epidermis (stratum granulosum and stratum corneum). In this study, we analyzed the expression of the CDSN gene in 16 human tissues. We confirmed the closely restricted expression pattern of CSDN. Indeed, apart from the skin, the mRNA was significantly detected only in the placenta and the thymus. As a step in elucidating the mechanisms of tissue-specific expression, transgenic mice bearing a 4.2 kb fragment of the human CSDN gene promoter linked to the LacZ gene were generated. The reporter-gene expression was detected in special areas of the inner root sheath of the hair follicles and the hair medulla but not in the epidermis. Induction of epidermis hyperproliferation however either by pharmacological agents or by wounding led to strong expression of the reporter gene in the keratinocytes of the stratum granulosum and the parakeratotic corneocytes of the stratum corneum. The data suggest that the genomic sequences and/or regulating factors responsible for the cell-specific expression of the human CDSN gene in the normal hair follicle as well as in the hyperproliferative epidermis are different from those necessary for expression in the normal epidermis. PMID:15086560

Gallinaro, Hélčne; Jonca, Nathalie; Langbein, Lutz; Vincent, Christian; Simon, Michel; Serre, Guy; Guerrin, Marina

2004-03-01

114

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

SciTech Connect

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

Haber, J. E.

2002-11-30

115

Gene therapy for trigeminal pain in mice  

PubMed Central

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

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

2014-01-01

116

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

PubMed

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

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

2013-05-01

117

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

PubMed

?-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. ?-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-?-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 ?-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 ?-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of ?-galactosidase played an important role in lac operon evolution. PMID:23486479

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

2013-05-01

118

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

PubMed Central

?-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. ?-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-?-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 ?-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 ?-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of ?-galactosidase played an important role in lac operon evolution. PMID:23486479

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

2013-01-01

119

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

PubMed Central

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

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

2013-01-01

120

Reconstruction of transcriptional dynamics from gene reporter data using differential equations  

E-print Network

¨arbel Finkenst¨adt 1, Elizabeth A. Heron 1,2,Michal Komorowski 1,2, Kieron Edwards 3,Sanyi Tang 2,Claire V of Manchester. ABSTRACT Motivation: Promoter driven reporter genes, notably luciferase (luc) and green.F.Finkenstadt@Warwick.ac.uk INTRODUCTION Imaging data from luciferase (LUC) and green fluorescent protein (GFP) reporters combined

Rand, David

121

Human Protamine-1 as an MRI Reporter Gene Based on Chemical Exchange  

PubMed Central

Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acid-long hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells. PMID:24138139

Bar-Shir, Amnon; Liu, Guanshu; Chan, Kannie W.Y.; Oskolkov, Nikita; Song, Xiaolei; Yadav, Nirbhay N.; Walczak, Piotr; McMahon, Michael T.; van Zijl, Peter C. M.; Bulte, Jeff W. M.; Gilad, Assaf A.

2014-01-01

122

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

PubMed

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

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

2003-05-01

123

Analysis of the Schwanniomyces occidentalis SWA2 gene promoter in Saccharomyces cerevisiae  

Microsoft Academic Search

The effect of different carbon sources on the expression in Saccharomyces cerevisiae of the SWA2 ?-amylase gene from Schwanniomyces occidentalis was studied from constructs containing its 5? region (?223 to +15), which were fused in-frame to the lacZ gene coding sequence. Maximal expression was achieved with the non-fermentable substrates ethanol and\\/or glycerol, whereas lower levels were found with maltose or

T. A Carmona; A Jiménez; M Fernández Lobato

2002-01-01

124

Organization and transcriptional regulation of Drosophila Na(+), K(+)-ATPase beta subunit genes: Nrv1 and Nrv2.  

PubMed

Drosophila melanogaster has two Na(+),K(+)-ATPase beta subunit genes (Nervana 1 and 2; Nrv), with tissue-specific expression patterns. Nrv1 produces a single beta subunit isoform expressed primarily in muscle tissue, whereas Nrv2 codes for two different isoforms (2.1 and 2.2) expressed in the nervous system. We have determined the complete molecular genomic organization for both Nrv genes. Only 3kb of DNA separate the 3' end of Nrv2 from Nrv1. The cDNAs from all three forms of Nrv have been mapped onto the genomic structure and all intron-exon junctions have been confirmed by direct sequencing. The genomic DNA positioned in the 5' flanking region of each Nrv gene has also been tested for tissue-specific transcriptional regulatory activity. P-element transformation vectors were constructed, which contained either 7.7kb of Nrv2 or 3.5kb Nrv1 5' flanking DNA driving expression of a lacZ reporter gene. Multiple transgenic Drosophila lines were established for each construct and analyzed for their beta-galactosidase expression pattern. The tissue-specific expression of each Nrv gene is independently regulated by the cis-element(s) present in the 5' flanking region. The Nrv2 5' flanking DNA directs expression exclusively to the nervous system, whereas Nrv1 5' flanking DNA directs expression primarily in muscle tissue. PMID:10452950

Xu, P; Sun, B; Salvaterra, P M

1999-08-20

125

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging.  

PubMed

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells. PMID:19116247

Wang, Fangjing; Dennis, James E; Awadallah, Amad; Solchaga, Luis A; Molter, Joseph; Kuang, Yu; Salem, Nicolas; Lin, Yuan; Tian, Haibin; Kolthammer, Jeffery A; Kim, Yunhui; Love, Zachary B; Gerson, Stanton L; Lee, Zhenghong

2009-03-01

126

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

SciTech Connect

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

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

1991-04-01

127

Genes  

NSDL National Science Digital Library

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

Excellence, Access

2005-03-12

128

Copyright0 1987 by the GeneticsSocietyof America A Tn10-lacZ-kanR-URA3Gene Fusion Transposon for Insertion  

E-print Network

Copyright0 1987 by the GeneticsSocietyof America A Tn10-lacZ-kanR-URA3Gene Fusion Transposon or translation start signals,an intact URA3 gene, and a RanR determinant. The lacZ gene can be activated be substituted for URA3 in the availableconstructs. The mini-Tn 10-LUKsystem has several important advantages. (1

Botstein, David

129

Phosphatidylglycerolphosphate synthase encoded by the PEL1\\/PGS1 gene in Saccharomyces cerevisiae is localized in mitochondria and its expression is regulated by phospholipid precursors  

Microsoft Academic Search

The PEL1\\/PGS1 gene of the yeast Saccharomyces cerevisiae is essential for the viability of rho\\u000a –\\/rho° mutants and the normal cardiolipin content of cells. The PEL1-GFP fusion gene has been found to complement the pel1\\/pgs1 mutation and its fluorescent protein was localized to mitochondria similarly to the ?-galactosidase activity of a protein encoded by the PEL1-lacZ fusion gene. The expression

Vladimíra Džugasová; Margita Obernauerová; Katarína Horváthová; Mariana Vachová; Martina Žáková; Július Šubík

1998-01-01

130

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

PubMed Central

The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP+ neurons, while the frequency of mEPSCs was not significantly different. Increasing neural activity by applying a GABAA receptor antagonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity. PMID:22408605

Delgado, Jary Y.; Owens, Geoffrey C.

2012-01-01

131

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

PubMed Central

BACKGROUND/AIMS—Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.?METHODS—An E1/E3 deleted recombinant adenovirus (denoted AdCMV?Gal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. ?-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.?RESULTS—Intravenous or intraperitoneal injection of AdCMV?Gal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMV?Gal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMV?Gal caused a higher ?-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with standard AdCMV?Gal vectors.?CONCLUSIONS—Local administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation.???Keywords: adenovirus; gene transfer; colitis; colon PMID:10323880

Wirtz, S; Galle, P; Neurath, M

1999-01-01

132

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

PubMed Central

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

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

1995-01-01

133

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

PubMed Central

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

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

2006-01-01

134

Regulation of photosynthesis genes in Rubrivivax gelatinosus: transcription factor PpsR is involved in both negative and positive control.  

PubMed

Induction of biosynthesis of the photosystem in anoxygenic photosynthetic bacteria occurs when the oxygen concentration drops. Control of this induction takes place primarily at the transcriptional level, with photosynthesis genes expressed preferentially under anaerobic conditions. Here, we report analysis of the transcriptional control of two photosynthesis promoters, pucBA and crtI, by the PpsR factor in Rubrivivax gelatinosus. This was accomplished by analyzing the photosystem production in the wild type and in the PPSRK (ppsR::Km) mutant grown under anaerobic and semiaerobic conditions and by assessing the beta-galactosidase activity of lacZ transcriptionally fused to promoters possessing the putative PpsR-binding consensus sequences. It was found that under semiaerobic conditions, inactivation of the ppsR gene resulted in overproduction of carotenoid and bacteriochlorophyll pigments, while the production of LH2 was drastically reduced. The beta-galactosidase activity showed that, in contrast to what has been found previously for Rhodobacter species, PpsR acts in R. gelatinosus as an aerobic repressor of the crtI gene while it acts as an activator for the expression of pucBA. Inspection of the putative PpsR-binding consensus sequences revealed significant differences that may explain the different levels of expression of the two genes studied. PMID:15126475

Steunou, Anne-Soisig; Astier, Chantal; Ouchane, Soufian

2004-05-01

135

Toxin gene expression by shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response.  

PubMed Central

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosome-based stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease. PMID:10998375

Kimmitt, P. T.; Harwood, C. R.; Barer, M. R.

2000-01-01

136

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

Microsoft Academic Search

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

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

2007-01-01

137

Yeast Sequencing Report A 38 kb segment containing the cdc2 gene from the  

E-print Network

Yeast Sequencing Report A 38 kb segment containing the cdc2 gene from the left arm of ®ssion yeast-8566, Japan. E-mail: machida@nibh.go.jp YEAST Yeast 2000; 16: 71±80. Received 3 May 1999 Accepted 18 September

138

Brief Genetics Report Variation in the Calpain-10 Gene Affects Blood Glucose  

E-print Network

insulin concentrations after a 75-g oral glucose tolerance test (OGTT). We have ex- amined the effect glucose tolerance test (OGTT), they had higher mean fasting plasma glucose (P 0.01, adjusted for age, sexBrief Genetics Report Variation in the Calpain-10 Gene Affects Blood Glucose Levels in the British

Cox, Nancy J.

139

Delivery and Inhibition of Reporter Genes by Small Interfering RNAs in a Mouse Skin Model  

Microsoft Academic Search

RNA interference offers the potential of a novel therapeutic approach for treating skin disorders. To this end, we investigated delivery of nucleic acids, including a plasmid expressing the reporter gene luciferase, to mouse skin by intradermal injection into footpads using in vivo bioluminescence imaging over multiple time points. In order to evaluate the ability of RNA interference to inhibit skin

Qian Wang; Heini Ilves; Pauline Chu; Christopher H. Contag; Devin Leake; Brian H. Johnston; Roger L. Kaspar

2007-01-01

140

Reconstruction of transcriptional dynamics from gene reporter data using differential equations  

Microsoft Academic Search

Motivation: Promoter driven reporter genes, notably luciferase (luc) and green fluorescent protein (gfp), provide a tool for the generation of a vast array of time-course data sets from living cells and organisms. The aim of this study is to introduce a modeling framework based on stochastic and ordinary differential equations that addresses the problem of reconstructing transcription time course profiles

Bärbel Finkenstädt; Elizabeth A. Heron; Michal Komorowski; Kieron D. Edwards; Sanyi Tang; Claire V. Harper; Julian R. E. Davis; Michael R. H. White; Andrew J. Millar; David A. Rand

2008-01-01

141

Circadian Rhythms in Prokaryotes: Luciferase as a Reporter of Circadian Gene Expression in Cyanobacteria  

Microsoft Academic Search

We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of

Takao Kondo; Carl A. Strayer; Resham D. Kulkarni; Walter Taylor; Masahiro Ishiura; Susan S. Golden; Carl Hirschie Johnson

1993-01-01

142

Research Report Steroidogenic enzyme gene expression in the brain of the  

E-print Network

Research Report Steroidogenic enzyme gene expression in the brain of the parthenogenetic whiptail. In this study, we indirectly test the hypothesis that the whiptail lizard brain is capable of de novo lizard, Cnemidophorus uniparens Brian George Diasa , Sonia Grace Chinb , David Crewsb, a Institute

Crews, David

143

Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection  

Microsoft Academic Search

Our aim is to develop a simple gene transfer method into egg cylinder and mid-gestational murine embryos. We examined whether plasmid\\/lipid complexes injected into the tail veins of pregnant transgenic mice can be transferred to fetuses at E 4.5–13.5. When pregnant CETZ-17 mice carrying a transgene consisting of a ubiquitous promoter, floxed EGFP\\/CAT and the LacZ gene, were injected with

N Kikuchi; S Nakamura; M Ohtsuka; M Kimura; M Sato

2002-01-01

144

Protein made by breast cancer gene purified http://www.innovations-report.com/html/reports/life_sciences/protein_made_breast_cancer_gene_purified_160190.html[8/24/2010 4:16:00 PM  

E-print Network

Protein made by breast cancer gene purified http://www.innovations-report.com/html/reports/life_sciences/protein_made_breast Sciences Content Protein made by breast cancer gene purified 23.08.2010 A key step in understanding the origins of familial breast cancer has been made by two teams of scientists at the University of California

Kowalczykowski, Stephen C.

145

Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase.  

PubMed

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions. Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant. An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified. Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S. typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene. The hemN gene was mapped to 87.3 min of the E. coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project. Complementation of S. typhimurium hemF hemN double mutants with the E. coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN. The previously cloned E. coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions. Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site. DNA sequences with homology to a sigma 70-dependent promoter were detected. Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions. Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions. Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression. Subsequent addition of iron restored normal expression. PMID:7768836

Troup, B; Hungerer, C; Jahn, D

1995-06-01

146

Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase.  

PubMed Central

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions. Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant. An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified. Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S. typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene. The hemN gene was mapped to 87.3 min of the E. coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project. Complementation of S. typhimurium hemF hemN double mutants with the E. coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN. The previously cloned E. coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions. Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site. DNA sequences with homology to a sigma 70-dependent promoter were detected. Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions. Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions. Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression. Subsequent addition of iron restored normal expression. PMID:7768836

Troup, B; Hungerer, C; Jahn, D

1995-01-01

147

Copy Number Related Transgene Expression and Mosaic Somatic Expression in Hemizygous and Homozygous Transgenic Tilapia (Oreochromis Niloticus)  

Microsoft Academic Search

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7?kb 5' regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these

M. Azizur Rahman; Gyu-Lin Hwang; Shaharudin Abdul Razak; Frédéric Sohm; Norman Maclean

2000-01-01

148

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

PubMed Central

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

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

2014-01-01

149

An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene?  

PubMed Central

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus. PMID:17142377

Janus, Danielle; Hoff, Birgit; Hofmann, Eckhard; Kuck, Ulrich

2007-01-01

150

A recombinant reporter system for monitoring reactivation of an endogenously DNA hypermethylated gene.  

PubMed

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 used remained basally silent, like the unaltered allele, and it was activated only by drug treatments that derepress gene silencing by reversing DNA hypermethylation. We used the established DNA methyltransferase inhibitor (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 derepress 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 reexpress 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

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

2014-07-15

151

The Anthrax Toxin Activator Gene atxA Is Associated with CO2Enhanced Non-Toxin Gene Expression in Bacillus anthracis  

Microsoft Academic Search

tions from atxA1 and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electro- phoresis. 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

ALEX R. HOFFMASTER; THERESA M. KOEHLER

1997-01-01

152

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

PubMed Central

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

Chadha, Radhika; Shah, Ritika; Mani, Shalini

2014-01-01

153

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

SciTech Connect

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.

Wessler, S.

1990-12-31

154

Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis.  

PubMed

In Neisseria meningitidis, the reversible expression of surface antigens, i.e. phase variation, results from changes within repeated simple sequence motifs located in coding or promoter regions of the genes involved in their biosynthesis. The mutation rates of these simple sequences, which have a major influence on the generation of phenotypic diversity, can affect the fitness of the population. The aim of the present study was to investigate the involvement of genetic factors involved (mutS and dam) and not yet analysed (drg and dinB) in the regulation of phase variation frequencies of genes associated with a variety of repeat tracts. The frequency of frameshifts occurring in the polycytidine (polyC) tracts associated with siaD, spr and lgtG and in the tetranucleotide (TAAA) repeat tract associated with nadA was determined by colony immunoblotting or using the lacZ gene as a reporter. Inactivation of mutS increased the frequency of phase variation of genes presenting homopolymeric tracts of diverse length. Overexpression of dinB enhanced the instability of the homopolymeric tract associated with siaD. Investigation of the dam locus in a population of genetically distinct N. meningitidis strains revealed that 27 % of strains associated with invasive disease contained the dam gene. In all strains where a Dam function was absent, the drg gene had been inserted into the dam locus. Disruption of dam and drg in strains representative of each genotype, i.e. dam(+)/drg and dam/drg(+), did not modify phase variation frequencies. In contrast to the effects of certain genes on homopolymeric tracts, none of the genetic factors investigated affected the stability of tetranucleotide repeat tracts. PMID:15347758

Martin, Patricia; Sun, Li; Hood, Derek W; Moxon, E Richard

2004-09-01

155

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

PubMed Central

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFP?nsp4 and pVaXJ-GLuc?nsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents. PMID:22427930

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

2012-01-01

156

Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor.  

PubMed Central

Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta-galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer. PMID:3006051

Hagen, D C; McCaffrey, G; Sprague, G F

1986-01-01

157

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors.  

PubMed

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying the Neo(r) selectable marker and the Escherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%. Neo(r) and lacZ genes were transcribed in vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny. PMID:7581517

Thoraval, P; Afanassieff, M; Cosset, F L; Lasserre, F; Verdier, G; Coudert, F; Dambrine, G

1995-11-01

158

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

PubMed

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

Hanig, Sacha; Entzeroth, Rolf; Kurth, Michael

2012-09-01

159

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

PubMed

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human ?-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium. PMID:23529929

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

2013-05-23

160

Transcriptional Regulation and Characteristics of a Novel N-Acetylmuramoyl-l-Alanine Amidase Gene Involved in Bacillus thuringiensis Mother Cell Lysis  

PubMed Central

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, PcwlA, 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 PcwlA was controlled by ?K. Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis. PMID:23603740

Yang, Jingni; Peng, Qi; Chen, Zhen; Deng, Chao; Shu, Changlong; Huang, Dafang

2013-01-01

161

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

PubMed

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

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

2013-06-01

162

Hir1p and Hir2p function as transcriptional corepressors to regulate histone gene transcription in the Saccharomyces cerevisiae cell cycle.  

PubMed Central

The HIR/HPC (histone regulation/histone periodic control) negative regulators play important roles in the transcription of six of the eight core histone genes during the Saccharomyces cerevisiae cell cycle. The phenotypes of hir1 and hir2 mutants suggested that the wild-type HIR1 and HIR2 genes encode transcriptional repressors that function in the absence of direct DNA binding. When Hir1p and Hir2p were artificially tethered to yeast promoters, each protein repressed transcription, suggesting that they represent a new class of transcriptional corepressors. The two proteins might function as a complex in vivo: Hir2p required both Hir1p and another Hir protein, Hir3p, to repress transcription when it was tethered to an HTA1-lacZ reporter gene, and Hir1p and Hir2p could be coimmunoprecipitated from yeast cell extracts. Tethered Hir1p also directed the periodic transcription of the HTA1 gene and repressed HTA1 transcription in response to two cell cycle regulatory signals. Thus, it represents the first example of a transcriptional corepressor with a direct role in cell cycle-regulated transcription. PMID:9001207

Spector, M S; Raff, A; DeSilva, H; Lee, K; Osley, M A

1997-01-01

163

Reporter gene transformation of the trunk disease pathogen Phaeomoniella chlamydospora and biological control agent Trichoderma harzianum  

Microsoft Academic Search

The economically important trunk disease pathogen Phaeomoniella chlamydospora causes Petri disease in Vitis vinifera and is also associated with the Esca trunk disease complex. Not much is known about the pathogen’s epidemiology and interactions\\u000a with the grapevine host, other trunk disease pathogens and biological control agents such as Trichoderma harzianum. Reporter gene labelling of plant pathogens and biocontrol agents can

T. McLean; P. H. Fourie; A. McLeod

2009-01-01

164

Brief Genetics Report Fine-Mapping Gene-by-Diet Interactions on Chromosome  

E-print Network

intercross line (AIL) from the SM/J and LG/J inbred strains. Half of our sample was fed a low-fat (15% energyBrief Genetics Report Fine-Mapping Gene-by-Diet Interactions on Chromosome 13 in a LG/J SM/J Murine strains reveals locus-by-diet interactions for all previously mapped loci. Adip7, located on proximal

Hrbek, Tomas - Department of Biology, Universidad de Puerto Rico

165

Potential usefulness of baculovirus-mediated sodium-iodide symporter reporter gene as non-invasively gene therapy monitoring in liver cancer cells: an in vitro evaluation.  

