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

NASA Astrophysics Data System (ADS)

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

Karttunen, Jaana; Shastri, Nilabh



Synthesis and Characterization of Novel lacZ Gene Reporter Molecules: Detection of ?-Galactosidase Activity Using 19F NMR of Polyglycosylated Fluorinated Vitamin B6  

PubMed Central

Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection, in particular, the spatial extent of expression in the target tissue and the longevity of expression. Thus, the development of non-invasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. We have previously demonstrated the ability to detect ?-gal activity based on 19F NMR chemical shift associated with release of fluorophenyl aglycones from galactopyranoside conjugates. Use of fluoropyridoxol as the aglycone provides a potential less toxic alternative and we now report the design, synthesis and structural analysis of a series of novel polyglycosylated fluorinated vitamin B6 derivatives as 19F NMR sensitive aglycones for detection of lacZ gene expression. In particular, we report the activity of 3, ?4, ?5-tri-O-(?-D-galactopyranosyl)-6-fluoropyridoxol 4, 3-O-(?-D-galactopyranosyl)-?4, ?5-di-O-(?-D-glucopyranosyl)-6-fluoropyridoxol 12 and 3-O-(?-D-galactopyranosyl)-?4, ?5-di-O-(?-D-mannopyranosyl)-6-fluoropyridoxol 13. 4, 12, and 13 all show promising characteristics including highly sensitive 19F NMR response to ?-gal activity (?? = 9.0 ~ 9.4 ppm), minimal toxicity for substrate or aglycone, and good water solubility. However, the differential glycosylation of 12 and 13 appears more advantageous for assessing lacZ gene expression in vivo.

Yu, Jianxin; Mason, Ralph P.



Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae  

Microsoft Academic Search

A translational lacZ reporter system to study gene regulation in Neisseria gonorrhoeae (Ng) was developed. The pUC18-based vector pLES94 transforms Ng and recombines into the Ng chromosome at the site of the proAB genes. The vector contains a restriction site for cloning promoters that will result in a lacZ gene fusion. Initial cloning and characterization of promoters can be done

Lin E. Silver; Virginia L. Clark



Non-invasive in vivo optical imaging of the lacZ and luc gene expression in mice  

Microsoft Academic Search

The bacterial lacZ gene encoding for ?-galactosidase (?-gal) is a common reporter gene used in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression. We used far-red fluorescence for imaging ?-Gal expression in live cells in vitro or in vivo. The 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl) ?-D-galactopyranoside substrate was used

V Josserand; I Texier-Nogues; P Huber; M-C Favrot; J-L Coll



Photoacoustic imaging of lacZ gene expression in vivo  

Microsoft Academic Search

In the postgenomic era, imaging techniques are playing an important role in visualizing gene expression in vivo. This work represents the first demonstration of pho- toacoustic tomography PAT for reporter gene imaging. Rats inoculated with 9L\\/lacZ gliosarcoma tumor cells are imaged with PAT before and after injection of X-gal, a colorimetric assay for the lacZ-encoded enzyme -galactosidase. Using far-red optical

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



Fluorescence-Activated Sorting of Totipotent Embryonic Stem Cells Expressing Developmentally Regulated lacZ Fusion Genes  

Microsoft Academic Search

Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ^+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS

Sita Reddy; Helen Rayburn; Harald von Melchner; H. Earl Ruley



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

SciTech Connect

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

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



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

PubMed Central

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

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



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


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

Fried, Luitpold; Lassak, Jürgen; Jung, Kirsten



lacZ fusions to genes that specify exported proteins: A general technique  

Microsoft Academic Search

We have devised a general, one-step technique for isolation of strains in which the gene coding for an exported protein is fused to the gene for ß-galactosidase (lacZ). These fusions specify a hybrid protein comprised of an NH2-terminal portion of the exported protein and a large functional COOH-terminal portion of ß-galactosidase. The fusions are constructed with a derivative of the

E. Tapio Palva; Thomas J. Silhavy



Transgenic LacZ under control of Hec-6st regulatory sequences recapitulates endogenous gene expression on high endothelial venules  

PubMed Central

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro. The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st (Chst4) gene with flanking sequences. Transgenic mice were generated with the ?-galactosidase (LacZ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LT?R-Ig (lymphotoxin-? receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity.