PubMed

Primary liver cancer has one of the highest mortality rates of all cancers, and the main current treatments have a poor prognosis. This study aims to examine the efficiency of baculovirus vectors for transducing target gene into liver cancer cells and to evaluate the feasibility of using baculovirus vectors to deliver the sodium-iodide symporter (NIS) gene as a reporter gene through co-vector administration approach to monitor the expression of the target therapeutic gene in liver cancer gene therapy. We constructed (green fluorescent protein) GFP- and NIS-expressing baculovirus vectors (Bac-GFP and Bac-NIS), and measured the baculovirus transduction efficiency in HepG2 cells and other tumor cells (A549, SW1116 and 8505C), and it showed that the transduction efficiency and target gene expression level rose with increasing viral multiplicity of infection (MOI) in HepG2 cells, and HepG2 cells had a significantly higher transduction efficiency (60.8% at MOI = 200) than other tumor cells. Moreover, the baculovirus transduction was not cytotoxic to HepG2 cells at a higher MOI (MOI 5 400). We also performed dynamic iodide uptake trials, and found that Bac-NIS-transduced HepG2 cells exhibited efficient iodide uptake which could be inhibited by sodium perchlorate (NaClO?). And we measured the correlation of fluorescent intensities and 125I uptake amount in HepG2 cells after co-vector administration with Bac-NIS and Bac-GFP at different MOIs, and found a high correlation coefficient (r(2) = 0.8447), which provides a good basis for successfully evaluating the feasibility of baculovirus-mediated NIS reporter gene monitoring target gene expression in liver cancer therapy. Therefore, this study indicates that baculovirus vector is a potential vehicle for delivering therapeutic genes in studying liver cancer cells. And it is feasible to use a baculovirus vector to deliver NIS gene as a reporter gene to monitor the expression of target genes. It therefore provides an effective approach and a good basis for future baculovirus-mediated therapeutic gene delivering or therapeutic gene expression monitoring in liver cancer cells studies. PMID:23919394

Pan, Yu; Wu, Haifei; Liu, Shuai; Zhou, Xiang; Yin, Hongyan; Li, Biao; Zhang, Yifan

2014-04-01

166

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

PubMed Central

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

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

2011-01-01

167

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

SciTech Connect

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

VanEtten, H.

1997-06-01

168

Analysis of the Schwanniomyces occidentalis SWA2 gene promoter in Saccharomyces cerevisiae.  

PubMed

The effect of different carbon sources on the expression in Saccharomyces cerevisiae of the SWA2 alpha-amylase gene from Schwanniomyces occidentalis was studied from constructs containing its 5' region (-223 to +15), which were fused in-frame to the lacZ gene coding sequence. Maximal expression was achieved with the non-fermentable substrates ethanol and/or glycerol, whereas lower levels were found with maltose or galactose. In contrast, glucose repressed it, even in the presence of any of these other carbon sources. Deletion analyses of the -233 to -85 SWA2 promoter region permitted the identification of two fragments involved in both glucose repression and ethanol activation. A possible region required for cAMP regulation was localised. The SWA2 promoter contains a MIG1-binding GC box whose deletion caused a five-fold increase in the glucose-repressed reporter expression. Despite this, expression of the SWA2 promoter was not MIG1-dependent. PMID:11886753

Carmona, T A; Jiménez, A; Fernández Lobato, M

2002-01-22

169

The first report of the vanC 1 gene in Enterococcus faecium isolated from a human clinical specimen  

PubMed Central

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1 gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1 and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1 gene. However, this study is the first to report the presence of the vanC1 gene in E. faecium of human origin. Additionally, our research showed the vanC1 gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1 gene from different species. PMID:25317698

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

2014-01-01

170

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

PubMed Central

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. Images PMID:2306085

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

1990-01-01

171

Msx genes define a population of mural cell precursors required for head blood vessel maturation.  

PubMed

Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22?-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs. PMID:21693521

Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

2011-07-01

172

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

PubMed

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

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

173

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

PubMed Central

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

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

2012-01-01

174

SHORT REPORTS Mutational analysis of the transcriptional activation domains of v-Myb  

E-print Network

was isolated from individual replica-plated yeast colonies, cloned by transformation into E. coli, then ampli CYC1 promoter and the E. coli lacZ reporter (Chen and Lipsick, 1993; Ness et al., 1989). We used transcriptional activation and oncogenic transformation. Oncogene (2002) 21, 1611 ± 1615. DOI: 10.1038/sj/ onc

Lipsick, Joseph S.

175

Vaccinia reporter viruses for quantifying viral function at all stages of gene expression.  

PubMed

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology. PMID:24894622

Rozelle, Daniel K; Filone, Claire Marie; Dower, Ken; Connor, John H

2014-01-01

176

Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas.  

PubMed

The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5' untranslated regions of the atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera. PMID:10589828

Minko, I; Holloway, S P; Nikaido, S; Carter, M; Odom, O W; Johnson, C H; Herrin, D L

1999-10-01

177

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

SciTech Connect

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

Kuchka, M.R.

1992-08-01

178

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

PubMed Central

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

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

2014-01-01

179

Differential Expression of Virulence Genes and Motility in Ralstonia (Pseudomonas) solanacearum during Exponential Growth  

PubMed Central

A complex network regulates virulence in Ralstonia solanacearum (formerly Pseudomonas solanacearum); central to this system is PhcA, a LysR-type transcriptional regulator. We report here that two PhcA-regulated virulence factors, endoglucanase (Egl) and acidic exopolysaccharide I (EPS I), and motility are expressed differentially during exponential growth in batch cultures. Tests with strains carrying lacZ fusions in a wild-type genetic background revealed that expression (on a per-cell basis) of phcA was constant but expression of egl and epsB increased 20- to 50-fold during multiplication from 1 x 10(sup7) to 5 x 10(sup8) CFU/ml. Expression of xpsR, an intermediate regulator downstream of PhcA in the regulatory cascade for eps expression, was similar to that of epsB and egl. Motility track photography revealed that all strains were essentially nonmotile at 10(sup6) CFU/ml. As cell density increased, 30 to 50% of wild-type cells were motile between 10(sup7) and 10(sup8) CFU/ml, but this population was again nonmotile at 10(sup9) CFU/ml. In contrast, about 60% of the cells of phcB and phcA mutants remained motile at 10(sup9) CFU/ml. Expression of phcB, which is not positively regulated by PhcA, was the inverse of epsB, egl, and xpsR (i.e., it decreased 20-fold at high cell density). PhcB is essential for production of an extracellular factor, tentatively identified as 3-hydroxypalmitic acid methyl ester (3-OH PAME), that might act as an exponential-phase signal to activate motility or expression of virulence genes. However, growth of the lacZ fusion strains in medium containing excess 3-OH PAME did not result in motility or expression of virulence genes at dramatically lower cell densities, suggesting that 3-OH PAME is not the only factor controlling these traits. PMID:16535550

Clough, S. J.; Flavier, A. B.; Schell, M. A.; Denny, T. P.

1997-01-01

180

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

SciTech Connect

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

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

2006-03-10

181

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

PubMed

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

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

2014-08-01

182

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

PubMed Central

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

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

183

A reporter gene assay for the detection of phytoestrogens in traditional Chinese medicine.  

PubMed

Bupleurum & Peony Formula (Jia Wei Xiao Yao San) is a herbal formula which possesses a clinical history for the treatment of menopausal syndrome and menstrual irregularity. The present investigation reports the ability to monitor the formula's phytoestrogen content that will allow for the implementation of a standardization protocol that is based on a quantifiable biological response. Utilizing an oestrogen-sensitive chimeric receptor/reporter gene element which has been stably transfected into HeLa cells, the botanical formula was shown to induce the expression of the reporter gene, luciferase, in a dose dependent manner. Pretreatment of the HeLa cells with the botanical formula produced a 5-fold increase in bioluminescence compared with the control. Additionally, our studies showed that the response of the cells, when challenged by the botanical formula, was oestrogen specific. Pretreatment of the cells with tamoxifen effectively blocked the activation of the chimeric oestrogen receptor by the botanical formula. The cell line provides a sensitive assay that can easily detect the presence of phytoestrogens in complex botanical formulas. PMID:11536376

Miller-Martini, D M; Chan, R Y; Ip, N Y; Sheu, S J; Wong, Y H

2001-09-01

184

T-box proteins differentially activate the expression of the endogenous interferon gene versus transfected reporter genes  

E-print Network

, histone deacetylase complex; HSVtk, herpes simplex thymidine kinase promoter; IFN, interferon gamma; IFNG, interferon gamma gene; IRF-1, interferon response factor 1; luc, luciferase; murA, muristerone A; NFATT-box proteins differentially activate the expression of the endogenous interferon gene versus

Gronostajski, Richard M.

185

Modeling of cell growth and phoA -directed expression of cloned genes in recombinant Escherichia coli  

Microsoft Academic Search

A mathematical model was formulated to describe growth and cloned protein production in the recombinantEscherichia coli cells containingphoA-directed expression systems. Kinetic parameters for the strains with two fusion genes (phoA- lacZ either on the chromosome or on a multicopy plasmid andphoA- amyE on a multicopy plasmid) were estimated and compared to analyze the effects of cloning site (chromosome and plasmid),

Pyong Kyun Shin; Ja Hyup Koo; Woo Jong Lee; Jin Ho Seo

1996-01-01

186

Development of fluorescent reporter tagged RIB gene cassettes for replicative transformation, early expression, and enhanced riboflavin production in Eremothecium ashbyi.  

PubMed

Eremothecium ashbyi is a riboflavin overproducing filamentous fungus in which the metabolic pathways have not been genetically characterized. Two genes of the riboflavin biosynthetic (RIB) pathway, RIB1 and RIB3, which encode GTP-cyclohydrolase II (GCH II) and 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase respectively, were selected for the present study. The two RIB genes under their native promoters were obtained from Ashbya gossypii genomic library. Yeast enhanced green fluorescent protein (yEGFP) and mCherry genes were tagged to the C-terminal ends of RIB1 and RIB3 genes to analyse the functionality of the RIB transgenes in E. ashbyi. Shuttle vectors with the reporter tagged RIB genes contained the Escherichia coli kan(R) gene and Saccharomyces cerevisiae ARS element. On transformation with these plasmids, the ARS element was found to be functional in E. ashbyi. The E. ashbyi transcription factors could recognize the Ashbya RIB gene promoters and express the reporter tagged RIB genes as cytoplasmic proteins, in early cell development. Replicative transformants carrying RIB1-mCherry plasmids showed 2.95 times more GCH II activity and 2.44 times more riboflavin production when compared to untransformed. This is the first report of genetic transformation of E. ashbyi and is of significance as the first step towards genetic engineering of this genus. PMID:23063183

Sengupta, Sudeshna; Kaufmann, Andreas; Chandra, T S

2012-10-01

187

Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency.  

PubMed

The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression. PMID:8875233

Makarova, O; Gorneva, G; Wu, F; Farutin, V; Villeponteau, B; Poliani, L; Fink, D; Levine, M

1996-09-01

188

ovo, a Drosophila gene required for ovarian development, is specifically expressed in the germline and shares most of its coding sequences with shavenbaby, a gene involved in embryo patterning.  

PubMed

Genetic analyses of Drosophila oogenesis have revealed the central role of ovo, a gene required for differentiation of the female germline. A number of recessive ovo mutations also affect the shavenbaby (svb) function required for late embryo patterning, suggesting a tight structural link between ovo and svb. By using various genomic probes for in situ hybridization to wild type and mutant embryos, we show that ovo indeed shares most of its coding sequences with svb. svb expression is detected early in the presumptive head region and later in each segment. It requires control elements located upstream of the ovo genomic region. ovo expresses abundant maternal RNAs which are uniformly distributed in early cleavage embryos. A fraction that lacks an alternative ovo-specific protein coding region (ORF 2b) is detected in pole cells. Expression of an ovo-specific lacZ reporter gene (ovoB) shows that ovo encodes a nuclear protein present in the germline of both sexes. Zygotic ovoB expression is first detected in embryos at around stage 17 and persists up to the adult stage. Our data show that the germline specific expression of ovo in females correlates with its function in oogenesis. This expression, however, is also observed in males in which ovo is not required. PMID:7748792

Mével-Ninio, M; Terracol, R; Salles, C; Vincent, A; Payre, F

1995-01-01

189

Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.  

PubMed Central

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp. Images PMID:1729203

Haney, S A; Platko, J V; Oxender, D L; Calvo, J M

1992-01-01

190

Growth phase and metal-dependent regulation of the dpsA gene in Synechococcus sp. strain PCC 7942, USA.  

PubMed

The Synechococcus sp. strain PCC 7942 dpsA gene encodes a stress-inducible DNA-binding protein whose transcription increases in the stationary phase. Such transcription is likely under the control of an alternative sigma factor. Our current work indicated that dpsA transcription is also important under metal-ion limitation, because dpsA mRNA levels increased 12-fold under low-iron conditions, and that dpsA function is essential for growth under iron-limiting conditions. Promoter activity of the dpsA-promoter-lacZ reporter gene constructs implied that a region of dyad symmetry centered 28 nucleotides from the transcription start is required for metal-dependent repression, as judged by the level of lacZ induction following treatment of cultures with the chelator 2,2'-dipyridyl. This potential operator sequence is distinct from the site recognized by the cyanobacterial Fur repressor homologue. No other nutrient stresses (nitrogen, sulfur, phosphorus) yielded the high level of induction seen following chelator treatment. These studies suggest that there may be more than one class of metal-dependent repressor in cyanobacteria. PMID:10896214

Sen, A; Dwivedi, K; Rice, K A; Bullerjahn, G S

2000-01-01

191

Dual Transcriptional Regulation of the Escherichia coli Phosphate-Starvation-Inducible psiE Gene of the Phosphate Regulon by PhoB and the Cyclic AMP (cAMP)cAMP Receptor Protein Complex  

Microsoft Academic Search

We have shown that the Escherichia coli phosphate-starvation-inducible psiE gene is regulated by both phosphate and the carbon source by using both lacZ and chloramphenicol acetyltransferase gene (cat) fusions. Yet, under all conditions tested, a single transcriptional start site lying 7 bp downstream of a predicted 210 region was revealed by primer extension analysis. DNase I footprinting showed that the

SOO-KI KIM; SIGENOBU KIMURA; HIDEO SHINAGAWA; ATSUO NAKATA; KI-SUNG LEE; BARRY L. WANNER; KOZO MAKINO

2000-01-01

192

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

PubMed

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

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

2011-08-26

193

A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists  

PubMed Central

Intracellular calcium response and resulting calcium signaling to an agonist-GPCR interaction are important for the measurement of compound activity in the GPCR drug development. The increase in cytosol calcium concentration can be measured by the fluorescent calcium indicator dye such as Fluo-4 in a quick assay (in 3-5 minutes) using the fluorescence imaging plate reader. The calcium signaling through the transcription factors such as NFAT that induces gene expression can be measured by the reporter gene assay that links to the expression of reporter enzyme such as the beta-lactamase that requires 5-hour incubation. We have evaluated a multiplexed assay that sequentially measures the calcium response to a GPCR agonist in a rapid fluorescent calcium dye assay, followed by a NFAT beta-lactamase assay, and compared them in the single assay format. We found that the agonist activity determined in the multiplexed assay were comparable with these determined in the single assay format and the Z’ factors were all >0.5. Five active compounds were identified that were active in both calcium dye assay and beta-lactamase assay. Therefore, our results demonstrated the utility of this multiplexed calcium assay for screening of GPCR compounds that can cross validate the primary hits and help to eliminate the false positive compounds. PMID:24396729

Sheth, Heeral; Gorey, Colleen; Roush, Nicole; Smallman, Shelly; Collantes, Elizabeth; Santoro, Maxine; Olson, Barbara; Fitzgerald, Laura; Lee, Paul H; Shen, Xiqiang John

2013-01-01

194

Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992.  

National Technical Information Service (NTIS)

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...

R. Michelmore

1994-01-01

195

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

PubMed

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

Gabriel, Gabriele V M; Viviani, Vadim R

2014-12-12

196

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

PubMed

Polaromonas naphthalenivorans CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (C.-O. Jeon et al., Proc. Natl. Acad. Sci. USA 100:13591-13596, 2003), 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 andadditional 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 (nagRAaGHAbAcAdBFCQEDJI'ORF1tnpA) is bounded by a LysR-type regulator (nagR). The small cluster (nagR2ORF2I"KL) 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. PMID:16461653

Jeon, Che Ok; Park, Minjeong; Ro, Hyun-Su; Park, Woojun; Madsen, Eugene L

2006-02-01

197

Gene transfer in human skin with different pseudotyped HIV-based vectors.  

PubMed

Pseudotyping lentiviral vector with other viral surface proteins could be applied for treating genetic anomalies in human skin. In this study, the modification of HIV vector tropism by pseudotyping with the envelope glycoprotein from vesicular stomatitis virus (VSV), the Zaire Ebola (EboZ) virus, murine leukemia virus (MuLV), lymphocytic choriomeningitis virus (LCMV), Rabies or the rabies-related Mokola virus encoding LacZ as a reporter gene was evaluated qualitatively and quantitatively in human skin xenografts. High transgene expression was detected in dermal fibroblasts transduced with VSV-G-, EboZ- or MuLV-pseudotyped HIV vector with tissue irregularities in the dermal compartments following repeated injections of EboZ- or LCMV-pseudotyped vectors. Four weeks after transduction, double-labeling immunofluorescence of beta-galactosidase and involucrin or integrin beta1 demonstrated that VSV-G-, EboZ- or MuLV-pseudotyped HIV vector effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies. Among the six different pseudotyped HIV-based vectors evaluated, VSV-G-pseudotyped vector was found to be the most efficient viral glycoprotein for cutaneous transduction as demonstrated by the highest level of beta-galactosidase expression and genome copy number evaluated by TaqMan PCR. PMID:17268532

Hachiya, A; Sriwiriyanont, P; Patel, A; Saito, N; Ohuchi, A; Kitahara, T; Takema, Y; Tsuboi, R; Boissy, R E; Visscher, M O; Wilson, J M; James, W M; Kobinger, G P

2007-04-01

198

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

PubMed Central

Introduction About 20% of the cases of human severe combined immunodeficiency are the result of the child being homozygous for defective genes encoding the enzyme adenosine deaminase. To our knowledge, the mutation pattern in Arab patients with severe combined immunodeficiency has never been reported previously. Case presentation A 14-month-old Arab boy had clinical features typical of severe combined immunodeficiency. His clinical picture and flow cytometric analysis raised the diagnosis of adenosine deaminase deficiency and prompted us to screen the adenosine deaminase gene for mutation(s). We detected a novel mutation in exon 9 of the adenosine deaminase gene (p.Arg282>Gln), which we believe is the cause of the severe combined immunodeficiency phenotype observed in our patient. Conclusion This is the first report of adenosine deaminase mutation in an Arab patient with severe combined immunodeficiency due to a novel pathogenic mutation in the adenosine deaminase gene. PMID:19830125

2009-01-01

199

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

PubMed Central

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

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

2012-01-01

200

Involvement of the BDNF Gene in Loneliness in Adolescence: A Report of Opposite Gene Effects in Boys and Girls  

PubMed Central

Previous research has shown that loneliness has a heritable component and that genes within the serotonin-, dopamine-, and oxytocin systems are related to loneliness in adolescence. In the present study, the relation between the BDNF Val66Met polymorphism and loneliness in adolescent boys and girls was examined in a longitudinal study spanning five annual waves (N?=?305). Latent growth curve modeling (LGCM) was used to examine the baseline level and the change in loneliness over time. The main finding was that the BDNF gene was not related to loneliness in the total sample. A BDNF by sex interaction was found, in that Met carrying girls had the highest levels of loneliness at baseline, whereas in boys the ValVal genotype was related to higher levels of loneliness. Our results underline the importance of sex-stratified analyses when examining effects of the BDNF genotype and the necessity of conducting gene studies to intermediate phenotypes of loneliness. PMID:24647525

Verhagen, Maaike; van Roekel, Eeske; Engels, Rutger C. M. E.

2014-01-01

201

Differential Expression of Virulence Genes and Motility in Ralstonia(Pseudomonas)solanacearumduring Exponential Growth  

Microsoft Academic Search

A complex network regulates virulence in Ralstonia solanacearum (formerly Pseudomonas solanacearum); central to this system is PhcA, a LysR-type transcriptional regulator. We report here that two PhcA-regulated virulence factors, endoglucanase (Egl) and acidic exopolysaccharide I (EPS I), and motility are expressed differentially during exponential growth in batch cultures. Tests with strains carrying lacZ fusions in a wild-typegeneticbackgroundrevealedthatexpression(onaper-cellbasis)ofphcAwasconstantbutexpression ofeglandepsBincreased 20- to

STEVEN J. CLOUGH; ALBERT B. FLAVIER; MARK A. SCHELL; ANDTIMOTHY P. DENNY

1997-01-01

202

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters  

PubMed Central

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems. PMID:23052244

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

2012-01-01

203

vttRA and vttRB Encode ToxR Family Proteins That Mediate Bile-Induced Expression of Type Three Secretion System Genes in a Non-O1/Non-O139 Vibrio cholerae Strain?  

PubMed Central

Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttRA (locus tag A33_1664) and vttRB (locus tag A33_1675), are predicted to encode proteins that show similarity to the transcriptional regulator ToxR, which is found in all strains of V. cholerae. Strains with a deletion of vttRA or vttRB showed attenuated colonization in vivo, indicating that the T3SS-encoded regulatory proteins play a role in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the T3SS structural components identified growth in the presence of bile as a condition that modulates gene expression. Under this condition, VttRA and VttRB were necessary for maximal gene expression. In contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene, which encodes an effector protein. Increased vttRB reporter fusion activity was observed in a ?vttRB strain background, suggesting that VttRB may regulate its own expression. The collective results are consistent with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the expression of selected T3SS genes. PMID:20385759

Alam, Ashfaqul; Tam, Vincent; Hamilton, Elaine; Dziejman, Michelle

2010-01-01

204

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

PubMed

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

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

1996-01-01

205

No effect of high fat diet-induced obesity on spontaneous reporter gene mutations in gpt delta mice.  

PubMed

A large number of epidemiological studies have demonstrated that obesity is a risk factor for several human cancers. Several animal studies using rodents with diet-induced or genetic obesity have also demonstrated that obesity can promote tumor development. However, the effects of obesity on the early stages of carcinogenesis, and especially on the spontaneous occurrence of somatic gene mutations, remain unclear. To investigate the effects of obesity on the rate of spontaneous gene mutations, we performed reporter gene mutation assays in liver, kidney, and colon, organs in which obesity appears to be associated with cancer development on the basis of epidemiological or animal studies, in mice with high fat diet (HFD)-induced obesity. Six-week-old male and female C57BL/6 gpt delta mice were fed HFD or standard diet (STD) for 13 or 26 weeks. At the end of the experiments, reporter gene mutation assays of liver, kidney, and colon were performed. Final body weights and serum leptin levels of male and female mice fed HFD for 13 or 26 weeks were significantly increased compared with corresponding STD-fed groups. Reporter gene mutation assays of liver, kidney, and colon revealed that there were no significant differences in gpt or Spi- mutant frequencies between STD- and HFD-fed mice in either the 13-week or 26-week groups. These results indicate that HFD treatment and consequent obesity does not appear to influence the spontaneous occurrence of somatic gene mutations. PMID:25227805

Takasu, Shinji; Ishii, Yuji; Matsushita, Kohei; Kuroda, Ken; Kijima, Aki; Kodama, Yukio; Ogawa, Kumiko; Umemura, Takashi

2014-01-01

206

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

PubMed

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

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

2012-05-01

207

A Polymorphism in the 3' Untranslated Region of the Gene for Tumor Necrosis Factor Receptor 2 Modulates Reporter Gene Expression  

Microsoft Academic Search

The gene encoding the human TNF receptor (TNFR) 2 con- tains polymorphisms in the 3untranslated region (UTR). Pre- vious studies have shown that some variant alleles in this region are associated with obesity and insulin resistance. However, the effect of these polymorphisms on the expression of TNFR2 has not been studied to date. To examine the role played by different

Irene Puga; Begona Lainez; JoseManuel Fernandez-Real; Maria Buxade; Montserrat Broch; Joan Vendrell; Enric Espel

2005-01-01

208

Structure and regulation of methanogen genes: Progress report, July 1, 1987February 29, 1988  

Microsoft Academic Search

We are characterizing the histidine biosynthetic genes (hisA and hisI) and methyl reductase (mcrBDCGA) encoding genes from Methanococcus vannielii and a purine biosynthetic gene (purE) and methyl-viologen reducing hydrogenase (mvh) encoding genes from Methanobacterium thermoautotrophicum. The primary structures of most of these genes have been determined. DNA-dependent RNA polymerases purified from M. vannielii and M. thermoautotrophicum have been used in

Reeve

1988-01-01

209

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

PubMed Central

The retinoblastoma tumor suppressor (Rb) pathway is mutated in most, if not all human tumors. In the G0/G1 phase, Rb and its family members p107 and p130 inhibit the E2F family of transcription factors. In response to mitogenic signals, Cyclin-dependent kinases (CDKs) phosphorylate Rb family members, which results in the disruption of complexes between Rb and E2F family members and in the transcription of genes essential for S phase progression. Beyond this role in early cell cycle decisions, Rb family members regulate DNA replication and mitosis, chromatin structure, metabolism, cellular differentiation, and cell death. While the RB pathway has been extensively studied in the past three decades, new investigations continue to provide novel insights into basic mechanisms of cancer development and, beyond cancer, help better understand fundamental cellular processes, from plants to mammals. This meeting report summarizes research presented at the recently held 3rd International Rb Meeting. PMID:24257515

2013-01-01

210

Human Pharmacokinetic and Dosimetry Studies of (18F)FHBG: A Reporter Probe for Imaging Herpes Simplex Virus Type1 Thymidine Kinase Reporter Gene Expression  

Microsoft Academic Search

9-(4-(18F)fluoro-3-(hydroxymethyl)butyl)guanine ((18F)FHBG) has been used as a reporter probe to image expression of herpes simplex virus type-1 thymidine kinase (HSV1-tk) reporter gene in living animals. Our aim was to study the kinetics, biodistribu- tion, stability, dosimetry, and safety of (18F)FHBG in healthy human volunteers, preparatory to imaging patients undergoing HSV1-tk gene therapy. Methods: (18F)FHBG was synthesized with a specific activity

Shahriar Yaghoubi; Jorge R. Barrio; Magnus Dahlbom; Meera Iyer; Mohammad Namavari; Nagichettiar Satyamurthy; Robin Goldman; Harvey R. Herschman; Michael E. Phelps; Sanjiv S. Gambhir

211

Reliable Gene Expression Analysis by Reverse Transcription-Quantitative PCR: Reporting and Minimizing the Uncertainty in Data Accuracy.  

PubMed

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

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

2014-10-01

212

Carbon source-dependent regulation of the Schizosaccharomyces pombe pbh1 gene.  

PubMed

Pbh1, from the fission yeast Schizosaccharomyces pombe, is a baculoviral inhibitor of apoptosis (IAP) repeat (BIR) domain-containing protein. Its unique encoding gene was previously found to be regulated by nitric oxide and nitrogen starvation. In the current work, the Pbh1-lacZ fusion gene was used to elucidate the transcriptional regulation of the pbh1 gene under various carbon sources. When fermentable carbon sources, such as glucose (at a low concentration of 0.2%), sucrose (2.0%) and lactose (2.0%), were the sole carbon source, the synthesis of beta-galactosidase from the Pbh1-lacZ fusion gene was reasonably enhanced. However, the induction by these fermentable carbon sources was abolished in the Pap1-negative S. pombe cells, implying that this type of induction of the pbh1 gene is mediated by Pap1. Ethanol (2.0%), a nonfermentable carbon source, was also able to enhance the synthesis of beta-galactosidase from the fusion gene in wild-type cells but not in Pap1-negative cells. The results indicate that the S. pombe pbh1 gene is up-regulated under metabolic oxidative stress in a Pap1-dependent manner. PMID:17205051

Kim, Su-Jung; Cho, Nam-Chul; Ryu, In Wang; Kim, Kyunghoon; Park, Eun-Hee; Lim, Chang-Jin

2006-12-01

213

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

PubMed Central

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

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

2000-01-01

214

Efficiency of Ferritin as an MRI Reporter Gene in NPC Cells Is Enhanced by Iron Supplementation  

PubMed Central

Background. An emerging MRI reporter, ferritin heavy chain (FTH1), is recently applied to enhance the contrast and increase the sensitivity of MRI in the monitoring of solid tumors. However, FTH1-overexpression-related cytotoxicity is required to be explored. Methods. By using the Tet-Off system, FTH1 overexpression was semi-quantitativiely and dynamicly regulated by doxycycline in a NPC cell line. Effects of FTH1 overexpression on the proliferation, cytotoxicity, apoptosis and migration of NPC cells were investigated in vitro, and MR relaxation rate was measured in vitro and in vivo. Results. In vitro and in vivo overexpression of FTH1 significantly increased the transverse relaxivity (R2), which could be enhanced by iron supplementation. In vitro, overexpression of FTH1 reduced cell growth and migration, which were not reduced by iron supplementation. Furthermore, cells were subcutaneously inoculated into the nude mice. Results showed FTH1 overexpression decreased tumor growth in the absence of iron supplementation but not in the presence of iron supplementation. Conclusion. To maximize R2 and minimize the potential adverse effects, supplementation of iron at appropriate dose is recommended during the application of FTH1 as a reporter gene in the monitoring of NPC by MRI. PMID:22536021

Feng, Yupeng; Liu, Qicai; Zhu, Junfeng; Xie, Fukang; Li, Li

2012-01-01

215

A novel reporter gene assay for Recombinant Human Erythropoietin (rHuEPO) pharmaceutical products.  

PubMed

Accurate determination of in vitro biological activity of therapeutic erythropoietin is essential in quality control of recombinant human erythropoietin (rHuEPO) pharmaceutical products. However, most of currently-used methods leave much to be desired so that a simpler, quicker and more accurate method is urgently needed. The bioassay described here utilizes a sub clone of UT-7/epo cell line stably transfected with luciferase gene under the control of sis inducible element and interferon ?-activated sequence element promoter. Active erythropoietin could induce the expression of luciferase by signaling through the erythropoietin receptor and the dose-response curve showed good linearity, yielding a coefficient of determination of 0.99 or higher. The optimized assay was simpler with the operation completed within 24h and more sensitive with EC50 being 0.077IU/mL. The accuracy estimates ranged from 81.7% to 102.4%, and both intra-assay and inter-assay precision was below 15.0%. The robustness of the assay was demonstrated by no effect of passage levels of the cells on the performance of the assay (p values: 0.772 for sample 1 and 0.943 for sample 2). Besides, Bland-Altman analysis showed a high consistency of the new assay with in vivo reticulocyte assay in results. These results suggested that the new reporter gene assay can be a viable supplement to the traditional reticulocyte assay and employed in potency determination of rHuEPO pharmaceutical products. PMID:25194345

Yang, Yushuai; Zhou, Yong; Yu, Lei; Li, Xiang; Shi, Xinchang; Qin, Xi; Rao, Chunming; Wang, Junzhi

2014-11-01

216

Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System  

PubMed Central

The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future. PMID:24416725

Yang, Chao-Hsun; Kuo, Wan-Ting; Chuang, Yun-Ting; Chen, Cheng-Yu

2013-01-01

217

Report on the 7(th) international workshop on the CCN family of genes : October 16-19, 2013-Nice, France.  

PubMed

In this report, chairs of the 7th International Workshop on the CCN family of Genes, review the progress made in understanding the biological functions of CCN proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) with a particular focus on their implications in various pathological conditions, including cancer, fibrosis, diabetes, and cardiovascular diseases. PMID:24553917

Perbal, B; Trackman, P; Castellot, J; Brigstock, D; Takigawa, M; Lau, L; Leask, A

2014-03-01

218

Genotyping for polymorphisms in interferon-?, interleukin-10, transforming growth factor-?1 and tumour necrosis factor-? genes: a technical report  

Microsoft Academic Search

Polymorphic variants of cytokine genes are associated with acute and chronic transplant rejection. In this technical report, the methods currently used in our centre to genotype individuals for interferon-?, interleukin-10, transforming growth facgor-?1 and tumour necrosis factor-? are described in detail. The DNA sequences of primers and probes, and conditions for polymerase chain reactions are given, and the allele and

Chris Perrey; Vera Pravica; Paul J Sinnott; Ian V Hutchinson

1998-01-01

219

Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme  

E-print Network

C reporter gene fusion was expressed in vegetative cells and not in heterocysts, whereas GFP driven from Phet conditions of energy limitation such as low light or limiting phosphate. They can withstand long periods of desiccation or cold, and germinate back into 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights

Summers, Michael L.

220

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

PubMed Central

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

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

2011-01-01

221

Selective suicide gene therapy of colon cancer exploiting the urokinase plasminogen activator receptor promoter.  

PubMed

Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process. PMID:20199127

Teimoori-Toolabi, Ladan; Azadmanesh, Kayhan; Amanzadeh, Amir; Zeinali, Sirous

2010-04-01

222

Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay.  

PubMed

A stable luciferase reporter gene assay was developed based on the thyroid hormone responsive rat pituitary tumor GH3 cell line that constitutively expresses both thyroid hormone receptor isoforms. Stable transfection of the pGL4CP-SV40-2xtaDR4 construct into the GH3 cells resulted in a highly sensitive cell line (GH3.TRE-Luc), which was further optimized into an assay that allowed the detection of Triiodothyronine (T(3)) and Thyroxine (T(4)) concentrations in the picomolar range after only 24 h of exposure. The greater than 20-fold induction of T(3) relative to the solvent control is illustrative of the high responsiveness of the system. The assay was validated by the quantification of the agonistic effect of the natural hormones (T(3) and T(4)), the acetic acid derivatives of T(3) (triiodothyroaceticacid, or Triac) and T(4) (tetraiodothyroacetic acid, or Tetrac), hydroxy polybrominated diphenylethers (OH-PBDEs), hydroxy polychlorinated biphenyls (OH-PCBs) and the antagonistic action of sodium arsenite (NaAsO(2)). The putative antagonist Amiodarone, Bisphenol A (BPA) and its halogenated derivatives (TCBPA and TBBPA) for which effects reported in the literature are not consistent, showed comparable dose-response curves with a slight agonistic effect (5% of T(3)-max) followed by a slight antagonistic effect. The magnitude and reproducibility of the responses to various compounds confirms this assay as a promising tool for the identification and quantification of specific thyroid hormone receptor disrupting potency of compounds. PMID:20732405

Freitas, Jaime; Cano, Patricia; Craig-Veit, Christina; Goodson, Michael L; Furlow, J David; Murk, Albertinka J

2011-02-01

223

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

PubMed

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

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

2010-07-01

224

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

NASA Astrophysics Data System (ADS)

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.

Paproski, Robert J.; Zemp, Roger J.

2012-02-01

225

Structure and regulation of methanogen genes: Progress report, July 1, 1987-February 29, 1988  

SciTech Connect

We are characterizing the histidine biosynthetic genes (hisA and hisI) and methyl reductase (mcrBDCGA) encoding genes from Methanococcus vannielii and a purine biosynthetic gene (purE) and methyl-viologen reducing hydrogenase (mvh) encoding genes from Methanobacterium thermoautotrophicum. The primary structures of most of these genes have been determined. DNA-dependent RNA polymerases purified from M. vannielii and M. thermoautotrophicum have been used in DNA binding-footprinting experiments to identify promoter sequences. Sites of transcription initiation in vivo have been shown to correlate well with the sites identified in vitro as RNA polymerase binding sites. Current work is aimed at determining how expression of these methanogen genes is regulated in vivo. 5 refs.

Reeve, J.N.

1988-01-01

226

The use of the luxA gene of the bacterial luciferase operon as a reporter gene  

Microsoft Academic Search

Bacterial luciferase can be assayed rapidly and with high sensitivity both in vivo and in vitro. Here we demonstrate that the N-terminal hydrophobic domain of the a catalytic subunit of the luciferase enzyme is indispensable for enzyme activity, although N-terminal translational fusions with full luciferase activity can be obtained. Bacterial luciferase is therefore ideally suited as a reporter enzyme for

Olof Olsson; Csaba Koncz; Aladar A. Szalay

1988-01-01

227

Structure and regulation of methanogen genes: Progress report, March 1, 1988February 15, 1989  

Microsoft Academic Search

The goals of this project were initially to obtain the primary structures of several cloned methanogen genes and subsequently to determine the mechanism(s) by which these genes are transcribed and how their expression is regulated. Studies have focused on both housekeeping genes encoding enzymes involved in the biosynthesis of amino acids (hisA, hisI, argG) and purines (purE) and on methane

Reeve

1989-01-01

228

Cloning, sequencing, and expression of bacteriophage BF23 late genes 24 and 25 encoding tail proteins.  

PubMed Central

Two bacteriophage BF23 late genes, genes 24 and 25, were isolated on a 7.4-kb PstI fragment from the phage DNA, and their nucleotide sequences were determined. Gene 24 encodes a minor tail protein with the expected M(r) of 34,309, and gene 25 located 4 bp upstream of gene 24 encodes a major tail protein with the expected M(r) of 50,329. When total cellular RNA isolated from either phage-infected cells or cells bearing the cloned genes was analyzed by the primer extension method using the primers specific to either gene 25 or gene 24, we identified a possible late gene promoter, designated P25, in the 5'-flanking region of gene 25. This promoter was similar in structure to Escherichia coli promoters for sigma 70. Studies of the translational gene 25- and gene 24-lacZ fusions in the cloned gene system revealed that the promoter P25 was responsible for the expression of both genes 25 and 24 even in the absence of the regulatory genes which were absolutely required for late gene expression in the normal phage-infected cells. These results indicate that the two genes constitute an operon under the control of P25 and that the regulatory gene products of BF23 do not participate directly in specifying the late gene promoter. Images PMID:7961500

Nakayama, S; Kaneko, T; Ishimaru, H; Moriwaki, H; Mizobuchi, K

1994-01-01

229

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

PubMed Central

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. Images PMID:2506437

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

1989-01-01

230

Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence  

Microsoft Academic Search

Agrobacterium-mediatedinfection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petuniagenes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment

Jen-Chih Chen; Cai-Zhong Jiang; Timothy E. Gookin; Donald A. Hunter; David G. Clark; Michael S. Reid

2004-01-01

231

Case report: IN SILICO ANALYSIS OF TERPENE SYNTHASE GENES IN ARABIDOPSIS THALIANA  

Microsoft Academic Search

Terpenes are defense chemicals found in wide groups of plants. Terpenoids play a large role in plant development and stress response. The terpene synthase family comprises a diverse set of genes, all which contribute to production of terpenoids. We have used tools of bioinformatics and performed an in silico analysis of developmental and tissue specific terpene synthase gene expression in

Sam Zwenger; Chhandak Basu

232

Identical Mutation in SH3BP2 Gene Causes Clinical Phenotypes with Different Severity in Mother and Daughter - Case Report.  

PubMed

Cherubism is a particular form of fibrous dysplasia of the jaws. Familial occurrence was reported in most cases. The condition is a rare hereditary disorder with autosomal dominant inheritance, with complete penetrance in males and incomplete penetrance in females and variable expressivity. It is known to be caused by mutations in the gene encoding SH3-domain binding protein 2, SH3BP2 gene. Major diagnostic criteria are cherubic facial appearance, painless hard enlargement of the jaws, and frequently associated dental abnormalities. The aim of the study was to analyze clinical and genetic features of cherubism in a family with 3 daughters in which the youngest one was affected. Clinical and radiographic examinations, hematological and biochemical evaluations and biopsy were performed. Molecular genetic analysis consisted of PCR amplification and direct sequencing of selected exons of the SH3BP2 gene. Cherubism was suspected based on clinical and radiographic examinations of the 9-year-old daughter. She presented asymmetrical enlargement of the mandible, speech and swallowing problems and dental abnormalities on the lower jaw. There was no history of similar clinical findings in any of the daughters or the parents of the affected girl. Abnormal results were obtained by genetic analysis. A c.1244G>A mutation was identified in exon 9 of the SH3BP2 gene in the asymptomatic mother and her affected daughter. The identified mutation in the SH3BP2 gene is probably disease-causing. The asymptomatic mother transmitted the gene mutation to her affected daughter. Our results confirm the reduced penetrance and variable expression of the gene mutation. PMID:21045962

Preda, L; Dinca, O; Bucur, A; Dragomir, C; Severin, E

2010-01-01

233

On the Use of Gene Ontology Annotations to Assess Functional Similarity among Orthologs and Paralogs: A Short Report.  