Liao, Shan; Bentley, Kevin; Lebrun, Marielle; Lesslauer, Werner; Ruddle, Frank H.; Ruddle, Nancy H.



Expression Cloning of Recombinant Escherichia coli lacZ Genes Encoding Cytoplasmic and Nuclear ?-galactosidase Variants  

PubMed Central

Objective(s) Nonviral vector can be an attractive alternative to gene delivery in experimental study. In spite of some advantages in comparison with the viral vectors, there are still some limitations for efficiency of gene delivery in nonviral vectors. To determine the effective expression, the recombinant Escherichia coli lacZ genes were cloned into the different variants of pcDNA3.1 and then the mammalian cells were transfected. Methods and Materials The coding sequences of cytoplasmic and nuclear variants of lacZ gene were inserted downstream of the human cytomegalovirus immediate-early gene promoter of plasmid pcDNA3.1/myc-His C. The new cytoplasmic and nuclear constricts of E. coli ?-galactosidase-coding sequences were introduced into HeLa cells with the aid of linear polyethylenimine and at 2 days post-transfection the cells were stained using 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal). Results Restriction enzyme analyses revealed the proper insertion of E. coli ?-galactosidase-coding sequences into the multiple cloning site of pcDNA3.1/myc-His C. The functionality of the resulting constructs designated pcDNA3.1-cyt.lacZ and pcDNA3.1-nls.lacZ(+) was confirmed by X-gal staining of HeLa cells transfected with these recombinant plasmids. While pcDNA3.1-cyt.lacZ directed the synthesis of cytoplasmically located ?-galactosidase molecules, the ?-galactosidase protein encoded by pcDNA3.1-nls.lacZ(+) was predominantly detected in the cell nucleus. Conclusion The expression of cytoplasmic and nuclear variant of LacZ gene confirmed the ability of pcDNA3.1 as versatility nonviral vector for the experimental gene delivery study in mammalian cells

Naderian, Homayoun; Rezvani, Zahra; Atlasi, Mohammad Ali; Nikzad, Hossein; Antoine, AF de Vries



Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system.  


Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system. PMID:14654437

Hansen, Jřrgen; Felding, Troels; Johannesen, Pia Francke; Piskur, Jure; Christensen, Christina Lund; Olesen, Kjeld



FlhD\\/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions  

Microsoft Academic Search

The Escherichia coli transcriptional regulatory complex FlhD\\/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD\\/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol

Birgit M Prüß; Xiaojin Liu; William Hendrickson; Philip Matsumura



A new vector-host system for construction of lacZ transcriptional fusions where only low-level gene expression is desirable  

Microsoft Academic Search

We improved a multicopy vector, pRS415 [Simons et al., Gene 53 (1987) 85-96], for use in operon fusion constructions by introducing a new multiple cloning site (MCS) containing eight unique restriction sites upstream from the promoterless reporter gene lacZ. In order to reduce plasmid copy number, a new Escherichia coli strain SP2 (pcnB, ?lac, recA) was constructed. This strain permits

Sergey M. Podkovyrov; Timothy J. Larson



Isolation of a Marek's disease virus (MDV) recombinant containing the lacZ gene of Escherichia coli stably inserted within the MDV US2 gene.  

PubMed Central

We have isolated a stable, recombinant Marek's disease virus (MDV) containing the lacZ gene of Escherichia coli inserted into the unique short region of the genome. The nucleotide sequence of the insertion site indicates that it lies within a sequence homologous to the US2 gene of herpes simplex virus. Stable insertion of the lacZ gene into the MDV US2 gene indicates that the site is nonessential for MDV growth in cell culture. Images

Cantello, J L; Anderson, A S; Francesconi, A; Morgan, R W



Genetic System for Reversible Integration of DNA Constructs and lacZ Gene Fusions into the Escherichia coli Chromosome  

Microsoft Academic Search

A plasmid system for site-specific integration into and excision and recovery of gene constructs and lacZ gene fusions from the Escherichia coli chromosome was developed. Plasmid suicide vectors utilizing the origin of replication of R6K plasmids and containing the attP sequence of bacteriophage ?, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the E. coli chromosome by

Ratree Platt; Christopher Drescher; Sei-Kyoung Park; Gregory J. Phillips



Tumour progression of human neuroblastoma cells tagged with a lacZ marker gene: earliest events at ectopic injection sites.  