PubMed

A recent paper (Nehrt et al., PLoS Comput. Biol. 7:e1002073, 2011) has proposed a metric for the "functional similarity" between two genes that uses only the Gene Ontology (GO) annotations directly derived from published experimental results. Applying this metric, the authors concluded that paralogous genes within the mouse genome or the human genome are more functionally similar on average than orthologous genes between these genomes, an unexpected result with broad implications if true. We suggest, based on both theoretical and empirical considerations, that this proposed metric should not be interpreted as a functional similarity, and therefore cannot be used to support any conclusions about the "ortholog conjecture" (or, more properly, the "ortholog functional conservation hypothesis"). First, we reexamine the case studies presented by Nehrt et al. as examples of orthologs with divergent functions, and come to a very different conclusion: they actually exemplify how GO annotations for orthologous genes provide complementary information about conserved biological functions. We then show that there is a global ascertainment bias in the experiment-based GO annotations for human and mouse genes: particular types of experiments tend to be performed in different model organisms. We conclude that the reported statistical differences in annotations between pairs of orthologous genes do not reflect differences in biological function, but rather complementarity in experimental approaches. Our results underscore two general considerations for researchers proposing novel types of analysis based on the GO: 1) that GO annotations are often incomplete, potentially in a biased manner, and subject to an "open world assumption" (absence of an annotation does not imply absence of a function), and 2) that conclusions drawn from a novel, large-scale GO analysis should whenever possible be supported by careful, in-depth examination of examples, to help ensure the conclusions have a justifiable biological basis. PMID:22359495

Thomas, Paul D; Wood, Valerie; Mungall, Christopher J; Lewis, Suzanna E; Blake, Judith A

2012-01-01

234

The first report of a Pelecaniformes defensin cluster: Characterization of ?-defensin genes in the crested ibis based on BAC libraries.  

PubMed

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 ?-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

Lan, Hong; Chen, Hui; Chen, Li-Cheng; Wang, Bei-Bing; Sun, Li; Ma, Mei-Ying; Fang, Sheng-Guo; Wan, Qiu-Hong

2014-01-01

235

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

PubMed Central

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

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

1993-01-01

236

Isolation and characterization of B-glucosidase gene and B-glucosidase of Trichoderma viride. Progress report  

SciTech Connect

The goal is to clone and characterize each of the cellulase genes from Trichoderma. This report is principally concerned with B-glucosidase. The induction of the Trichoderma cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. B-glucosidase has been isolated and purified to homogeneity. The enzyme contains significant amounts of carbohydrate and has a molecular weight greater than bovine serum albumin (68,000). (ACR)

Stafford, D.W.; Lundblad, R.L.

1982-03-25

237

Biodistribution of an adenoviral vector carrying the luciferase reporter gene following intravesical or intravenous administration to a mouse  

Microsoft Academic Search

The biodistribution and resulting pattern of transgene expression were determined following intravesical administration of an adenoviral vector carrying the luciferase reporter gene (AdLuc). Female BALB\\/c mice were subjected to intravesical instillation of 1 × 109 or 5 × 109 plaque-forming units of AdLuc. After sacrifice, transgene expression was detected in tissues using luciferase assays; vector DNA was detected by vector-specific

Mark Wood; Paul Perrotte; Eric Onishi; Mary E Harper; Colin Dinney; Lance Pagliaro; Deborah R Wilson

1999-01-01

238

Transposon tagging of disease resistance genes. Final report, May 1, 1988--April 30, 1993  

SciTech Connect

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.

Michelmore, R.

1994-09-01

239

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

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

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

2013-01-01

240

Structure and regulation of methanogen genes: Progress report, March 1, 1988--February 15, 1989  

SciTech Connect

The goals of this project were initially to obtain the primary structures of several cloned methanogen genes and subsequently to determine the mechanism(s) by which these genes are transcribed and how their expression is regulated. Studies have focused on both housekeeping genes encoding enzymes involved in the biosynthesis of amino acids (hisA, hisI, argG) and purines (purE) and on methane genes (mcrBDCGA; mvhDGAB) which encode enzymes (methyl coenzyme M reductase, methyl viologen-reducing hydrogenase) directly involved in the biosynthesis of methane. DNA-dependent-RNA-polymerases (RNAPs) have been purified from Methanococcus vannielii and Methanobacterium thermoautotrophicum and used in DNA-binding and footprinting studies to identify promoter sequences. Sites of transcription initiation in vivo have been shown to correlate well with the sites identified in vitro as RNAP binding sites. The mcr and mvh gene clusters contain open-reading frames (ORFs) which presumably encode polypeptides important in methyl reduction (mcrD and mcrC) and hydrogenase (mvhB) activity, which have yet to be identified in methanogenic cells. The mvhB gene appears to encode a new type of electron-transport protein, a poly-ferredoxin. 18 refs., 1 fig.

Reeve, J.N.

1989-01-01

241

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

SciTech Connect

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

Hofmann, A.; Garfinkel, M.D.; Meyerowitz, E.M. (California Inst. of Tech., Pasadena, CA (United States))

1991-06-01

242

Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements.  

PubMed

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes. PMID:1280329

Hamernik, D L; Keri, R A; Clay, C M; Clay, J N; Sherman, G B; Sawyer, H R; Nett, T M; Nilson, J H

1992-10-01

243

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

PubMed

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

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

1987-08-01

244

The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.  

PubMed

The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation. PMID:20221733

Souza, André L F; Invitti, Adriana L; Rego, Fabiane G M; Monteiro, Rose A; Klassen, Giseli; Souza, Emanuel M; Chubatsu, Leda S; Pedrosa, Fábio O; Rigo, Liu U

2010-02-01

245

A Fugu-Human Genome Synteny Viewer: web software for graphical display and annotation reports of synteny between Fugu genomic sequence and human genes  

Microsoft Academic Search

A web server has been developed to access anno- tation and graphical reports of synteny and gene order between the Fugu genome and human genes. In this system, the assembled Fugu genomic sequences (also known as scaffolds) are annotated. The annotations for each Fugu scaffold are com- puted, stored and made publicly available. The annotations describe matches to human homo-

Mark Halling-Brown; Clare Sansom; David S. Moss; Greg Elgar; Yvonne J. K. Edwards

2004-01-01

246

Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana  

PubMed Central

Background Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. Results We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. Conclusion We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation. PMID:22676624

2012-01-01

247

Developmental expression patterns of Arabidopsis XTH genes reported by transgenes and Genevestigator  

E-print Network

requires sophistication of construction and dy- namic remodeling of the plant cell wall. The major, xyloglucan endotransglucosylase/hydrolase Abstract The plant cell wall is the structural basis of cellular. XTHs are encoded by large gene families in plants; the Arabidopsis genome encodes 33 XTHs. To gain

Braam, Janet

248

Traits, Genes, Particles and Information: Re-Visiting Students' Understandings of Genetics. Research Report  

ERIC Educational Resources Information Center

Findings from a study of 10 German students aged 15-19, using problem-centred interviews, suggest that many students hold an 'everyday' conception of genes as small, trait-bearing, particles. Analysis of this notion identified a number of ways in which such a view might restrict the ability of students to develop an understanding of the scientific…

Lewis, Jenny; Kattmann, Ulrich

2004-01-01

249

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

PubMed Central

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

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

2014-01-01

250

Comprehensive Luciferase-Based Reporter Gene Assay Reveals Previously Masked Up-Regulatory Effects of miRNAs  

PubMed Central

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region) target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs. PMID:25192285

Campos-Melo, Danae; Droppelmann, Cristian A.; Volkening, Kathryn; Strong, Michael J.

2014-01-01

251

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

PubMed Central

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

2010-01-01

252

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 Central

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

Rainbow, Andrew J.

2013-01-01

253

Identification and functional analysis of a developmentally regulated extracellular signal-regulated kinase gene in Dictyostelium discoideum.  

PubMed Central

We have cloned a developmentally regulated mitogen-activated protein kinase (extracellular signal-regulated kinase) from Dictyostelium discoideum designated ERK1. Using anti-pTyr antibodies, we show that ERK1 is phosphorylated on tyrosine in vivo and that it will phosphorylate myelin basic protein. The gene expresses two transcripts, one that is preferentially expressed during vegetative growth and early development and one that is induced during the multicellular stages. Developmental Western blots (immunoblots) using anti-ERK1 antibodies indicate that ERK1 is present throughout development. ERK1/lacZ reporter constructs suggest that, in the multicellular stages, the gene is preferentially expressed in a subpopulation of cells scattered throughout the organism, similar to the pattern seen with anterior-like cell markers. Antisense mutagenesis from a derepressible promoter indicates that ERK1 is essential for vegetative growth. Overexpression of ERK1 from either the Actin 15 promoter or the ERK1 promoter results in abnormal morphogenesis starting at the slug stage. Overexpression of ERK1 in null mutants of the phosphotyrosine phosphatase PTP2 results in the production of large aggregation streams and subsequent abnormal morphogenesis that indicate a genetic interaction between ERK1 and PTP2. These cells produce very large aggregation streams that break up into very small mounds that undergo abnormal morphogenesis. The genetic interaction between ERK1 and PTP2 appears to be specific since overexpression of ERK1 in a ptp1- null mutant does not produce the same phenotype. Our results indicate that ERK1 plays an essential role during the growth and differentiation of D. discoideum. Images PMID:7935416

Gaskins, C; Maeda, M; Firtel, R A

1994-01-01

254

Estrogenicity ofFissure Sealants andAdhesive Resins Determined byReporter GeneAssay  

Microsoft Academic Search

Itiscontroversial whether thedental resinous materials containing 2,2-bis(4-(2-hydroxy-3- methacryloyloxypropoxy)phenyl)propane (Bis- GMA),whichissynthesized fromtheestrogenic compound bisphenol A (BPA), include unreacted BPA and\\/or canmimictheeffects ofnatural steroid hormones. Inthepresent study, the estrogenic activities of3fissure sealants and5 adhesive resins, whichwereallunpolymerized, weredetermined bymeansofareporter gene assay, andtherelevance ofthecomponents tothe estrogenicity wasinvestigated. Twocommercially available sealants wereconfirmed tohave estrogenic activity, although noneofthetested materials contained BPA.Incontrast, hydrophobic monomerbisphenol A dimethacrylate (BPA-

M. Matsuo

255

[Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report  

SciTech Connect

We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

Guerinot, M.L.

1992-06-01

256

Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of the gapA Gene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase of Corynebacterium glutamicum? †  

PubMed Central

SugR, RamA, GlxR, GntR1, and a MarR-type transcriptional regulator bind to the promoter region of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), essential for glycolysis in Corynebacterium glutamicum. We previously showed that SugR, a transcriptional repressor of phosphotransferase system genes for the sugar transport system, is involved in the downregulation of gapA expression in the absence of sugar. In this study, the role of RamA in the expression of the gapA gene was examined. Comparing the gapA expression and GAPDH activity of a ramA mutant with those of the wild type revealed that RamA is involved in upregulation of gapA expression in glucose-grown cells. DNase I footprint analyses and electrophoretic mobility shift assays revealed that RamA binds with different affinities to three sites in the gapA promoter. lacZ reporter assays with mutated RamA binding sites in the gapA promoter showed that the middle binding site is the most important for RamA to activate gapA expression and that binding of RamA to the gapA promoter activates the gene expression not only in glucose-grown cells but also in acetate-grown cells. Furthermore, RamA also directly activates sugR expression, indicating that two global regulators, RamA and SugR, are coordinately involved in the complex regulation of gapA expression in C. glutamicum. PMID:19047347

Toyoda, Koichi; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

2009-01-01

257

Characterization of three mnp genes of Fomitiporia mediterranea and report of additional class II peroxidases in the order hymenochaetales.  

PubMed

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

Morgenstern, Ingo; Robertson, Deborah L; Hibbett, David S

2010-10-01

258

Characterization of Three mnp Genes of Fomitiporia mediterranea and Report of Additional Class II Peroxidases in the Order Hymenochaetales ? †  

PubMed Central

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

Morgenstern, Ingo; Robertson, Deborah L.; Hibbett, David S.

2010-01-01

259

Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines.  

PubMed

It is important to obtain structure-activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N(4),N(9)-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N(4),N(9)-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N(4),N(9)-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000. PMID:22129427

Metwally, Abdelkader A; Reelfs, Olivier; Pourzand, Charareh; Blagbrough, Ian S

2012-07-01

260

Patient with Mutation in the Matrix Metalloproteinase 2 (MMP2) Gene - A Case Report and Review of the Literature  

PubMed Central

Torg and Winchester syndromes and patients reported by Al-AqeelSawairi as well as nodulosis-arthropathy-osteolysis (NAO) patients, patients with multicentric NAO share autosomal recessive inheritance. The common presenting symptomatology includes progressive osteolysis chiefly affecting the carpal, tarsal and interphalangeal joints. Here, we report a patient with Torg syndrome. Torg syndrome is caused by homozygous or compound heterozygous mutations in the matrix metalloproteinase 2 (MMP2) gene. MMP2 codes for a gelatinase that cleaves type IV collagen, a major component of basement membrane. The clinical presentation of our patient included moderate osteolysis of the small joints of the hands and knees, hirsutism, nodulosis sparing the palms and soles, corneal opacities and mild facial dysmorphism without gum hypertrophy. Genetic analysis showed that the patient was homozygous for a novel base variant c538 G>A (p.D180N) in the MMP2 gene. Both parents were carriers of the same mutated variant. Our patient had some previously unreported endocrine manifestations such as premature thelarche and elevated follicle-stimulating hormone levels. PMID:24637309

Ekbote, Alka V.; Danda, Sumita; Zankl, Andreas; Mandal, Kausik; Maguire, Tina; Ungerer, Kobus

2014-01-01

261

Generation of transgenic mesenchymal stem cells expressing green fluorescent protein as reporter gene using no viral vector in caprine.  

PubMed

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. While there are many ways to label and track cells each with strengths and weakness, the green fluorescent protein (GFP) is a reporter gene commonly employed. In the present study, caprine MSC were collected from bone marrow and cells were characterised with MSC specific markers. Passage 10 (P10) MSC cells were transfected using plasmid vector containing GFP as reporter gene with different concentrations of DNA and lipofectamine. Six different concentrations of DNA and lipofectamine as 1 microg DNA: 2 microL lipofectamine, 1 microg DNA: 2.5 microL lipofectamine, 1.2 microg DNA: 2.2 microL lipofectamine, 1.2 microg DNA: 2.5 microL lipofectamine, 1.5 microg DNA: 2.5 microL lipofectamine, 1.5 microg DNA: 3 microL lipofectamine were used. After 24 h and 48 h of transfection, caprine MSC were observed under florescent microscope. Highest transfection rate indicating green flourecscent MSC were found when the cells were transfected with 1.2 microg DNA: 2.2 microL lipofectamine and 1.5 microg DNA: 2.5 microL lipofectamine than other combinations. These cells have been propagated beyond 4th passage maintaining GFP expression. The results indicated that stable GFP positive MSC cells can be generated using the above protocol. These cells are being used for transplantation studies. PMID:23898548

Kumar, Manish; Yasotha, T; Singh, R K; Singh, Renu; Kumar, Kuldeep; Ranjan, R; Meshram, Chetan D; Das, B C; Bag, Sadhan

2013-07-01

262

Molecular Analysis of Bardet-Biedl Syndrome Families: Report of 21 Novel Mutations in 10 Genes  

PubMed Central

Purpose. Bardet-Biedl syndrome (BBS) is genetically heterogeneous with 15 BBS genes currently identified, accounting for approximately 70% of cases. The aim of our study was to define further the spectrum of BBS mutations in a cohort of 44 European-derived American, 8 Tunisian, 1 Arabic, and 2 Pakistani families (55 families in total) with BBS. Methods. A total of 142 exons of the first 12 BBS-causing genes were screened by dideoxy sequencing. Cases in which no mutations were found were then screened for BBS13, BBS14, BBS15, RPGRIP1L, CC2D2A, NPHP3, TMEM67, and INPP5E. Results. Forty-three mutations, including 8 frameshift mutations, 10 nonsense mutations, 4 splice site mutations, 1 deletion, and 20 potentially or probably pathogenic missense variations, were identified in 46 of the 55 families studied (84%). Of these, 21 (2 frameshift mutations, 4 nonsense mutations, 4 splice site mutations, 1 deletion, and 10 missense variations) were novel. The molecular genetic findings raised the possibility of triallelic inheritance in 7 Caucasian families, 1 Arabian family, and 1 Tunisian patient. No mutations were detected for BBS4, BBS11, BBS13, BBS14, BBS15, RPGRIP1L, CC2D2A, NPHP3, TMEM67, or INPP5E. Conclusions. This mutational analysis extends the spectrum of known BBS mutations. Identification of 21 novel mutations highlights the genetic heterogeneity of this disorder. Differences in European and Tunisian patients, including the high frequency of the M390R mutation in Europeans, emphasize the population specificity of BBS mutations with potential diagnostic implications. The existence of some BBS cases without mutations in any currently identified BBS genes suggests further genetic heterogeneity. PMID:21642631

Chen, Jianjun; Smaoui, Nizar; Hammer, Monia Ben Hamed; Jiao, Xiaodong; Riazuddin, S. Amer; Harper, Shyana; Katsanis, Nicholas; Riazuddin, Sheikh; Chaabouni, Habiba; Berson, Eliot L.

2011-01-01

263

Report on the second international workshop on the CCN family of genes  

PubMed Central

For the second time, researchers from leading laboratories in the CCN field gathered in Saint-Malo, France, to participate in the Second International Workshop on the CCN family of genes. In addition to the regular research communications, meeting highlights included the inauguration of the first CCN newsletter (http://ccnnewsletter.free.fr) and the recognition of the International CCN Society (http://www.ccnsociety.jussieu.fr) as an important medium for the exchange of scientific knowledge and resources in the CCN field. Once more, the high quality of scientific communications and individual interactions set the stage for an extremely fruitful meeting. PMID:12665625

Perbal, B; Brigstock, D R; Lau, L F

2003-01-01

264

Primary gene products as plant defenses. Technical progress report, July 1, 1982-June 30, 1983  

SciTech Connect

The Bowman-Birk proteinase inhibitor (BBI) and its related family of isoinhibitors (PI I-IV) from soybean represent a group of polypeptides that are closely related in their amino acid sequences. Interest in the proteins comes from their high content of sulfur amine acids and the potential use of their proteinase inhibitor function in natural plant protection. The major focus of this study was to obtain a cDNA probe for the BBI and PI I-IV by standard recombinant DNA techniques. These probes were used to characterize the messenger RNA and to isolate and characterize the genes coding for these polypeptides. (ACR)

Not Available

1983-01-01

265

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

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.

Dutta, S.K.

1988-03-01

266

Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coli.  

PubMed Central

Expression of the Saccharomyces cerevisiae PIS gene encoding phosphatidylinositol synthase in Escherichia coli was achieved by inserting its coding sequence into lacZ on pUC8. The fused gene encoded a phosphatidylinositol synthase whose amino-terminal three amino acids had been replaced by the amino-terminal five amino acids of E. coli beta-galactosidase. E. coli cells bearing this recombinant plasmid produced a significant level of phosphatidylinositol synthase in the presence of a lacZ inducer, isopropylthio-beta-D-galactopyranoside. When the culture medium was supplemented with myo-inositol and isopropylthio-beta-D-galactopyranoside, the cells accumulated a substantial amount of phosphatidylinositol in their membranes. When a saturating level of myo-inositol was added, phosphatidylinositol constituted about 4% of the total phospholipids. Phosphatidylinositol accumulation occurred at the expense of phosphatidylglycerol. The ratio of phosphatidylethanolamine to total acidic phospholipids remained constant. The growth rate of phosphatidylinositol-containing E. coli cells did not differ significantly from that of cells with the normal phospholipid composition. Images PMID:2844726

Nikawa, J; Kodaki, T; Yamashita, S

1988-01-01

267

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

PubMed

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

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

2013-12-17

268

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

SciTech Connect

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.

Jirtle, Randy L.

2012-10-11

269

Neonatal Lethality, Dwarfism, and Abnormal Brain Development in Dmbx1 Mutant Mice  

Microsoft Academic Search

Dmbx1 encodes a paired-like homeodomain protein that is expressed in developing neural tissues during mouse embryogenesis. To elucidate the in vivo role of Dmbx1, we generated two Dmbx1 mutant alleles. Dmbx1 lacks the homeobox and Dmbx1z is an insertion of a lacZ reporter gene. Dmbx1z appears to be a faithful reporter of Dmbx1 expression during embryogenesis and after birth. Dmbx1-lacZ

Akihira Ohtoshi; Richard R. Behringer

2004-01-01

270

Phenylalanine hydroxylase gene mutations in the United States: report from the Maternal PKU Collaborative Study.  

PubMed

The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g-->a, and Y414C, accounting for 18.7%, 7.8%, and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies < or = 1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment of mutations has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. PMID:8659548

Guldberg, P; Levy, H L; Hanley, W B; Koch, R; Matalon, R; Rouse, B M; Trefz, F; de la Cruz, F; Henriksen, K F; Güttler, F

1996-07-01

271

Phenylalanine hydroxylase gene mutations in the United States: report from the Maternal PKU Collaborative Study.  

PubMed Central

The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g-->a, and Y414C, accounting for 18.7%, 7.8%, and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies < or = 1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment of mutations has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. Images Figure 1 PMID:8659548

Guldberg, P.; Levy, H. L.; Hanley, W. B.; Koch, R.; Matalon, R.; Rouse, B. M.; Trefz, F.; de la Cruz, F.; Henriksen, K. F.; Guttler, F.

1996-01-01

272

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

SciTech Connect

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.

Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others

1996-07-01

273

716. The Human Norepinephrine Transporter and [11C]-mHED, a Novel Reporter Gene-Tracer Combination for PET Imaging of Gene Therapy  

Microsoft Academic Search

Currently many clinical protocols for gene therapy are being evaluated for use in the treatment of human disease. In the majority of these protocols however, it is difficult to determine the exact fate of the vector, or to determine the location and extend of expression of the introduced gene. Data about the expression of transgenes is not only invaluable in

Antoine M. J. Beerens; Anne Rixt Buursma; Marianne G. Rots; Aren van Waarde; Hidde J. Haisma; Erik F. J. de Vries

2004-01-01

274

Connexin 47 (Cx47)Deficient Mice with Enhanced Green Fluorescent Protein Reporter Gene Reveal Predominant Oligodendrocytic Expression of Cx47 and Display Vacuolized Myelin in the CNS  

Microsoft Academic Search

To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no obvious morphological or behavioral abnormalities. Colocaliza- tion of EGFP fluorescence

Benjamin Odermatt; Kerstin Wellershaus; Anke Wallraff; Gerald Seifert; Joachim Degen; Carsten Euwens; Babette Fuss; Heinrich Bussow; Karl Schilling; Christian Steinhauser; Klaus Willecke

275

An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.  

PubMed

The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli. PMID:19214389

Seo, Jeong-Woo; Hong, Won-kyung; Rairakhwada, Dina; Seo, Pil-Soo; Choi, Min Ho; Song, Ki-Bang; Rhee, Sang-Ki; Kim, Chul Ho

2009-06-01

276

A report of a national mutation testing service for the MEN1 gene: clinical presentations and implications for mutation testing  

PubMed Central

Introduction: Mutation testing for the MEN1 gene is a useful method to diagnose and predict individuals who either have or will develop multiple endocrine neoplasia type 1 (MEN 1). Clinical selection criteria to identify patients who should be tested are needed, as mutation analysis is costly and time consuming. This study is a report of an Australian national mutation testing service for the MEN1 gene from referred patients with classical MEN 1 and various MEN 1-like conditions. Results: All 55 MEN1 mutation positive patients had a family history of hyperparathyroidism, had hyperparathyroidism with one other MEN1 related tumour, or had hyperparathyroidism with multiglandular hyperplasia at a young age. We found 42 separate mutations and six recurring mutations from unrelated families, and evidence for a founder effect in five families with the same mutation. Discussion: Our results indicate that mutations in genes other than MEN1 may cause familial isolated hyperparathyroidism and familial isolated pituitary tumours. Conclusions: We therefore suggest that routine germline MEN1 mutation testing of all cases of "classical" MEN1, familial hyperparathyroidism, and sporadic hyperparathyroidism with one other MEN1 related condition is justified by national testing services. We do not recommend routine sequencing of the promoter region between nucleotides 1234 and 1758 (Genbank accession no. U93237) as we could not detect any sequence variations within this region in any familial or sporadic cases of MEN1 related conditions lacking a MEN1 mutation. We also suggest that testing be considered for patients <30 years old with sporadic hyperparathyroidism and multigland hyperplasia. PMID:15635078

Cardinal, J; Bergman, L; Hayward, N; Sweet, A; Warner, J; Marks, L; Learoyd, D; Dwight, T; Robinson, B; Epstein, M; Smith, M; Teh, B; Cameron, D; Prins, J

2005-01-01

277

Tomato ringspot virus coat protein binds to ARGONAUTE 1 and suppresses the translation repression of a reporter gene.  

PubMed

RNA silencing regulates plant gene expression and antiviral defenses and functions by cleaving target RNAs or repressing translation. As a counter defense, many plant viruses encode suppressor proteins that sequester small RNAs or inactivate Argonaute (AGO) proteins. All known plant virus silencing suppressor activities eventually inhibit the degradation of target mRNAs. Using a transiently expressed green fluorescent protein (GFP) reporter gene, we show that Tomato ringspot virus (ToRSV) coat protein (CP) is a suppressor of RNA silencing that enhances GFP expression but does not prevent the degradation of the GFP mRNA or the accumulation of GFP small interfering RNAs (siRNAs). Coexpression of the CP with GFP resulted in increased association of residual GFP mRNAs with polysome fractions and reduced association of GFP siRNAs with monosome fractions. AGO1 was co-immunoprecipitated with the CP and CP expression destabilized AGO1. A WG motif within the CP was critical for the enhanced GFP expression, AGO1 interaction, and AGO1 destabilization, suggesting that the ToRSV CP acts as an AGO-hook protein and competes for AGO binding with a plant cellular GW/WG protein involved in translation repression. PMID:24804809

Karran, Rajita A; Sanfaçon, Hélčne

2014-09-01

278

In Vivo Bioluminescence Tumor Imaging of RGD Peptide-modified Adenoviral Vector Encoding Firefly Luciferase Reporter Gene  

PubMed Central

Purpose The goal of this study is to demonstrate the feasibility of chemically modified human adenovirus (Ad) vectors for tumor retargeting. Procedures E1- and E3-deleted Ad vectors carrying firefly luciferase reporter gene under cytomegalovirus promoter (AdLuc) was surface-modified with cyclic arginine–glycine–aspartic acid (RGD) peptides through a bifunctional poly(ethyleneglycol) linker (RGD-PEG-AdLuc) for integrin ?v?3 specific delivery. The Coxsackie and adenovirus viral receptor (CAR) and integrin ?v?3 expression in various tumor cell lines was determined by reverse transcriptase PCR and fluorescence-activated cell sorting. Bioluminescence imaging was performed in vitro and in vivo to evaluate RGD-modified AdLuc infectivity. Results RGD-PEG-AdLuc abrogated the native CAR tropism and exhibited significantly enhanced transduction efficiency of integrin-positive tumors than AdLuc through intravenous administration. Conclusion This approach provides a robust platform for site-specific gene delivery and noninvasive monitoring of the transgene delivery efficacy and homing. PMID:17297551

Niu, Gang; Xiong, Zhengming; Cheng, Zhen; Cai, Weibo; Gambhir, Sanjiv S.; Xing, Lei; Chen, Xiaoyuan

2014-01-01

279

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

PubMed Central

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

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

1988-01-01

280

Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system.  

PubMed

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format. PMID:18655041

Nanda, Kamna; Chatterjee, Mou; Arya, Ranjana; Mukherjee, Shohini; Saini, Kulvinder Singh; Dastidar, Sunanda; Ray, Abhijit

2008-10-01

281

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

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

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

2014-07-01

282

Reliable Gene Expression Analysis by Reverse Transcription-Quantitative PCR: Reporting and Minimizing the Uncertainty in Data Accuracy[W][OPEN  

PubMed Central

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

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

2014-01-01

283

A dynamic FRET reporter of gene expression improved by functional screening.  

PubMed

Here, we describe a reporter system that consists of a FRET biosensor and its corresponding aptamer. The FRET biosensor employs the synthetic aptamer binding peptide Rsg1.2 sandwiched between mutants of the Green Fluorescent Protein and undergoes FRET when binding its corresponding Rev Responsive Element (RRE) RNA aptamer. We developed a novel approach to engineer FRET biosensors by linker extension and screening to improve signal strength of the biosensor which we called VAmPIRe (Viral Aptamer binding Peptide based Indicator for RNA detection). We demonstrate that the system is quantitative, reversible and works with high specificity in vitro and in vivo in living bacteria and mammalian cells. Thus, VAmPIRe may become valuable for RNA localizations and as a dynamic RNA-based reporter for live cell imaging. Moreover, functional screening of large libraries as demonstrated here may become applicable to optimize some of the many FRET biosensors of cellular signaling. PMID:22946509

Schifferer, Martina; Griesbeck, Oliver

2012-09-19

284

Estrogenicity of Fissure Sealants and Adhesive Resins Determined by Reporter Gene Assay  

Microsoft Academic Search

It is controversial whether the dental resinous materials containing 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)phenyl] propane (BisGMA), which is synthesized from the estrogenic compound bisphenol A (BPA), include unreacted BPA and\\/or can mimic the effects of natural steroid hormones. In the present study, the estrogenic activities of 3 fissure sealants and 5 adhesive resins, which were all unpolymerized, were determined by means of a reporter

H. Tarumi; S. Imazato; M. Narimatsu; M. Matsuo; S. Ebisu

2000-01-01

285

Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter  

NASA Astrophysics Data System (ADS)

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 ?l s.c., on the ventral side of the left thigh. Then mouse was given 250 ?l 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.

Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

2012-03-01

286

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

PubMed Central

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 = 1.27–2.59, 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 = 1.46–2.96, P = 0.0001; GSTT1: controlled OR = 2.80, 95% CI = 1.65–4.75, 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

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

2007-01-01

287

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

Microsoft Academic Search

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicansACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of\\u000a the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous,

J. Morschhäuser; S. Michel; J. Hacker

1998-01-01

288

Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle.  

PubMed

Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.Molecular Therapy - Nucleic Acids (2013) 2, e86; doi:10.1038/mtna.2013.16; published online 16 April 2013. PMID:23591809

Wallace, Lindsay M; Moreo, Andrew; Clark, K Reed; Harper, Scott Q

2013-01-01

289

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns  

PubMed Central

Background Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. Results A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. Conclusions Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community. PMID:20687964

2010-01-01

290

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis  

PubMed Central

Background The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. Results We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in ?-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Conclusion Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in ?-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2. PMID:19552825

2009-01-01

291

The PBP 5 synthesis repressor ( psr) gene of Enterococcus hirae ATCC 9790 is substantially longer than previously reported  

Microsoft Academic Search

A reexamination of the nucleotide sequence of the psr gene of Enterococcus hirae revealed the presence of two additional nucleotides at residues 1190 and 1191. As a result, instead of a stop codon after 148 aa, the psr gene product would contain 293 aa residues. The revised size of the gene product was confirmed by subsequently cloning and expressing the

Orietta Massidda; Olivier Dardenne; Michael B. Whalen; Willy Zorzi; jacques Coyette; Gerald D. Shockman; Lolita Daneo-moore

1998-01-01

292

The Vibrio cholerae fatty acid regulatory protein, FadR, represses transcription of plsB, the gene encoding the first enzyme of membrane phospholipid biosynthesis.  

PubMed

Glycerol-3-phosphate (sn-glycerol-3-P, G3P) acyltransferase catalyses the first committed step in the biosynthesis of membrane phospholipids, the acylation of G3P to form 1-acyl G3P (lysophosphatidic acid). The paradigm G3P acyltransferase is the Escherichia coli plsB gene product which acylates position-1 of G3P using fatty acids in thioester linkage to either acyl carrier protein (ACP) or CoA as acyl donors. Although the E. coli plsB gene was discovered about 30 years ago, no evidence for transcriptional control of its expression has been reported. However A.E. Kazakov and co-workers (J Bacteriol 2009; 191: 52-64) reported the presence of a putative FadR binding site upstream of the candidate plsB genes of Vibrio cholerae and three other Vibrio species suggesting that plsB might be regulated by FadR, a GntR family transcription factor thus far known only to regulate fatty acid synthesis and degradation. We report that the V. cholerae plsB homologue restored growth of E. coli strain BB26-36 which is a G3P auxotroph due to an altered G3P acyltransferase activity. The plsB promoter was also mapped and the predicted FadR-binding palindrome was found to span positions -19 to -35, upstream of the transcription start site. Gel shift assays confirmed that both V. cholerae FadR and E. coli FadR bound the V. cholerae plsB promoter region and binding was reversed upon addition of long-chain fatty acyl-CoA thioesters. The expression level of the V. cholerae plsB gene was elevated two- to threefold in an E. coli fadR null mutant strain indicating that FadR acts as a repressor of V. cholerae plsB expression. In both E. coli and V. cholerae the ?-galactosidase activity of transcriptional fusions of the V. cholerae plsB promoter to lacZ increased two- to threefold upon supplementation of growth media with oleic acid. Therefore, V. cholerae co-ordinates fatty acid metabolism with 1-acyl G3P synthesis. PMID:21771112

Feng, Youjun; Cronan, John E

2011-08-01

293

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

PubMed

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

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

2013-12-01

294

Use of optical reporter genes to assess sublethal cellular damage following skin ablation  

NASA Astrophysics Data System (ADS)

Numerous medical procedures utilize pulsed lasers to remove unwanted biological tissue. Mid-infrared wavelengths which preferentially target protein absorption bands ablate tissue more efficiently than wavelengths targeting water absorption. However, the mechanism responsible for this finding has not been established. In this report, we combine optical imaging and conventional techniques to assess lethal and sublethal collateral damage after ablative surgery with a Free Electron Laser (FEL). Heat shock protein expression was used to evaluate tissue damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and GFP expression (hsp70A1-L2G). To examine wavelength-dependence in the mid-IR, laser surgery was conducted on the hsp70A1-L2G mouse model using wavelengths targeting protein (amide II band, 6.45 ?m), both water and protein (amide I band, 6.10 ?m) and water (2.94 ?m). Hsp70-driven luciferase activity was used as a quantitative biomarker for intracellular damage, and histological analyses were conducted to measure the depth of thermal damage. For all of the wavelengths tested, the bioluminescent data showed that the magnitude of hsp70 expression was dose-dependent. Tissues treated at 6.45 µm had approximately 2x higher hsp70 expression than tissues treated at 6.10 ?m. Histology showed that immediate tissue injury at the 6.45 ?m wavelength was ~2x deeper than at 6.10 ?m. The 6.10 ?m wavelength generated the least amount of epidermal hyperplasia. Overall, the data suggests that 6.10 ?m is a superior wavelength for cutaneous laser ablation procedures.

Wilmink, Gerald J.; Opalenik, Susan R.; Davidson, Jeffrey M.; Jansen, E. Duco

2008-02-01

295

Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli  

PubMed Central

Background Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude. Results The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP). It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population. Conclusion The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. PMID:19243588

Delvigne, Frank; Boxus, Mathieu; Ingels, Sophie; Thonart, Philippe

2009-01-01

296

The Rok protein of Bacillus subtilis represses genes for cell surface and extracellular functions.  

PubMed

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens group by horizontal transmission. PMID:15743949

Albano, Mark; Smits, Wiep Klaas; Ho, Linh T Y; Kraigher, Barbara; Mandic-Mulec, Ines; Kuipers, Oscar P; Dubnau, David

2005-03-01

297

GCR3 encodes an acidic protein that is required for expression of glycolytic genes in Saccharomyces cerevisiae.  

PubMed Central

Screening of a mutagenized strain carrying a multicopy ENO1-'lacZ fusion plasmid revealed a new mutation affecting several glycolytic enzyme activities. The recessive single nuclear gene mutation, named gcr3, caused an extremely defective growth phenotype on fermentable carbon sources such as glucose, while growth on respiratory media was almost normal. The GCR3 gene was obtained by growth complementation from a genomic DNA library, and the complemented strains had normal enzyme levels. GCR3 gene was sequenced, and a 99,537-Da protein was predicted. The predicted GCR3 protein was fairly acidic (net charge, -34). The C-terminal region was highly charged, and an acidic stretch was found in it. Images PMID:1512188

Uemura, H; Jigami, Y

1992-01-01

298

Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems.  

PubMed

Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system. PMID:18310895

Satoh, Kanako; Nonaka, Ryouichi; Ohyama, Ken-ichi; Nagai, Fumiko; Ogata, Akio; Iida, Mitsuru

2008-03-01

299

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

PubMed

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

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

2014-07-01

300

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice  

PubMed Central

Background Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics. Results In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-?-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation. Conclusion We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy. PMID:19128466

Bergwerf, Irene; De Vocht, Nathalie; Tambuyzer, Bart; Verschueren, Jacob; Reekmans, Kristien; Daans, Jasmijn; Ibrahimi, Abdelilah; Van Tendeloo, Viggo; Chatterjee, Shyama; Goossens, Herman; Jorens, Philippe G; Baekelandt, Veerle; Ysebaert, Dirk; Van Marck, Eric; Berneman, Zwi N; Linden, Annemie Van Der; Ponsaerts, Peter

2009-01-01

301

Optic atrophy and a Leigh-like syndrome due to mutations in the c12orf65 gene: report of a novel mutation and review of the literature.  

PubMed

Combined oxidative phosphorylation deficiency type 7 (COXPD7) is a rare disorder of mitochondrial metabolism that results in optic atrophy and Leigh syndrome-like disease. We describe 2 siblings with compound heterozygous mutations in the recently identified C12orf65 gene who presented with optic atrophy and mild developmental delays and subsequently developed bilateral, symmetric lesions in the brainstem reminiscent of Leigh syndrome. Repeat neuroimaging demonstrated reversibility of the findings in 1 sibling and persistent metabolic stroke in the other. This article highlights the phenotypic manifestations from a novel mutation in the C12orf65 gene and reviews the clinical presentation of the 5 other individuals reported to date who carry mutations in this gene. PMID:24284555

Heidary, Gena; Calderwood, Laurel; Cox, Gerald F; Robson, Caroline D; Teot, Lisa A; Mullon, Jennifer; Anselm, Irina

2014-03-01

302

Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica  

Microsoft Academic Search

The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for

Gabriel Rinaldi; Maria E. Morales; Martín Cancela; Estela Castillo; Paul J. Brindley; José F. Tort

2008-01-01

303

Use of bgaH as a reporter gene for studying translation initiation in the archaeon Haloferax volcanii  

E-print Network

The bgaH gene isolated from Haloferax lucentensis codes for P-galactosidase. To study the function of initiator tRNAs in translation initiation in Haloferax volcanii, the initiator AUG codon of the bgaH gene was mutated ...

Sullivan, Eric L., S.M. Massachusetts Institute of Technology

2008-01-01

304

Combination of fluorescent reporters for simultaneous monitoring of root colonization and antifungal gene expression by a biocontrol pseudomonad on cereals with flow cytometry.  