PubMed Central

Human Platt neuroblastoma cells were transfected with the marker gene, bacterial lacZ, to track cells at the earliest stages after ectopic injection at two different sites in athymic nude mice. Three clones (LZPt-1,-2 and -3) of differing morphologies were analysed. All clones yielded large primary tumours subcutaneously or intradermally with similar latency. While LZPt-2 and -3 clones generated well-staining primary tumours, LZPt-1 cells yielded many non-staining tumours, indicating greater instability of lacZ expression for this clone in situ (stability of lacZ expression in culture was similar for all three clones). After s.c. or intradermal injections, tumour cells were tracked for 1 h to > 3 weeks (palpable) to evaluate the topology and population expansion characteristics at the earliest times. From 1 h to 2 days, tumour cells were concentrated in central masses with 'crinkly hair' distributions emanating from the periphery. Between 3 and 7 days, these 'crinkly hair' patterns were cleared from the tissue, leaving dense ovoid patterns of tumour cells. These concentrations of cells expanded collectively, not by division of one or a few cells, but by division of many cells. For clone LZPt-1, cells stained well with X-gal for 2-3 days; by 7 days, most cells were non-staining. Evidence suggests that lacZ expression is turned off in these tumour cells, rather than a lacZ- cell type clonally dominating the population. For all three clones, tumour cells remained rounded and did not spread in any tissue environment at all time points, indicating very different matrix adhesion mechanisms operating in situ compared with their distinctive spreading patterns in culture. Angioneogenesis near primary tumours became evident by 2-3 days, leading to extensive vascularisation by 1-2 weeks. Overall, these studies indicate common tumour progression characteristics for three different clones of human neuroblastoma, insight into lacZ instability mechanisms operating in one of these clones and the earliest events in primary tumour formation for this tumour at two different ectopic sites. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Kleinman, N. R.; Lewandowska, K.; Culp, L. A.



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

SciTech Connect

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

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



RpoS-Regulated Genes of Escherichia coli Identified by Random lacZ Fusion Mutagenesis  

Microsoft Academic Search

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant

Somalinga R. V. Vijayakumar; Mark G. Kirchhof; Cheryl L. Patten; Herb E. Schellhorn



Gene expression in Zymomonas mobilis: promoter structure and identification of membrane anchor sequences forming functional lacZ' fusion proteins.  

PubMed Central

We have described a procedure for the isolation of lacZ' fusion genes which contain anchor sequences conferring membrane association. This method was used to isolate fragments of DNA from Zymomonas mobilis which contain promoter activity and amino-terminal sequences. The sequences and transcriptional initiation sites of three of these were compared. Both Escherichia coli and Z. mobilis recognized similar regions of DNA for transcriptional initiation. Five to eight consecutive hydrophobic amino acids in the amino terminus served to anchor these hybrid proteins to the membrane in both E. coli and Z. mobilis. General features observed in the Z. mobilis fragments included partial sequence homology with the -35 region sequence of E. coli, repetitive and palindromic A + T-rich regions preceding and adjoining the -10 region, a sequence resembling the consensus sequence of E. coli in the -10 region, and a potential ribosomal-binding site (AGGA) 8 to 12 bases upstream from an in-frame start codon. The level of expression of fusion proteins was generally higher in E. coli than in Z. mobilis. This higher level of expression in E. coli may result from multiple sites of transcriptional initiation and higher plasmid copy number. Images

Conway, T; Osman, Y A; Ingram, L O



Genotoxicity evaluation of amorphous silica nanoparticles of different sizes using the micronucleus and the plasmid lacZ gene mutation assay.  


We investigated the potential of four well-characterized amorphous silica nanoparticles to induce chromosomal aberrations and gene mutations using two in vitro genotoxicity assays. Transmission electron microscopy (TEM) was used to verify the manufacturer's nominal size of 10, 30, 80 and 400 nm which showed actual sizes of 11, 34, 34 and 248 nm, respectively. The 80 (34) nm silica nanoparticles induced chromosomal aberrations in the micronucleus assay using 3T3-L1 mouse fibroblasts and the 30 (34) and 80 (34) nm silica nanoparticles induced gene mutations in mouse embryonic fibroblasts carrying the lacZ reporter gene. TEM imaging demonstrated that the majority of nanoparticles were localized in vacuoles and not in the nucleus of 3T3-L1 cells, indicating that the observed DNA damage was most likely a result of indirect mechanisms. Further studies are needed to reveal these mechanisms and to determine the biological relevance of the effects of these particular silica nanoparticles in vivo. PMID:20735203

Park, Margriet V D Z; Verharen, Henny W; Zwart, Edwin; Hernandez, Lya G; van Benthem, Jan; Elsaesser, Andreas; Barnes, Clifford; McKerr, George; Howard, C Vyvyan; Salvati, Anna; Lynch, Iseult; Dawson, Kenneth A; de Jong, Wim H



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


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

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



Contrasting Expression of Canonical Wnt Signaling Reporters TOPGAL, BATGAL and Axin2LacZ during Murine Lung Development and Repair  

PubMed Central

Canonical Wnt signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2LacZ. Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2LacZ expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2LacZ expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2LacZ are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

Al Alam, Denise; Green, Melissa; Tabatabai Irani, Reza; Parsa, Sara; Danopoulos, Soula; Sala, Frederic G.; Branch, Jonathan; El Agha, Elie; Tiozzo, Caterina; Voswinckel, Robert; Jesudason, Edwin C.; Warburton, David; Bellusci, Saverio



19F-NMR detection of lacZ gene expression via the enzymic hydrolysis of 2-fluoro-4-nitrophenyl beta-D-galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse.  


Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding beta-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing beta-galactosidase (beta-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl beta-D-galactopyranoside (OFPNPG) is readily hydrolyzed by beta-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach. PMID:17351127

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



Tissue specific expression of an ?-skeletal actin- lacZ fusion gene during development in transgenic mice  

Microsoft Academic Search

Transgenic mice carrying a chimaeric transgene containing 730 bp of the 5?-flanking sequences and the entire first intron of the rat ?-skeletal actin gene fused to thelacZ reporter gene have been produced by microinjection. ThelacZ reporter gene was used to verify the suitability of using the rat ?-actin promoter elements to target expression of genes of agricultural and therapeutic value

Emmanuel A. Asante; Jacqueline M. Boswell; David W. Burt; Grahame Bulfield



Tracking Adult Neovascularization during Ischemia and Inflammation Using Vegfr2-LacZ Reporter Mice  

Microsoft Academic Search

The vascular endothelial growth factor\\/vascular endothelial growth factor receptor 2 (VEGF\\/VEGFR-2) signal transduction system plays a key role during embryonic vascular development and adult neovascularization. In contrast to many endothelial genes, VEGFR-2 is expressed at low levels in most adult vessels but is strongly upregulated during neovascularization, leading to a pro-angiogenic response. Here, we analyzed the activity of regulatory sequences

Regina Heidenreich; Toshinori Murayama; Marcy Silver; Christine Essl; Takayuki Asahara; Martin Röcken; Georg Breier



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


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



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

PubMed Central

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

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



A modular set of Flp, FRT and lacZ fusion vectors for manipulating genes by site-specific recombination  

Microsoft Academic Search

Site-specific recombinases can serve as powerful tools to target genetic manipulations to specific cell populations in culture and in the organism. A series of vectors for engineering gene activation, deletion and integration in mammalian cells using Flp recombinase is described here. The vectors are modular in design so that specific cassettes can be linked depending on the application. Using these

Susan M. Dymecki



The influence of formamidopyrimidine-DNA glycosylase on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZ alpha gene.  