PubMed

Some root-associated pseudomonads sustain plant growth by suppressing root diseases caused by pathogenic fungi. We investigated to which extent select cereal cultivars influence expression of relevant biocontrol traits (i.e., root colonization efficacy and antifungal activity) in Pseudomonas fluorescens CHA0. In this representative plant-beneficial bacterium, the antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), and hydrogen cyanide (HCN) are required for biocontrol. To monitor host plant effects on the expression of biosynthetic genes for these compounds on roots, we developed fluorescent dual-color reporters suited for flow cytometric analysis using fluorescence-activated cell sorting (FACS). In the dual-label strains, the constitutively expressed red fluorescent protein mCherry served as a cell tag and marker for root colonization, whereas reporter fusions based on the green fluorescent protein allowed simultaneous recording of antifungal gene expression within the same cell. FACS analysis revealed that expression of DAPG and PRN biosynthetic genes was promoted in a cereal rhizosphere, whereas expression of PLT and HCN biosynthetic genes was markedly less sustained. When analyzing the response of the bacterial reporters on roots of a selection of wheat, spelt, and triticale cultivars, we were able to detect subtle species- and cultivar-dependent differences in colonization and DAPG and HCN gene expression levels. The expression of these biocontrol traits was particularly favored on roots of one spelt cultivar, suggesting that a careful choice of pseudomonad-cereal combinations might be beneficial to biocontrol. Our approach may be useful for selective single-cell level analysis of plant effects in other bacteria-root interactions. PMID:20521957

Rochat, Laurčne; Péchy-Tarr, Maria; Baehler, Eric; Maurhofer, Monika; Keel, Christoph

2010-07-01

305

Isolated duodenal myeloid sarcoma associated with the CBF?/MYH11 fusion gene followed by acute myeloid leukemia progression: A case report and literature review  

PubMed Central

Myeloid sarcoma (MS) is a rare disease that presents as an extramedullary tumorous mass of immature myeloid precursors. The majority of MS are identified in acute myeloid leukemia (AML) patients and rarely present as a primary isolated MS without AML. In addition, inversion of chromosome 16 [inv(16)] and the CBF?/MYH11 fusion gene are rarely associated with MS. The current study reports a female patient with an isolated duodenal MS, who developed AML-M4 associated with the CBF?/MYH11 fusion gene and 48,XX,inv(16),+13,+22. A review of previously reported cases of isolated MS with the CBF?/MYH11 fusion gene was also performed. Isolated MS with the CBF?/MYH11 fusion gene was often observed in abdominal lesions, with the intestinal tract being the predominantly involved site. In addition, patients with isolated MS with the CBF?/MYH11 fusion exhibited a high risk of developing systemic AML. The diagnosis of isolated MS may be particularly challenging and, therefore, determining the optimal standard treatment for isolated MS is required. PMID:25120702

HUANG, BO; YOU, PENG; ZHU, PING; DU, ZUNGUO; WU, BEIQIAN; XU, XIAOPING; CHEN, ZI

2014-01-01

306

Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.  

PubMed Central

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

Maciver, B; McHale, R H; Saul, D J; Bergquist, P L

1994-01-01

307

Inferring candidate genes for Attention Deficit Hyperactivity Disorder (ADHD) assessed by the World Health Organization Adult ADHD Self-Report Scale (ASRS)  

Microsoft Academic Search

Summary.  The present study tests the psychometric properties and validity of the German version of the World Health Organization Adult\\u000a Attention Deficit Hyperactivity Disorder (ADHD) Self-Report Scale (ASRS), which is a short screening instrument for use in\\u000a the general population. Furthermore, two candidate genes for ADHD, the COMT VAL158MET and the 5-HT2a T102C polymorphisms,\\u000a were tested for associations with the ASRS

M. Reuter; P. Kirsch; J. Hennig

2006-01-01

308

Interventional magnetic resonance imaging for guiding gene and cell transfer in the heart  

PubMed Central

Background: Interventional magnetic resonance imaging (iMRI) has the potential for guiding interventional cardiac procedures in real time. Objectives: To test the feasibility of iMRI guided gene and cell transfer to the heart and to monitor myocardial remodelling after myocardial infarction in a rat model. Methods: The MRI contrast agent GdDTPA, together with either Evans blue dye, or a recombinant adenovirus encoding the LacZ gene, or primary fibroblasts tagged by BrdU, were injected into the myocardium of rats under iMRI guidance. Rats were killed seven days after the injection and the hearts sectioned to identify the blue dye, LacZ expression, or fibroblast presence, respectively. In a parallel study, left ventricular area was measured before and after myocardial infarction and in sham operated rats by T1 weighted MRI and by echocardiography. Results: Location of GdDTPA enhancement observed with iMRI at the time of injection was correlated with Evans blue stain, ?-gal expression, and the primary fibroblast location in histological studies. iMRI and echocardiography measured a comparable increase in left ventricular area at seven and 30 days after myocardial infarction. A good correlation was found between the iMRI and echocardiographic assessment of left ventricular area (r ?=? 0.70; p < 0.0001) and change in left ventricular area with time (r ?=? 0.75; p < 0.0001). Conclusions: The results show the feasibility and efficiency of iMRI guided intramyocardial injections, and the ability to monitor heart remodelling using iMRI. Genes, proteins, or cells for tissue engineering could be injected accurately into the myocardial scar under iMRI guidance. PMID:14676253

Barbash, I M; Leor, J; Feinberg, M S; Tessone, A; Aboulafia-Etzion, S; Orenstein, A; Ruiz-Cabello, J; Cohen, J S; Mardor, Y

2004-01-01

309

SSTR2-based reporters for assessing gene transfer into non-small cell lung cancer: evaluation using an intrathoracic mouse model.  

PubMed

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

Singh, S P; Han, L; Murali, R; Solis, L; Roth, J; Ji, L; Wistuba, I; Kundra, V

2011-01-01

310

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

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.

Arranz, M.; Sharma, T.; Sham, P.; Kerwin, R. [Institute of Psychiatry, London (United Kingdom)] [and others

1995-10-09

311

Suicidal Behavior and Haplotypes of the Dopamine Receptor Gene (DRD2) and ANKK1 Gene Polymorphisms in Patients with Alcohol Dependence – Preliminary Report  

PubMed Central

Suicide is a significant public health issue and a major cause of death throughout the world. According to WHO it accounts for almost 2% of deaths worldwide. The etiology of suicidal behavior is complex but the results of many studies suggest that genetic determinants are of significant importance. In our study,- we have analyzed selected SNPs polymorphisms in the DRD2 and ANKK1 genes in patients with alcohol dependence syndrome (169 Caucasian subjects) including a subgroup of individuals (n?=?61) who have experienced at least one suicide attempt. The aim of the study was to verify if various haplotypes of selected genes, comprising Taq1A, Taq1B, and Taq1D single nucleotide polymorphisms (SNP), play any role in the development of alcohol dependence and suicidal behavior. The control group comprised 157 unrelated individuals matched for ethnicity, gender,- and age and included no individuals with mental disorders. All subjects were recruited in the North West region of Poland. The study showed that alcohol dependent subjects with a history of at least one suicidal attempt were characterized by a significantly higher frequency of the T-G-A2 haplotype when compared to individuals in whom alcohol dependence was not associated with suicidal behavior (p?=?0.006). It appears that studies based on identifying correlation between SNPs is the future for research on genetic risk factors that contribute to the development of alcohol addiction and other associated disorders. To sum up, there is a necessity to perform further research to explain dependencies between the dopaminergic system, alcohol use disorders and suicidal behavior. PMID:25415204

Jasiewicz, Andrzej; Samochowiec, Agnieszka; Samochowiec, Jerzy; Ma?ecka, Iwona; Suchanecka, Aleksandra; Grzywacz, Anna

2014-01-01

312

(The isolation and characterization of beta-glucosidase gene and beta-glucosidase of Trichoderma viride): Progress report  

SciTech Connect

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.

Stafford, D.W.

1983-01-01

313

Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1988--April 14, 1989  

SciTech Connect

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.

Okita, T.W.

1989-12-31

314

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report  

NASA Technical Reports Server (NTRS)

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.

Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

2001-01-01

315

Systematic analysis of RNAi reports identifies dismal commonality at gene-level and reveals an unprecedented enrichment in pooled shRNA screens.  

PubMed

RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes. PMID:23848309

Bhinder, Bhavneet; Djaballah, Hakim

2013-11-01

316

Systematic analysis of RNAi reports identifies dismal commonality at gene-level & reveals an unprecedented enrichment in pooled shRNA screens  

PubMed Central

RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes. PMID:23848309

Bhinder, Bhavneet; Djaballah, Hakim

2013-01-01

317

Mutations in the Spt4, Spt5, and Spt6 Genes Alter Transcription of a Subset of Histone Genes in Saccharomyces Cerevisiae  

PubMed Central

The SPT4, SPT5, and SPT6 gene products define a class of transcriptional repressors in Saccharomyces cerevisiae that are thought to function through their effects on chromatin assembly or stability. Mutations in these genes confer a similar range of phenotypes to mutations in HIR genes, which encode transcriptional repressors that regulate expression of many of the core histone genes. Here we show that mutations in the three SPT genes also affect transcription of the histone genes that reside at the HTA1-HTB1 locus. HTA1-lacZ transcription was reduced in each spt mutant background, an effect that required a negative site in the HTA1 promoter. The transcriptional effect could be reversed by the overproduction of histones H2A and H2B in an spt4 mutant and histones H3 and H4 in all three spt mutants. Suppression of the spt4 transcriptional defect was dependent on the overproduction of both histones H2A and H2B, and required the presence of N-terminal amino acids in both histones. The results are consistent with the idea that the effects of the spt mutations on nucleosome assembly and/or stability activate repressors of HTA1 transcription. PMID:8844144

Compagnone-Post, P. A.; Osley, M. A.

1996-01-01

318

Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum.  

PubMed

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 (oriC MG). 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, oriC MG, 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

Nieszner, Isolde; Vronka, Martin; Indikova, Ivana; Szostak, Michael P

2013-01-01

319

Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in Mycoplasma gallisepticum  

PubMed Central

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

Indikova, Ivana; Szostak, Michael P.

2013-01-01

320

Associations of ADH and ALDH2 gene variation with self report alcohol reactions, consumption and dependence: an integrated analysis  

PubMed Central

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

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

321

RNAprotein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening  

E-print Network

METHOD RNA­protein interactions in the yeast three-hybrid system: Affinity, sensitivity. However, the relationship between reporter gene activation and in vitro affinity of an RNA reporter genes as a function of the in vitro affinity of the interaction. HIS3 and LacZ expression levels

Wickens, Marv

322

Generation of a Stable Antioxidant Response Element-Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents  

Microsoft Academic Search

The NF-E2 p45-related factor 2 (Nrf2) regulates cytoprotective genes that contain an antioxidant response element (ARE) in their promoters. To investigate whether anticancer drugs can induce ARE-driven gene expression, we have developed a stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element. In these cells, luciferase activity

Xiu Jun Wang; John D. Hayes

2006-01-01

323

PAX5 fusion genes in t(7;9)(q11.2;p13) leukemia: a case report and review of the literature  

PubMed Central

Background B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by recurrent genetic alterations including chromosomal translocations. The transcription factor PAX5, which is pivotal for B-cell commitment and maintenance, is affected by rearrangements, which lead to the expression of in-frame fusion genes in about 2.5% of the cases. Results Using conventional cytogenetics, fluorescence in situ hybridization (FISH), and molecular methods, an additional case with a der(9)t(7;9)(q11.23;p13) resulting in the expression of a PAX5-ELN fusion gene was identified. Furthermore, a general review of leukemia harboring a t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which occurs more often in children than in adults and shows a remarkably high male preponderance, is given. These cytogenetically highly similar translocations lead to the expression of one of three different in frame PAX5-fusions, namely with AUTS2 (7q11.22), ELN (7q11.23), or POM121 (7q11.23), which constitute the only currently known cluster of PAX5 partner genes. Conclusion Our report underlines the recurrent involvement of PAX5 in different fusion genes resulting either from t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which cannot be distinguished cytogenetically and whose discrimination requires molecular analysis. PMID:24507461

2014-01-01

324

(Nuclear genes from nicotiana encoding the small subunit of ribulose-1,5-bisphosphate carboxylase). Progress report  

SciTech Connect

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.

Cashmore, A.R.

1985-01-01

325

Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii.  

PubMed

For the model organism Chlamydomonas reinhardtii, a codon-adapted gene variant of the extracellular luciferase of Gaussia princeps was generated as a sensitive molecular tool to study gene expression from the nuclear genome. In the past, monitoring promoter activity in Chlamydomonas employing the commonly used luciferase encoded by Renilla reniformis was hampered due to the detection limit of the reporter assay, especially if analyzing weak promoters. In this work, the expression of Gaussia-luciferase from such promoters resulted in an average luminescent activity at least 500 times higher than that detected for the Renilla enzyme. The wildtype signal peptide of Gaussia princeps efficiently mediated the export of the luciferase into the culture medium of Chlamydomonas strain cw15arg ( - ), and the characterization of the secreted protein showed an unexpected temperature instability, probably arising from post-translational modifications made by the algae. To further test the utility of Gaussia-luciferase, promoter sequences originating from different viral genomes were analyzed for their ability to drive transgene expression in Chlamydomonas. Solely, the 35S-promoter of the Cauliflower mosaic virus (CaMV) displayed a significant transcriptional activity and this happened only when the shunting region of the 5'-untranslated region of the 35S-sequence was omitted from the luciferase expression cassette. Gaussia-luciferase proved to be a superior quantifiable reporter gene for the analysis of constitutive promoter sequences in Chlamydomonas reinhardtii. PMID:18516621

Ruecker, Ovidiu; Zillner, Karina; Groebner-Ferreira, Regina; Heitzer, Markus

2008-08-01

326

Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1987--April 14, 1988  

SciTech Connect

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.

Okita, T.W.

1988-12-31

327

Luciferase Reporter Gene Assay on Human, Murine and Rat Histamine H4 Receptor Orthologs: Correlations and Discrepancies between Distal and Proximal Readouts  

PubMed Central

The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological tools. Such differences were extremely pronounced in case of proximal readouts, e. g. [32P]GTPase or [35S]GTP?S binding assays. To improve the predictability of in vitro investigations, the aim of this study was to establish a reporter gene assay for human, murine and rat H4Rs, using bioluminescence as a more distal readout. For this purpose a cAMP responsive element (CRE) controlled luciferase reporter gene assay was established in HEK293T cells, stably expressing the human (h), the mouse (m) or the rat (r) H4R. The potencies and efficacies of 23 selected ligands (agonists, inverse agonists and antagonists) were determined and compared with the results obtained from proximal readouts. The potencies of the examined ligands at the human H4R were consistent with reported data from [32P]GTPase or [35S]GTP?S binding assays, despite a tendency toward increased intrinsic efficacies of partial agonists. The differences in potencies of individual agonists at the three H4R orthologs were generally less pronounced compared to more proximal readouts. In conclusion, the established reporter gene assay is highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [32P]GTPase and [35S]GTP?S binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways. PMID:24023919

Nordemann, Uwe; Wifling, David; Schnell, David; Bernhardt, Gunther; Stark, Holger; Seifert, Roland; Buschauer, Armin

2013-01-01

328

An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level  

PubMed Central

RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker ( ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification. PMID:24741441

Kojima, Shin-ichiro

2014-01-01

329

Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes  

Microsoft Academic Search

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds\\u000a and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was

Isabel M. G. Padilla; Agnieszka Golis; Adele Gentile; Carmine Damiano; Ralph Scorza

2006-01-01

330

Interactions of Drosophila Ultrabithorax Regulatory Regions with Native and Foreign Promoters  

PubMed Central

The Ultrabithorax (Ubx) gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Two P-element ``enhancer traps'' have been recovered within the locus that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lacZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome. PMID:9017395

Casares, F.; Bender, W.; Merriam, J.; Sanchez-Herrero, E.

1997-01-01

331

New ex vivo reporter assay system reveals that ? factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants.  

PubMed

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 (?) factors, RpoD and FliA, that are homologous to Escherichia coli ?(70) and ?(28) , 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 ? 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 ? 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. PMID:23723081

Ishii, Yoshiko; Kakizawa, Shigeyuki; Oshima, Kenro

2013-08-01

332

Retrovector encoding a green fluorescent protein-herpes simplex virus thymidine kinase fusion protein serves as a versatile suicide/reporter for cell and gene therapy applications.  

PubMed

Expression vectors encoding herpes simplex virus thymidine kinase (HSVTK) have been extensively used in cell and gene therapy applications either as anticancer "suicide" or as "self-destruct" transgenes in adoptive immunotherapy applications. In both gene therapy applications, reliable detection of HSVTK transgene expression is required in genetically engineered cells. Direct fluorescent labeling of the HSVTK protein may be the remedy. We designed a retrovector encoding a chimeric GFP-HSVTK fusion protein that can serve as a bifunctional suicide and reporter transgene. The fusion gene was incorporated in a VSV G-pseudotyped retrovector (vGFPTKfus) and high-titer stable retroviral producer was generated ( approximately 3 x 10(6) retroparticles/ml). Tumor cell lines transduced at an MOI of 8 for 3 days led to >90% gene transfer efficiency. Southern blot analysis confirmed that unrearranged proviral genomes integrated in chromosomal DNA. Protein extract immunoblot with HSVTK antisera revealed the presence of a 70-kDa protein consistent with the predicted size of an HSVTK-GFP fusion protein. Fluorescence microscopy and FACS analysis revealed that GFPTKfus-mediated fluorescence was nuclear localized and was 30-fold greater than that observed in a bicistronic HSVTK-GFP vector. Growth of cell lines expressing vGFPTKfus was significantly suppressed in the presence of ganciclovir. The DA3 mouse mammary carcinoma cell line was transduced with vGFPTKfus and implanted in syngeneic BALB/c mice. Preestablished tumors completely regressed in seven of nine mice treated with ganciclovir. Normal human peripheral blood T lymphocytes were transduced with vGFPTKfus and nucleus-restricted green fluorescence was observed. Sorting of green fluorescent lymphocytes allowed for selection of engineered cells. In conclusion, we demonstrate the utility of vGFPTKfus as a suicide/reporter transgene in tumor cells in vitro and in vivo. Furthermore, its potential use as an analytical and therapeutic tool targeting human T lymphocytes is shown. PMID:11177538

Paquin, A; Jaalouk, D E; Galipeau, J

2001-01-01

333

Attempted Replication of 50 Reported Asthma Risk Genes Identifies a SNP in RAD50 as Associated with Childhood Atopic Asthma  

Microsoft Academic Search

Objectives: Asthma is a childhood disease that is strongly influenced by genetic factors. We sought to replicate an association between single nucleotide polymorphisms (SNPs) of the top-ranked candidate genes and childhood atopic asthma in Perinatal Risk of Asthma in Infants of Asthmatic Mothers (PRAM) study subjects. Methods: Using data from a systematic literature search and an exploratory genome-wide association study

William Murk; Kyle Walsh; Ling-I Hsu; Linlu Zhao; Michael B. Bracken; Andrew T. DeWan

2011-01-01

334

Local Gene Transfer of OPG Prevents Joint Damage and Disease Progression in Collagen-Induced Arthritis  

PubMed Central

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

Zhang, Qingguo; Gong, Weiming; Ning, Bin; Nie, Lin; Wooley, Paul H.

2013-01-01

335

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein  

SciTech Connect

A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio (Aichi Cancer Center Research Inst., Nagoya (Japan))

1991-10-01

336

Differences in enhancer activity in mouse and zebrafish reporter assays are often associated with changes in gene expression  

PubMed Central

Background Phenotypic evolution in animals is thought to be driven in large part by differences in gene expression patterns, which can result from sequence changes in cis-regulatory elements (cis-changes) or from changes in the expression pattern or function of transcription factors (trans-changes). While isolated examples of trans-changes have been identified, the scale of their overall contribution to regulatory and phenotypic evolution remains unclear. Results Here, we attempt to examine the prevalence of trans-effects and their potential impact on gene expression patterns in vertebrate evolution by comparing the function of identical human tissue-specific enhancer sequences in two highly divergent vertebrate model systems, mouse and zebrafish. Among 47 human conserved non-coding elements (CNEs) tested in transgenic mouse embryos and in stable zebrafish lines, at least one species-specific expression domain was observed in the majority (83%) of cases, and 36% presented dramatically different expression patterns between the two species. Although some of these discrepancies may be due to the use of different transgenesis systems in mouse and zebrafish, in some instances we found an association between differences in enhancer activity and changes in the endogenous gene expression patterns between mouse and zebrafish, suggesting a potential role for trans-changes in the evolution of gene expression. Conclusions In total, our results: (i) serve as a cautionary tale for studies investigating the role of human enhancers in different model organisms, and (ii) suggest that changes in the trans environment may play a significant role in the evolution of gene expression in vertebrates. PMID:23253453

2012-01-01

337

Genomic and Clinical Analysis of Fusion Gene Amplification in Rhabdomyosarcoma: a Report from the Children's Oncology Group  

PubMed Central

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of the myogenic lineage with frequent chromosomal translocations involving the PAX3 or PAX7 and FOXO1 genes. Based on previous studies indicating that the fusion genes are amplified in a subset of these cancers, we conducted a comprehensive molecular and clinical investigation of these amplification events. Using oligonucleotide arrays to localize amplicons, we found that the minimal 1p36 amplicon measured 0.13 Mb and only contained PAX7 whereas the minimal 13q14 amplicon measured 0.53 Mb and contained FOXO1 and the poorly characterized LOC646982 gene. Application of a fluorescence in situ hybridization assay to over 100 fusion-positive cases revealed that the fusion gene is amplified in 93% of PAX7-FOXO1-positive and 9% of PAX3-FOXO1-positive cases. While most cells in amplified PAX7-FOXO1-positive cases contained the amplicon, only a fraction of cells in the amplified PAX3-FOXO1-positive cases contained the amplicon. Expression studies demonstrated that the fusion transcripts were generally expressed at higher levels in amplified cases, and that the PAX7-FOXO1 fusion transcript was expressed at higher levels than the PAX3-FOXO1 fusion transcript. Finally, fusion gene amplification and PAX7-FOXO1 fusion status were each associated with significantly improved outcome; a multivariate analysis demonstrated that this predictive value was independent of other standard prognostic parameters. These findings therefore provide further evidence for a novel good prognosis subset of fusion-positive rhabdomyosarcoma. PMID:22447499

Duan, Fenghai; Smith, Lynette M.; Gustafson, Donna M.; Zhang, Chune; Dunlevy, Mandy J.; Gastier-Foster, Julie M.; Barr, Frederic G.