Base excision repair (BER) is a very important repair mechanism to cope with oxidative DNA damage. One of the most predominating oxidative DNA damages after exposure to ionizing radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This damage is repaired by formamidopyrimidine-DNA glycosylase (Fpg), a DNA glycosylase which is part of BER. Correct repair of 8oxoG is of great importance for cells, because 8oxoG has strong miscoding properties. Mispairing of 8oxoG with adenine instead of cytosine results in G:C to T:A transversion mutations. To determine the effect of a Fpg-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene, double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target, was irradiated with gamma-rays in aqueous solution under oxic conditions. Subsequently, the DNA was transfected into a wild-type Escherichia coli strain (JM105) and an isogenic Fpg-deficient E. coli strain (BH410). Although the overall spontaneous mutation spectra between JM105 and BH410 seemed similar, remarkable differences could be observed when the individual base pair substitutions were viewed. The amount of C to A transversions, which are most probably caused by unrepaired 8oxoG, has increased 3. 5-fold in the spontaneous BH410 spectrum. When the gamma-radiation-induced mutation spectra of JM105 and BH410 were compared, there was even a larger increase of C to A transversions in the BH410 strain (7-fold). We can therefore conclude that the straightforward approach used in this study confirms the importance of Fpg in repair of gamma-radiation-induced damage, and most probably especially in the repair of 8oxoG. PMID:10556594

Kuipers, G K; Poldervaart, H A; Slotman, B J; Lafleur, M V



Green fluorescent protein as a reporter of gene expression in transgenic mice  

Microsoft Academic Search

We used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a reporter of gene expression in transgenic mice. The GFP coding sequence was placed under the control of the human hemopexin and the mouse ?1 integrin promoter that were previously studied in transgenic mice using the lacZ reporter gene. We showed that GFP has a higher degree

Annalisa Chiocchetti; Emanuela Tolosano; Emilio Hirsch; Lorenzo Silengo; Fiorella Altruda



Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies  

Microsoft Academic Search

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins ?-galactosidase, GFP, luciferase, and ?-lactamase, respectively. Targeted transgenic expression of these reporter proteins is

Daniel J. Spergel; Ulrich Krüth; Derya R. Shimshek; Rolf Sprengel; Peter H. Seeburg



Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824  

Microsoft Academic Search

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter




recA + gene-dependent regulation of a urvD :: lacZ fusion in Escherichia coli K12  

Microsoft Academic Search

The expression of the Escherichia coli uvrD gene was studied with a uvrD::Mud(Aprlac) insertion mutant. The results indicate that it is inducible by DNA damaging agents in a recA+ gene-dependent manner.

Koji Nakayama; Nobuto Irino; Hiroaki Nakayama



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

NASA Technical Reports Server (NTRS)

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

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



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


Dentin sialoprotein (DSP) and phosphophoryn (PP) are two major dentin noncollagenous proteins that are encoded on a single DSP-PP transcript whose expression is tightly regulated during tooth dentinogenesis. The recent identification of this gene transcript in other tissues, including inner ear and jaw tissue, suggests that DSP and PP may have pleiotropic effects on other organs besides teeth. To identify candidate regulatory elements that control DSP-PP temporal and spatial expression, we constructed a -5 kb upstream region rat DSP-PP promoter into the beta-galactosidase expression vector pnLacF plasmid and used this construct to prepare DSP-PP-LacZ transgenic mice. Multiple mouse tissues including teeth, bone, and kidney obtained from the six resulting transgenic mouse lines displayed strong LacZ activity. This spatial distribution was confirmed in several of these tissues by in situ hybridization studies. LacZ activity was transiently expressed in preameloblasts and continuously expressed in odontoblasts demonstrating that this -5 kb rat promoter-dependent LacZ expression mimics reported DSP-PP mRNA expression patterns. Interestingly, this -5 kb rat promoter construct drives LacZ expression according to the rat developmental clock. Based on identified transcription factors present in this -5 kb promoter region, we have identified several probable cis-regulatory modules whose interaction with one another could account for the spatial and temporal distribution of DSP-PP transcripts in developing tissues. PMID:16310176

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



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

PubMed Central

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

Ma, Wenxin; Wang, Shu-Zhen



A set of Hansenula polymorpha integrative vectors to construct lacZ fusions  

Microsoft Academic Search

A set of YEp Saccharomyces cerevisiae-based, integrative Hansenula polymorpha plasmids was constructed to express lacZ gene under yeast gene promoters. The HpLEU2 and HpURA3 genes were used both as markers and to target the integration of plasmids into the corresponding H. polymorpha genome locus. The frequency of transformation reached with these plasmids linearised either in HpLEU2 or HpURA3 was around

N. Brito; M. D. Pérez; G. Perdomo; C. González; P. García-Lugo; J. M. Siverio



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

Microsoft Academic Search

By using three nonviral transfection methods, i.e., microparticle bombardment, lipofection and electroporation, the transfection efficiency and the expression intensity of a lacZ reporter gene were compared in developing chicken embryosin ovo.Of the three transfection methods employed, electroporation conferred the strongest expression of the bacterial lacZ gene with similar transfection efficiency. The results suggest that as far as transient gene expression

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



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


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

Microsoft Academic Search

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

Carsten Petersen



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



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.

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



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

PubMed Central

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

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



Use of a lacZ Gene Fusion to Determine the Dependence Pattern and the Spore Compartment Expression of Sporulation Operon spoVA in spo Mutants of Bacillus subtilis  

Microsoft Academic Search

A spo VAA : : lac2 gene fusion has been used to study expression of the spo VA operon during sporulation in Bacillus subtilis. fl-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of




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

PubMed Central

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

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



Bacterial species specificity in proU osmoinducibility and nptII and lacZ expression.  


Reporter gene-based transcriptional fusions are increasingly being used to address questions in microbial ecology, with constitutively expressed fusions enabling microbe tracking and inducible fusions reporting the presence of environmental signals. To more readily apply this technology to a variety of bacterial species, we examined species specificity in the expression of three promoters of interest. A comparison of two potentially constitutive promoters, each fused to the reporter gene gfp, showed that the nptII promoter (P(nptII)) was expressed in a broader range of species (100% of 11 tested) than the lacZ promoter (P(lacZ)) (75% of 11 tested), and thus has broader applicability for marking bacteria than P(lacZ). For the species that expressed P(lacZ), however,P(lacZ) was expressed 3-fold more than P(nptII), on average. The Escherichia coli proU promoter, which is induced by low water potential in E. coli, Salmonella typhimurium, Pantoea agglomerans, and Pseudomonas syringae, was shown to be similarly responsive to water potential in strains of Clavibacter michiganensis, Enterobacter aerogenes, Pseudomonas fluorescens, Pseudomonas putida, and Sinorhizobium meliloti, as well as mildly osmoresponsive in Agrobacterium tumefaciens, supporting its broad use as a reporter of water potential. Surprisingly, this promoter was not regulated by water potential in strains of Staphylococcus aureus and Erwinia amylovora, illustrating heretofore unrecognized species specificity in proU inducibility, as well as potential limitations in the species that can serve as bioreporters of water potential. PMID:16179797

Wright, Catherine A; Beattie, Gwyn A



Simultaneous Measurement of Benzo[a]pyrene-induced Pig-a and lacZ Mutations, Micronuclei and DNA Adducts in Muta(TM) Mouse  

PubMed Central

In this study we compared the response of the Pig-a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28-day repeat dose study. Muta™Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig-a mutants. BaP-derived DNA adducts were detected in all tissues examined and significant dose-dependent increases in mutant Pig-a phenotypes (i.e., RETCD24- and RBC CD24-) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig-a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig-a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose-related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig-a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)-anchor deficiencies detected in the Pig-a assay. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.

Lemieux, Christine L; Douglas, George R; Gingerich, John; Phonethepswath, Souk; Torous, Dorothea K; Dertinger, Stephen D; Phillips, David H; Arlt, Volker M; White, Paul A



Airway gene transfer in a non-human primate: lentiviral gene expression in marmoset lungs.  


Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials. PMID:23412644

Farrow, N; Miller, D; Cmielewski, P; Donnelley, M; Bright, R; Parsons, D W



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


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

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



Plasma Escherichia coli beta-galactosidase as a marker of tumor burden and response to experimental anti-neoplastic therapy in nude mice xenografted with lacZ transduced human tumor cells.  