2012-01-01

338

New ex vivo reporter assay system reveals that ? factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants  

PubMed Central

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 (?) factors, RpoD and FliA, that are homologous to Escherichia coli ?70 and ?28, 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 ? 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 ? 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

Ishii, Yoshiko; Kakizawa, Shigeyuki; Oshima, Kenro

2013-01-01

339

The Haemophilus influenzae dprABC genes constitute a competence-inducible operon that requires the product of the tfoX (sxy) gene for transcriptional activation.  

PubMed Central

We previously showed that dprA is required for efficient processing of linear DNA during cellular transformation in Haemophilus influenzae. In this study the transcriptional regulation of dprA and two downstream genes, dprB and dprC, is examined. We demonstrate by Northern blot analysis that the dprABC genes are transcriptionally coregulated and competence inducible. We used primer extension analysis to map the transcriptional start site of dprA and of rec-2, another transformation gene involved in DNA processing. Based upon these results, we were able to identify a 26-bp dyad symmetry element immediately upstream of the -35 regions of the predicted promoters of dprA, rec-2, and two other transformation genes, comA and pilA. Finally, using transcriptional fusions of dprA to the Escherichia coli lacZ gene, we show that expression of dprA::lacZ requires tfoX and that the presence of multiple copies of tfoX abolishes the temporal regulation of dprA, resulting in its constitutive expression. PMID:9244270

Karudapuram, S; Barcak, G J

1997-01-01

340

Silencing of Reporter Gene Expression in Skin Using siRNAs and Expression of Plasmid DNA Delivered by a Soluble Protrusion Array Device (PAD)  

PubMed Central

Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation. PMID:20571543

Gonzalez-Gonzalez, Emilio; Speaker, Tycho J; Hickerson, Robyn P; Spitler, Ryan; Flores, Manuel A; Leake, Devin; Contag, Christopher H; Kaspar, Roger L

2010-01-01

341

Silencing of reporter gene expression in skin using siRNAs and expression of plasmid DNA delivered by a soluble protrusion array device (PAD).  

PubMed

Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation. PMID:20571543

Gonzalez-Gonzalez, Emilio; Speaker, Tycho J; Hickerson, Robyn P; Spitler, Ryan; Flores, Manuel A; Leake, Devin; Contag, Christopher H; Kaspar, Roger L

2010-09-01

342

Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites.  

PubMed

Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research. PMID:22673932

Kümmerer, Beate Mareike; Grywna, Klaus; Gläsker, Sabine; Wieseler, Janett; Drosten, Christian

2012-09-01

343

Positive Darwinian Selection after Gene Duplication in Primate Ribonuclease Genes  

Microsoft Academic Search

Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and

Jianzhi Zhang; Helene F. Rosenberg; Masatoshi Nei

1998-01-01

344

Anomalous left coronary artery from the right coronary cusp with gene positive apical hypertrophic cardiomyopathy: a case report and literature review.  

PubMed

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

Georgekutty, Justin; Cross, Russell R; Rosenthal, Joanna B; Heath, Deneen M; Sinha, Pranava; John, Anitha S

2014-06-01

345

[Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, [March 15, 1989--April 14, 1990  

SciTech Connect

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.

Okita, T.W.

1990-12-31

346

Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of Dendrobium Sonia 17  

Microsoft Academic Search

Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with

C. Tee; M. Marziah; C. Tan; M. Abdullah

2003-01-01

347

Clinical and genetic characterization of chanarin-dorfman syndrome patients: first report of large deletions in the ABHD5 gene  

Microsoft Academic Search

BACKGROUND: Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disorder characterized by nonbullous congenital ichthyosiform erythroderma (NCIE) and an intracellular accumulation of triacylglycerol (TG) droplets in most tissues. The clinical phenotype involves multiple organs and systems, including liver, eyes, ears, skeletal muscle and central nervous system (CNS). Mutations in ABHD5\\/CGI58 gene are associated with CDS. METHODS: Eight CDS patients belonging

Chiara Redaelli; Rosalind A Coleman; Laura Moro; Catherine Dacou-Voutetakis; Solaf Mohamed Elsayed; Daniele Prati; Agostino Colli; Donatella Mela; Roberto Colombo; Daniela Tavian

2010-01-01

348

Polymorphisms in Stromal Genes and Susceptibility to Serous Epithelial Ovarian Cancer: A Report from the Ovarian Cancer Association Consortium  

Microsoft Academic Search

Alterations in stromal tissue components can inhibit or promote epithelial tumorigenesis. Decorin (DCN) and lumican (LUM) show reduced stromal expression in serous epithelial ovarian cancer (sEOC). We hypothesized that common variants in these genes associate with risk. Associations with sEOC among Caucasians were estimated with odds ratios (OR) among 397 cases and 920 controls in two U.S.-based studies (discovery set),

Ernest K. Amankwah; Qinggang Wang; Joellen M. Schildkraut; Ya-Yu Tsai; Susan J. Ramus; Brooke L. Fridley; Jonathan Beesley; Sharon E. Johnatty; Penelope M. Webb; Georgia Chenevix-Trench; Laura C. Dale; Diether Lambrechts; Frederic Amant; Evelyn Despierre; Ignace Vergote; Simon A. Gayther; Aleksandra Gentry-Maharaj; Usha Menon; Jenny Chang-Claude; Shan Wang-Gohrke; Hoda Anton-Culver; Argyrios Ziogas; Thilo Dörk; Matthias Dürst; Natalia Antonenkova; Natalia Bogdanova; Robert Brown; James M. Flanagan; Stanley B. Kaye; James Paul; Ralf Bützow; Heli Nevanlinna; Ian Campbell; Diana M. Eccles; Beth Y. Karlan; Jenny Gross; Christine Walsh; Paul D. P. Pharoah; Honglin Song; Susanne Krüger Kjćr; Estrid Hřgdall; Claus Hřgdall; Lene Lundvall; Lotte Nedergaard; Lambertus A. L. M. Kiemeney; Leon F. A. G. Massuger; Anne M. van Altena; Sita H. H. M. Vermeulen; Nhu D. Le; Angela Brooks-Wilson; Linda S. Cook; Catherine M. Phelan; Julie M. Cunningham; Celine M. Vachon; Robert A. Vierkant; Edwin S. Iversen; Andrew Berchuck; Ellen L. Goode; Thomas A. Sellers; Linda E. Kelemen; John D. Minna

2011-01-01

349

Vitamin D-Dependent Rickets Type II: Report of a Novel Mutation in the Vitamin D Receptor Gene  

Microsoft Academic Search

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

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

350

Associations of ADH and ALDH2 gene variation with self report alcohol reactions, consumption and dependence: an integrated analysis  

Microsoft Academic Search

Alcohol dependence (AD) is a complex disorder with environmental and genetic origins. The role of two gen- etic variants in ALDH2 and ADH1B in AD risk has been extensively investigated. This study tested for associ- ations 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

Stuart Macgregor; Penelope A. Lind; Kathleen K. Bucholz; Narelle K. Hansell; Pamela A. F. Madden; Melinda M. Richter; Grant W. Montgomery; Nicholas G. Martin; Andrew C. Heath; John B. Whitfield

2009-01-01

351

Excess congenital non-synonymous variation in leukemia-associated genes in MLL? infant leukemia: a Children's Oncology Group report  

PubMed Central

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

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

352

3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells  

SciTech Connect

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.

Moorthy, Bhagavatula [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States)]. E-mail: bmoorthy@bcm.tmc.edu; Muthiah, Kathirvel [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States); Fazili, Inayat S. [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States); Kondraganti, Sudha R. [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States); Wang Lihua [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States); Couroucli, Xanthi I. [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States); Jiang Weiwu [Department of Pediatrics, Baylor College of Medicine, 6621 Fannin, FC 530.01, Houston, TX 77030 (United States)

2007-03-23

353

LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.  

PubMed

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA. PMID:25172300

Ramirez, Tzutzuy; Mehling, Annette; Kolle, Susanne N; Wruck, Christoph J; Teubner, Wera; Eltze, Tobias; Aumann, Alexandra; Urbisch, Daniel; van Ravenzwaay, Ben; Landsiedel, Robert

2014-12-01

354

Asymmetrical N4,N9-diacyl spermines: SAR studies of nonviral lipopolyamine vectors for efficient siRNA delivery with silencing of EGFP reporter gene.  

PubMed

Our aim is to study the effects of varying the two acyl moieties in synthesized N(4),N(9)-diacyl spermines on siRNA formulations and their delivery efficiency in cell lines. Six novel asymmetrical lipopolyamines, [N(4)-cholesteryloxy-3-carbonyl-N(9)-oleoyl-, N(4)-decanoyl-N(9)-oleoyl-, N(4)-decanoyl-N(9)-stearoyl-, N(4)-lithocholoyl-N(9)-oleoyl-, N(4)-myristoleoyl-N(9)-myristoyl-, and N(4)-oleoyl-N(9)-stearoyl]-1,12-diamino-4,9-diazadodecane, were assessed for their abilities to bind to siRNA, studied using a RiboGreen intercalation assay, and to form nanoparticles. Their siRNA delivery efficiencies were quantified in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA) using a fluorescein-tagged siRNA, and compared with formulations of N(4),N(9)-dioleoyl-1,12-diamino-4,9-diazadodecane and of a leading transfecting agent, TransIT-TKO. Transfection was measured in terms of siRNA delivery and silencing of EGFP reporter gene in HeLa cells. By incorporating two different acyl moieties, changing their length and oxidation level in a controlled manner, we show efficient fluorescein-tagged siRNA formulation, delivery, and knock-down of EGFP reporter gene. N(4)-Oleoyl-N(9)-stearoyl spermine and N(4)-myristoleoyl-N(9)-myristoyl spermine are effective siRNA delivery vectors typically resulting in 89% cell delivery and gene silencing to 34% in the presence of serum, comparable with the results obtained with TransIT-TKO; adding a second lipid chain is better than incorporating a steroid moiety. PMID:22224453

Blagbrough, Ian S; Metwally, Abdelkader A; Ghonaim, Hassan M

2012-07-01

355

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering  

PubMed Central

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish. PMID:25293390

Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin-ichi

2014-01-01

356

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.  

PubMed

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish. PMID:25293390

Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin-ichi

2014-01-01

357

A novel ER?-mediated reporter gene assay for screening estrogenic/antiestrogenic chemicals based on LLC-MK2 cells.  

PubMed

Abstract Low concentration of endocrine-disrupting chemicals (EDCs) may lead to serious consequences in animals and human, so it is essential to develop an effective assay for EDCs detection. In this study, we developed a novel ER?-mediated reporter gene assay based on the LLC-MK2 cells by co-transfecting pERE-sv40-Luc, hER?-pcDNA3.1, and pRL-tk. Then we determined 17?-estradiol (E2) and some estrogenic/antiestrogenic chemicals to verify the validity of this assay. Data showed that the assay possesses a concentration-dependent responses to E2 and diethylstilbestrol (DES) from 10(-12?)M to 10(-8?)M with EC50 3.4?×?10(-10?)M and 5.9?×?10(-10?)M, and ICI 182,780 completely blocks the luciferase activity induced by 10(-9?)M E2. Bisphenol A (BPA), nonylphenol (NP), genistein (GS), and tamoxifen (TAM) also showed corresponding estrogenic or antiestrogenic activity at test concentrations. All evidences proved that the LLC-MK2 reporter gene assay was specific and sensitive to estrogen receptor (ER) agonistic and antagonistic chemicals. PMID:25045971

Huang, Xiaoming; Huang, Jie; Zhang, Lishi; Zhu, Yanfeng; Li, Yun

2014-12-01

358

A novel TRPS1 gene mutation causing trichorhinophalangeal syndrome with growth hormone responsive short stature: a case report and review of the literature  

PubMed Central

The role of growth hormone (GH) and its therapeutic supplementation in the trichorhinophalangeal syndrome type I (TRPS I) is not well delineated. TRPS I is a rare congenital syndrome, characterized by craniofacial and skeletal malformations including short stature, sparse, thin scalp hair and lateral eyebrows, pear-shaped nose, cone shaped epiphyses and hip dysplasia. It is inherited in an autosomal dominant manner and caused by haploinsufficiency of the TRPS1 gene. We report a family (Mother and 3 of her 4 children) with a novel mutation in the TRPS1 gene. The diagnosis was suspected only after meeting all family members and comparing affected and unaffected siblings since the features of this syndrome might be subtle. The eldest sibling, who had neither GH deficiency nor insensitivity, improved his growth velocity and height SDS after 2 years of treatment with exogenous GH. No change in growth velocity was observed in the untreated siblings during this same period. This report emphasizes the importance of examining all family members when suspecting a genetic syndrome. It also demonstrates the therapeutic effect of GH treatment in TRPS I despite normal GH-IGF1 axis. A review of the literature is included to address whether TRPS I is associated with: a) GH deficiency, b) GH resistance, or c) GH-responsive short stature. More studies are needed before recommending GH treatment for TRPS I but a trial should be considered on an individual basis. PMID:25177352

2014-01-01

359

Report of a Novel Mutation in MLH1 Gene in a Hispanic Family from Puerto Rico Fulfilling Classic Amsterdam Criteria for Lynch Syndrome  

PubMed Central

In Puerto Rico, colorectal cancer (CRC) represents the second leading cause of cancer in men and women. Familial CRC accounts for 10–15% of the total CRC cases, while Lynch syndrome accounts for approximately 2–4% of cases. Limited information is available about the prevalence, clinical manifestations, and genetic mutations of hereditary CRC in US Hispanic individuals. In this paper we report a novel mutation in the hMLH1 gene in a Puerto Rican Hispanic family with Lynch syndrome recruited through the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR). Our proband was identified by applying Amsterdam and Bethesda criteria for Lynch syndrome, analysis of protein expression by immunohistochemistry, and genetic sequencing of the mismatch repair genes. A novel mutation at c.2044_2045 in hMLH1 consisting of the deletion of two consecutive nucleotides (AT) at exon 18 was identified. This deletion causes a frameshift in the protein coding sequence at p.682 resulting in premature termination and a truncated MLH1 protein. To our knowledge, this mutation has not been previously reported in the literature. The detection of this novel mutation in MLH1 further emphasizes the need for genetic testing in at-risk patients for hereditary CRC from various ethnic and racial backgrounds. PMID:25389437

Marques-Lespier, Juan M.; Diaz-Algorri, Yaritza; Gonzalez-Pons, Maria

2014-01-01

360

Grimontia indica AK16T, sp. nov., Isolated from a Seawater Sample Reports the Presence of Pathogenic Genes Similar to Vibrio Genus  

PubMed Central

Grimontia indica strain AK16T sp. nov. is the type strain of G. indica sp. nov. a new species within the genus Grimontia. This strain, whose genome is described here, was isolated from seawater sample collected from southeast coast of Palk Bay, India. G. indica AK16T is a Gram-negative, facultative aerobic rod shaped bacterium. There are only two other strains in the genus Grimontia one of which, Grimontia hollisae CIP 101886T, is a reported human pathogen isolated from human stool sample while the other, ‘Grimontia marina IMCC5001T’, was isolated from a seawater sample. As compared to the pathogenic strain Grimontia hollisae CIP 101886T, the strain AK16T lacks some genes for pathogenesis like the accessory colonization factors AcfA and AcfD, which are required for the colonization of the bacterium in the host body. While it carries some pathogenesis genes like OmpU, which are related to pathogenesis of Vibrio strains. This suggests that the life cycle of AK16T may include some pathogenic interactions with marine animal(s), or it may be an opportunistic pathogen. Study of the Grimontia genus is important because of the severe pathogenic traits exhibited by a member of the genus with only three species reported in total. The study will provide some vital information which may be useful in future clinical studies on the genus. PMID:24465608

Singh, Aditya; Vaidya, Bhumika; Khatri, Indu; Srinivas, T. N. R.; Subramanian, Srikrishna; Korpole, Suresh; Pinnaka, Anil Kumar

2014-01-01

361

Prevalence of horB gene among the Helicobacter pylori strains isolated from dyspeptic patients: first report from Iran.  

PubMed

Helicobacter pylori (H. pylori) is globally accepted as an important cause of gastritis in human, and evidence strongly shows an etiological role for H. pylori in gastric cancer and peptic ulceration. In this study, we determined the relationship between digestive diseases and the horB gene of H. pylori infection. Fresh antral biopsy specimens were obtained from 140 dyspeptic patients (67 men and 73 women; mean age 41.5, aged 19-63 years). They were examined for presence of the horB gene of H. pylori clinical isolates. Bacterial DNA content was extracted directly from the antral biopsy. Statistical analysis was performed with SPSS version 16.0. Prevalence of the horB gene in H. pylori isolated from patients with gastric cancer, gastric ulcer, gastritis and duodenal ulcer is (5/32) 15.6%, (4/25) 16%, (30/43) 70%, and (9/40) 22.5%, respectively. No significant relationship is observed between age, pathologic findings and gender factors with respect to the four digestive diseases (P > 0.05). In our examination, a significant association was observed between a horB positive genotype of H. pylori and the occurrence of gastritis; in support of the protective theory. Studies with a higher sample size in different countries of the world should be conducted to obtain a thorough assessment as to whether horB has a role in the progress of gastritis (protective effect) or not. Further tests should be carried out to determine the exact role of horB in infection of H. pylori. PMID:21559747

Taghvaei, Tarang; Talebi Bezmin Abadi, Amin; Ghasemzadeh, Ali; Naderi, Behnam Kalali; Mohabbati Mobarez, Ashraf

2012-12-01

362

The isolation and characterization of beta-glucosidase gene and beta-glucosidase of Trichoderma viride: Progress report  

SciTech Connect

We have demonstrated the induction by sophorose of ..beta..-glucosidase and other cellulases in Trichoderma cultures, isolated large quantities of intact RNA from induced and noninduced cultures, isolated large quantities of high molecular weight DNA free from contaminating polysaccarides and obtained about 3000 recombinant clones containing inserts, presumably of restriction enzyme cleaved Trichoderma DNA. Additionally we have examined the effect of sophorose on the ..beta..-glucosidase of Sporotrichum pulverentum, established an in vitro translation system, and prepared enzymes from the bacterium Oerskovia which will aid in attempts to insert ethanol producing genes into Trichoderma. 28 refs., 2 figs.

Stafford, D.W.; Lunblad, R.L.

1981-05-01

363

Analysis of Multiple Association Studies Provides Evidence of an Expression QTL Hub in Gene-Gene  

E-print Network

-Gene Interaction Network Affecting HDL Cholesterol Levels Li Ma1ďż˝ , Christie Ballantyne2 , Ariel Brautbar2 report an analysis of gene-gene interactions affecting HDL cholesterol (HDL-C) levels in a candidate gene Hub in Gene-Gene Interaction Network Affecting HDL Cholesterol Levels. PLoS ONE 9(3): e92469. doi:10

Keinan, Alon

364

A newly identified mutation in the complement factor I gene not associated with early post-transplant recurrence of atypical hemolytic-uremic syndrome: a case report.  