Genetic labeling of tumor cells with the Escherichia coli lacZ reporter gene, encoding the enzyme beta-galactosidase, is widely used for histochemical detection of micrometastases in mice. Recently, we have developed a novel, highly sensitive and specific immunocapture chemiluminescence assay for the quantitation of E. coli beta-galactosidase. This assay achieved a detection limit of 0.01 mU of E. coli beta-galactosidase per milliliter, and 97% signal recovery of purified enzyme added to mouse plasma. LacZ transduced MDA-MB-231 BAG human breast cancer cells grown in vitro released soluble beta-galactosidase into the culture medium, and the concentration found correlated with cell density. Growth of the same cells in nude mice produced readily measurable levels of E. coli beta-galactosidase enzyme activity in host plasma and a highly significant correlation could be demonstrated between the size of primary tumor xenografts and the host plasma level of E. coli beta-galactosidase activity. When mice bearing MDA-MB-231 BAG tumor xenografts were treated intravenously with a single injection of doxorubicin (5 mg/kg), the mean tumor volume after 16 days was reduced 4-fold in the group of doxorubicin-treated mice compared with saline-treated control mice, and the mean level of plasma E. coli beta-galactosidase was correspondingly reduced 3.8-fold in the doxorubicin-treated mice compared with control mice. Sensitive and specific measurement of soluble E. coli beta-galactosidase in blood, using an immunocapture chemiluminescence assay, thus provides objective assessment of tumor burden in mice xenografted with lacZ transduced human tumors. This assay may have important applications as a tool for determining the efficacy of new experimental anti-tumor agents. PMID:10830782

Holst-Hansen, C; Stephens, R W; Johannessen, B E; Jensen, P B; Brünner, N



A GAL4HP1 fusion protein targeted near heterochromatin promotes gene silencing  

Microsoft Academic Search

We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have

Carole Seum; Anne Spierer; Marion Delattre; Daniel Pauli; Pierre Spierer



Deletion of Yeast CWP Genes Enhances Cell Permeability to Genotoxic Agents  

Microsoft Academic Search

We have previously reported the development of a novel geno- toxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in micro- organisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects

Min Zhang; Yuping Liang; Xiaohua Zhang; Ying Xu; Heping Dai; Wei Xiao



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

Microsoft Academic Search

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

Robert W. West; Brian Kruger; Sean Thomas; Junli Ma; Elena Milgrom



Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus.  


A promoterless trp-lac fusion fragment was inserted near one end of the bacterial transposon Tn5 in the correct orientation to fuse lacZ gene expression to promoters outside Tn5. The resulting transposon, Tn5 lac, retains the kanamycin-resistance gene of Tn5 and transposes in Escherichia coli at 6% the frequency of Tn5 to many different sites in a bacteriophage lambda target. Expression of beta-galactosidase, the product of the lacZ gene, from Tn5 lac insertions in phage lambda depends both on insertion into a transcription unit in the correct orientation and on the regulation of the promoter of the transcription unit, verifying that by transposition Tn5 lac can fuse lacZ expression to outside promoters. An insertion of Tn5 lac in bacteriophage P1 was isolated and used to introduce Tn5 lac into Myxococcus xanthus, a bacterium that undergoes multicellular development. Stable kanamycin-resistant transductants are obtained that contain no P1 DNA sequences but have Tn5 lac inserted at different sites in the Myxococcus chromosome. Individual transductants express different levels of beta-galactosidase. A chromogenic substrate of beta-galactosidase, 5-bromo-4-chloro-3-indolyl beta-D-galactoside, is toxic in Myxococcus when cleaved in large amounts. In principle, Tn5 lac could be used to assay transcription in any bacterium in which Tn5 can transpose and beta-galactosidase can be measured. PMID:6091110

Kroos, L; Kaiser, D



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

PubMed Central

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

Douglas, Grace L.; Klaenhammer, Todd R.



The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.  

PubMed Central

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo. Images

Ghattas, I R; Sanes, J R; Majors, J E



Method for Screening Compounds That Influence Virulence Gene Expression in Staphylococcus aureus?  

PubMed Central

We present a simple assay to examine effects of compounds on virulence gene expression in the human pathogen Staphylococcus aureus. The assay employs transcriptional reporter strains carrying lacZ fused to central virulence genes. Compounds affecting virulence gene expression and activity of the agr locus are scored based on color change in the presence of a chromogenic ?-galactosidase substrate. The assay can be used to screen for novel antivirulence compounds from many different sources, such as fungi, as demonstrated here.