PubMed

Atypical hemolytic uremic syndrome (aHUS), which can recur after renal transplantation, is associated with poor graft outcomes. The underlying genetic defect, namely, mutations in genes coding for the complement factor H, I (CFI), or membrane cofactor protein, greatly impacts the risk of aHUS recurrence. We report here the case of a patient with chronic renal failure due to aHUS in which screening for complement mutations, performed before wait-listing for kidney transplantation, showed a never described previously heterozygous mutation in the exon II of the CFI gene. Specifically, this mutation leads to a substitution of cytosine for guanosine at nucleotide 148, resulting in the change at amino acid 50 from arginine to proline. Subsequently, he received a renal allograft from deceased donor. Good graft function was established immediately, without clinical features of aHUS. Due to a lack of data on this mutation, we avoided prophylactic treatment for aHUS but closely monitored biochemical markers of aHUS to treat a possible recurrence. Immunosuppressive treatment was based on basiliximab, tacrolimus, steroids, and mycophenolic acid. At the time of discharge the serum creatinine was 1.4 mg/dL. Ten months after transplantation the patient is doing well without evidence of aHUS. Our case suggested that a heterozygous mutation in exon II of the CFI gene was not associated with a risk of early post-transplant aHUs recurrence adding new knowledge on complement mutations implicated in aHUS post-transplant recurrences. PMID:24034049

Ranghino, A; Tognarelli, G; Basso, E; Messina, M; Manzione, A M; Daidola, G; Segoloni, G P

2013-09-01

365

The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells.  

PubMed Central

P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract. We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constructs in which the promoter area of the pgp genes was fused to the coding region of lacZ. Expression of pgp-1 and pgp-3, as inferred from pgp-lacZ transgenic nematodes, was confined to the intestinal cells. The expression patterns of both genes were virtually indistinguishable. Quantitative analysis of pgp mRNA levels during development showed that pgp-1, -2, and -3 were expressed throughout the life cycle of C.elegans, albeit with some variation indicating developmental regulation. The expression of P-glycoprotein genes in intestinal cells is an evolutionarily conserved feature of these genes, consistent with the hypothesis that P-glycoproteins provide a mechanism of protection against environmental toxins. Images PMID:8096815

Lincke, C R; Broeks, A; The, I; Plasterk, R H; Borst, P

1993-01-01

366

Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans.  

PubMed

Endo-beta-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subtilis PAP115 endo-beta-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, S1 nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ alpha-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum celB endoglucanase sequence but very little similarity to endoglucanases from other Bacillus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding. PMID:2307659

Baird, S D; Johnson, D A; Seligy, V L

1990-03-01

367

Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995  

SciTech Connect

Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

Marton, L.

1996-02-01

368

A TYROSINE HYDROXYLASE-YELLOW FLUORESCENT PROTEIN KNOCK-IN REPORTER SYSTEM LABELING DOPAMINERGIC NEURONS REVEALS POTENTIAL REGULATORY ROLE FOR THE FIRST INTRON OF THE RODENT TYROSINE HYDROXYLASE GENE  

PubMed Central

Degeneration of the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson’s disease. To facilitate the study of the differentiation and maintenance of this population of dopaminergic neurons both in vivo and in vitro, we generated a knock-in reporter line in which the yellow fluorescent protein (YFP) replaced the first exon and the first intron of the tyrosine hydroxylase (TH) gene in one allele by homologous recombination. Expression of YFP under the direct control of the entire endogenous 5? upstream region of the TH gene was predicted to closely match expression of TH from the wild type allele, thus marking functional dopaminergic neurons. We found that YFP was expressed in dopaminergic neurons differentiated in vitro from the knock-in mouse embryonic stem cell line and in dopaminergic brain regions in knock-in mice. Surprisingly, however, YFP expression did not overlap completely with TH expression, and the degree of overlap varied in different TH-expressing brain regions. Thus, the reporter gene did not identify functional TH-expressing cells with complete accuracy. A DNaseI hypersensitivity assay revealed a cluster of hypersensitivity sites in the first intron of the TH gene, which was deleted by insertion of the reporter gene, suggesting that this region may contain cis-acting regulatory sequences. Our results suggest that the first intron of the rodent TH gene may be important for accurate expression of TH. PMID:16876957

KELLY, B. B.; HEDLUND, E.; KIM, C.; ISHIGURO, H.; ISACSON, O.; CHIKARAISHI, D. M.; KIM, K.-S.; FENG, G.

2008-01-01

369

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

PubMed Central

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 -211 and -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 -133 to -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 (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region. PMID:1903838

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

1991-01-01

370

Induction of Rhizobium meliloti nodC gene by Alnus incana compounds.  

PubMed

An expression of nodC promoter of R. meliloti 1021, cloned in front of the Escherichia coli lacZ gene, was used to study the presence of R. meliloti nodC gene-inducing compound(s) in extracts and exudates of A. incana seeds and roots. The regulatory gene nodD was expressed at comparable level in bacterial culture of R. meliloti with and without the studied plant extracts and exudates, whereas the nodC-lacZ fusion expression was increased 4 times by seed exudates of grey alder and 2 times by seed extracts and root ingredients in comparison to the nodC-lacZ fusion expression in R. meliloti grown in a medium free of plant compounds. Induction of R. meliloti nodC gene expression by A. incana substances was also supported in plant test. Sterile filtrate of coculture of R. meliloti with seed exudates of A. incana induced root hair deformations and nodule-like structures on Medicago sativa. PMID:8914264

Ma?ek, W

1996-01-01

371

Search for Proteins Required for Accurate Gene Expression under Oxidative Stress  

PubMed Central

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base is oxidized to 8-oxo-7,8-dihydroguanine, which can pair with adenine as well as cytosine. This mispairing causes alterations in gene expression, and cells possess mechanisms to prevent such outcomes. In Escherichia coli, 8-oxo-7,8-dihydroguanine-related phenotypic suppression of lacZ amber is enhanced by mutations in genes related to the prevention of abnormal protein synthesis under oxidative stress. A genome-wide search for the genes responsible, followed by DNA sequence determination, revealed that specific amino acid changes in guanylate kinase and in the ? and ?? subunits of RNA polymerase cause elevated levels of phenotypic suppression, specifically under aerobic conditions. The involvement of the DnaB, DnaN, and MsbA proteins, which are involved in DNA replication and in preserving the membrane structure, was also noted. Interactions of these proteins with each other and also with other molecules may be important for preventing errors in gene expression. PMID:24097971

Inokuchi, Hachiro; Ito, Riyoko; Sekiguchi, Takeshi; Sekiguchi, Mutsuo

2013-01-01

372

Pyrrolo[2,3-b]quinoxalines as inhibitors of firefly luciferase: their Cu-mediated synthesis and evaluation as false positives in a reporter gene assay.  

PubMed

2-Substituted pyrrolo[2,3-b]quinoxalines having free NH were prepared directly from 3-alkynyl-2-chloroquinoxalines in a single pot by using readily available and inexpensive methane sulfonamide (or p-toluene sulfonamide) as an ammonia surrogate. The reaction proceeded in the presence of Cu(OAc)(2) affording the desired product in moderate yield. The crystal structure analysis of a representative compound and its supramolecular interactions are presented. Some of the compounds synthesized exhibited inhibitory activities against luciferase that was supported by the predictive binding mode of these compounds with luciferase enzyme through molecular docking studies. The key observations disclosed here can alert users of luciferase reporter gene assays for possible false positive results due to the direct inhibition of luciferase. PMID:22981335

Nakhi, Ali; Rahman, Md Shafiqur; Kishore, Ravada; Meda, Chandana Lakshmi T; Deora, Girdhar Singh; Parsa, Kishore V L; Pal, Manojit

2012-10-15

373

Cloning and expression of the sulfoxide/sulfone/sulfonate/sulfate genes in Pseudomonads and Thiobacillae. Tenth quarterly report  

SciTech Connect

The original conception of the work was that genetic determinants of the sulfoxide/sulfone/sulfonate/sulfate (``4S``) pathway in Pseudomonas spp. would be cloned in vivo and then transferred to Thiobacillus spp. This ambition remains an appealing prospect; however, fulfilling that ambition has been confounded by an instability observed in the DbtS{sup +} phenotype in Pseudomonas spp. But the persisting interest in the phenotype has lead to isolation of fresh strains which have a DbtS{sup +} phenotype. One strain in particular, N1-36, has been the focus of extensive characterizations in long-term cultures. During the present quarter, seven cultures maintained in a ``fermentor`` for a week or longer have been run to determine rate and extent of growth, extent of conversion of dibenzothiophene (DBT) or dibenzosulfone (DBTO{sub 2}) to monohydroxybiphenyl (OH-BP), effect of pH maintained at 6.0, and the effect of adding glucose to cultures in which the amount of glucose had been diminished by bacterial consumption. In addition, a study of the effectiveness of using R68.445 as a vehicle for in vivo cloning of genes was completed this semester, and introduction of DbtS{sup +} determinants into Thiobacillus spp. continues to be an important goal.

Krawiec, S.

1992-02-07

374

Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: comparisons with AHR1-mediated reporter gene activity and in ovo toxicity.  

PubMed

Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. PMID:23142756

Manning, Gillian E; Mundy, Lukas J; Crump, Doug; Jones, Stephanie P; Chiu, Suzanne; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W

2013-01-01

375

Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters  

Microsoft Academic Search

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium

Paul Carroll; Lise J. Schreuder; Julian Muwanguzi-Karugaba; Siouxsie Wiles; Brian D. Robertson; Jorge Ripoll; Theresa H. Ward; Gregory J. Bancroft; Ulrich E. Schaible; Tanya Parish; Olivier Neyrolles

2010-01-01

376

Gene Cloning  

NSDL National Science Digital Library

This lesson covers the utilization of gene cloning to isolate and copy a specific gene of interest. The transformation of bacteria with plasmids containing antibiotic resistance genes to make gene libraries and the selection of bacteria colonies that contain the specific gene of interest are described.

377

Bilateral pulmonary embolism in twins with PAI-1 4G/5G gene polymorphism: a case report.  

PubMed

The aetiology of venous thromboembolism in adolescents is frequently associated with hereditary abnormalities of the coagulation system, autoimmune disorders or malignancies. The advent of specific laboratory tests has refined the identification of genetic traits. In this case report, we describe the occurrence of pulmonary embolism in young twins. Intensive tumour screening remained unremarkable. Evaluation of established risk factors for a clotting disorder remained negative, with the exception of a plasminogen activator inhibitor-1 4G/5G polymorphism. Despite the mild association accompanied by the presence of the 4G allele, this polymorphism might predispose to venous thromboembolism in some cases in general and in our case in particular. PMID:24249316

Kronbichler, Andreas; Oelzner, Peter; Syrbe, Günter; Lopatta, Eric; Wolf, Gunter; Neumann, Thomas

2014-01-01

378

Ectopic Cushing syndrome associated with thymic carcinoid tumor as the first presentation of MEN1 syndrome-report of a family with MEN1 gene mutation.  

PubMed

Multiple endocrine neoplasia type 1(MEN1) is an autosomal dominant syndrome. Although thymic carcinoid tumor is recognized as a part of MEN1 syndrome but functioning thymic carcinoid tumor as the first presentation of the MEN1 seems to be very rare. In this report, we present a 29-year-old male who developed ectopic Cushing syndrome secondary to thymic carcinoid tumor and was diagnosed as MEN1 syndrome 2 years later. Further evaluation revealed the presence of carcinoid tumor and other MEN 1 manifestations in several other member of family. Genetic evaluation showed presence of a previously reported mutation in exon 10(R527X) of MEN1 gene in these patients. This presentation showed that thymic neuroendocrine tumor could be the first manifestation of the MEN1 syndrome and it might be diagnosed as a dominant manifestation of this syndrome in a family. We suggest biochemical or genetic screening for MEN-1 syndrome for patients with thymic carcinoid. PMID:24218143

Hasani-Ranjbar, Shirin; Rahmanian, Masoud; Ebrahim-Habibi, Azadeh; Soltani, Akbar; Soltanzade, Akbar; Mahrampour, Elnaz; Amoli, Mahsa M

2014-06-01

379

A new mutation of the PCNT gene in a Colombian patient with microcephalic osteodysplastic primordial dwarfism type II: a case report  

PubMed Central

Introduction Microcephalic osteodysplastic primordial dwarfism is a syndrome characterized by the presence of intrauterine growth restriction, post-natal growth deficiency and microcephaly. Microcephalic osteodysplastic primordial dwarfism type II is the most distinctive syndrome in this group of entities. Individuals affected by this disease present at an adult height of less than 100cm, a post-pubertal head circumference of 40cm or less, mild mental retardation, an outgoing personality and bone dysplasia. Case presentation We report the first case of a five-year-old Colombian boy of mixed race ancestry (mestizo), with clinical features of microcephaly, prominent and narrow nose, arched palate, amelogenesis imperfecta, short stature, tall and narrow pelvis, disproportionate shortening of fore-arms and legs, and mild coxa vara. Analysis of the PCNT gene by sequencing showed the presence of a nucleotide change in exon 10, c. 1468C>T, evidencing a new mutation not reported in the literature for microcephalic osteodysplastic primordial dwarfism. Conclusion The new mutation identified in this case could be associated with the severity of the phenotypic expression of the disease, resulting in the extreme short stature of the patient. Further studies are required to reach an explanation that can justify such findings, and it is vital to emphasize the importance of detection and follow-up by the epidemiological surveillance groups in birth defects and rare diseases. PMID:24928221

2014-01-01

380

5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis.  

PubMed Central

The regulation of the levansucrase gene sacB was studied in Bacillus subtilis strains. Fusions were constructed in which genes of cytoplasmic proteins such as lacZ were placed immediately downstream from sacR, the regulatory region located upstream from sacB. These fusions were introduced in mutants affected in sacB regulation. In all cases the marker gene was affected in the same way as sacB by the genetic context. This result is of particular interest for the sacU pleiotropic mutations, which affect sacB expression and other cellular functions such as the synthesis of several exocellular enzymes. We also showed that strains harboring sacU+ or sacU-hyperproducing alleles contained different amounts of sacB mRNA, which was proportional to their levansucrase secretion. We concluded that the sacU gene does not affect sacB expression at the level of secretion but acts on a target within sacR. We discuss the possibility that sacU acts on a part of sacR, a homologous copy of which was found upstream from the gene of another sacU-dependent secreted enzyme of B. subtilis, beta-glucanase. Images PMID:3086292

Aymerich, S; Gonzy-Treboul, G; Steinmetz, M

1986-01-01

381

Orgyia pseudotsugata baculovirus p10 and polyhedron envelope protein genes: analysis of their relative expression levels and role in polyhedron structure.  

PubMed

To investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the beta-glucuronidase (GUS) or beta-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10- viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10-GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra. PMID:8176372

Gross, C H; Russell, R L; Rohrmann, G F

1994-05-01

382

A case report of epithelioid inflammatory myofibroblastic sarcoma with RANBP2-ALK fusion gene treated with the ALK inhibitor, crizotinib.  

PubMed

Epithelioid inflammatory myofibroblastic sarcoma is a variant of inflammatory myofibroblastic tumor with aggressive clinical course associated with RANBP2-ALK fusion. The present report describes a case of a 22-year-old Japanese man with a pelvic mesenchymal neoplasm. The feature of the neoplasms, including epithelioid morphology, anaplastic lymphoma kinase staining on the nuclear membrane, and results from the reverse transcriptase-polymerase chain reaction, led to diagnosis of epithelioid inflammatory myofibroblastic sarcoma with RANBP2-ALK fusion. Despite two surgical excision procedures, local recurrence rapidly occurred, and the tumor developed resistance to conventional chemotherapy with doxorubicin. Subsequent administration of crizotinib, an oral anaplastic lymphoma kinase inhibitor, resulted in tumor shrinkage. Distinguishing epithelioid inflammatory myofibroblastic sarcoma from conventional inflammatory myofibroblastic tumor is important, and crizotinib is a promising treatment for this aggressive tumor. PMID:25028698

Kimbara, Shiro; Takeda, Koji; Fukushima, Hiroko; Inoue, Toru; Okada, Hideaki; Shibata, Yumi; Katsushima, Utae; Tsuya, Asuka; Tokunaga, Shinya; Daga, Haruko; Okuno, Takahiro; Inoue, Takeshi

2014-09-01

383

Gene Duplication within the Family Salmonidae: Disomic Inheritance of Two Loci Reported to Be Tetrasomic in Rainbow Trout  

PubMed Central

We describe our studies of the genetics of allelic variation for NADP-dependent isocitrate dehydrogenase (IDH) in rainbow trout (Salmo gairdneri). Five populations of rainbow trout were studied to determine the phenotypic distribution of IDH; 453 progeny from a number of controlled matings were examined to determine the nature of inheritance of these alleles. The variation was found to be the result of four alleles producing protein subunits of differing electrophoretic mobilities. Progeny from crosses clearly demonstrated the presence of two disomic loci controlling the variation, rather than one tetrasomic locus as had been previously reported. These findings support our contention that the hypothesis of a tetraploid event in salmonid evolution should not be uncritically accepted. PMID:17248635

Allendorf, Frederick W.; Utter, Fred M.

1973-01-01

384

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns  

Microsoft Academic Search

BACKGROUND: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in

Yong-Li Xiao; Julia C Redman; Erin L Monaghan; Jun Zhuang; Beverly A Underwood; William A Moskal; Wei Wang; Hank C Wu; Christopher D Town

2010-01-01

385

Sclerosing epithelioid fibrosarcoma - a report of two cases with cytogenetic analysis of FUS gene rearrangement by FISH technique.  

PubMed

Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue sarcoma. Recently, a link has been suggested between SEF and low-grade fibromyxoid sarcoma (LGFMS) on the basis of the finding of the characteristic translocation t(7;16) (FUS-CREB3L2) of LGFMS in a small number of studied cases of SEF. The frequency of this translocation in SEF is still unknown. We present 2 cases of SEF with cytogenetic analysis for FUS rearrangement. The tumors occurred in 12 and 58 year old patients, respectively and consisted of a well to partially circumscribed, non-encapsulated mass, comprising monomorphic, polygonal cells arranged in aggregates, cords and single file arrays in a variably sclerotic stroma. The cells exhibited minimal nuclear atypia with moderate amount of clear to eosinophilic cytoplasm and rare mitotic figures. One case also showed bland spindle cell areas with myxoid change, as seen in LGFMS. By immunohistochemistry (IHC), the tumor cells were diffusely positive for vimentin, focally for S-100 in 1 case and negative for cytokeratin (CK), epithelial membrane antigen (EMA), HMB-45, desmin, smooth muscle actin (SMA), H-caldesmon, Myo D-1, CD34 and CD 168. By fluorescent in-situ hybridization (FISH) technique, the case with mixed SEF and LGFMS histology was positive for FUS rearrangement. Our study reinforces the previously reported relationship between SEF and LGFMS, and suggests that SEF may represent a variant of LGFMS in at least some cases, rather than an entirely distinct fibrosarcoma variant. PMID:20499220

Rekhi, Bharat; Folpe, Andrew L; Deshmukh, Mahesh; Jambhekar, Nirmala Ajit

2011-03-01

386

Characterization of Two Bacillus thuringiensis Genes Identified by In Vivo Screening of Virulence Factors  

PubMed Central

Bacillus thuringiensis vegetative cells are known to be highly pathogenic when injected into the hemocoel of susceptible insect larvae. This pathogenicity is due to the capacity of B. thuringiensis to cause septicemia in the host. We screened a B. thuringiensis mini-Tn10 insertion library for loss of virulence against Bombyx mori larvae on injection into the hemocoel. Three clones with attenuated virulence were isolated, corresponding to two different mini-Tn10 insertions mapping to the yqgB/yqfZ locus. Single disruptions of the yqgB and yqfZ genes did not affect virulence against B. mori. In contrast, the inactivation of both genes simultaneously reproduced the effect of the mini-Tn10 insertion and resulted in a significant delay to infection. The double ?yqgB ?yqfZ mutant was also nonmotile, and its growth was affected at 25°C. We analyzed lacZ transcriptional fusions and detected promoter activity upstream from yqgB at 25 and 37°C. Overall, our findings suggest that the yqgB and yqfZ genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host. PMID:15294815

Fedhila, Sinda; Guillemet, Elisabeth; Nel, Patricia; Lereclus, Didier

2004-01-01

387

Drosophila Immunity: Analysis of Larval Hemocytes by P-Element-Mediated Enhancer Trap  

PubMed Central

Our aim was to identify new genes involved in the cellular aspects of defense mechanisms of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for ex