Nielsen, Anita; Nielsen, Kristian F.; Frees, Dorte; Larsen, Thomas O.; Ingmer, Hanne



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.

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



Development of new inbred transgenic strains of rats with LacZ or GFP  

Microsoft Academic Search

The ideal goal of regeneration medicine is to restore form and function to damaged tissues. While stem cell transplantation is considered a promising therapeutic approach, knowing the fate of transplanted cells using appropriate markers is essential. We developed new inbred transgenic rat strains with lacZ and GFP based on the transgenic (Tg) animal technique in rats. These Tg animals expressed

Hirokazu Inoue; Ichiro Ohsawa; Takashi Murakami; Atsushi Kimura; Yoji Hakamata; Yuki Sato; Takashi Kaneko; Masafumi Takahashi; Takashi Okada; Keiya Ozawa; Jeremy Francis; Paola Leone; Eiji Kobayashi



Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.  

PubMed Central

Muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and Duchene's muscular dystrophy. Previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. We introduced recombinant adeno-associated virus vector (rAAV) carrying a lacZ reporter into muscle tissue of immunocompetent mice. The lacZ reporter gene was efficiently transduced and expressed with no evidence of a cellular immune response. Moreover, gene expression persisted for more than 1.5 years. Molecular characterization of rAAV vector DNA suggests a mechanism for persistence, since vector episomes convert to high-molecular-weight genomic DNA. These data provide the first report for establishing long-term gene transduction into mammalian muscle cells in vivo without the need for immune modulation of the organism.

Xiao, X; Li, J; Samulski, R J



The tomato nia gene promoter functions in fission yeast but not in budding yeast  

Microsoft Academic Search

A fragment comprising 1 kb of the 5' region and the 81 first nucleotides of the coding region of the tomato nitrate reductase nia gene was placed in translational fusion with the lacZ reporter gene. This construct was introduced in budding and in fission yeast using a derivative of the Saccharomyces cerevisiae\\/Schizosaccharomyces pombe autonomously replicating vector pUZL. ß-galactosidase activity was

Hoai-Nam Truong; Michel Caboche; Françoise Daniel-Vedele



Zero background yeast reporter plasmids.  


UAS-less reporter plasmids are widespread and powerful tools for the identification and analysis of binding sites for transcriptional activators. The common reporter plasmids for the yeast Saccharomyces cerevisiae are multicopy (2mu) vectors with the CYC1 core promoter upstream of the lacZ gene. Insertion of putative or known activator binding sites upstream of the core promoter puts lacZ (beta-galactosidase) expression under the control of the corresponding activator. Although these constructs have proved to work well for most purposes, they have certain limitations: (1) they give significant and carbon-source-dependent lacZ background expression; (2) unlike most other yeast promoters, the CYC1 upstream region has a partially open chromatin structure with an accessible TATA box; (3) they use only a single, moderately sensitive reporter; and (4) the use of multicopy vectors can result in activator titration. Here, we introduce novel reporter plasmids based on the yeast MEL1 (alpha-galactosidase) gene that can overcome all of these limitations. It is also shown that background expression is due to fortuitous activator binding sites within the plasmid backbones that are insufficiently shielded from the core promoters in the common CYC1 reporter plasmids. PMID:10773444

Melcher, K; Sharma, B; Ding, W V; Nolden, M



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


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. Environ. Mol. Mutagen. 55:500-509, 2014. © 2014 Wiley Periodicals, Inc. 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



Construction and application of mycobacterial reporter transposons.  


The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria. PMID:10925203

Machowski, E E; McAdam, R A; Derbyshire, K M; Mizrahi, V



Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal  

Microsoft Academic Search

BACKGROUND: Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP) from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a

Anna-Katerina Hadjantonakis; Suzanne Macmaster; Andras Nagy



Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system  

Microsoft Academic Search

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also

Jřrgen Hansen; Troels Felding; Pia Francke Johannesen; Jure Piskur; Christina Lund Christensen; Kjeld Olesen



Gene delivery by the hSP-B promoter to lung alveolar type II epithelial cells in LAL-knockout mice through bone marrow mesenchymal stem cells  

Microsoft Academic Search

Tissue damage and inflammation promote bone marrow stem cells (BMSCs) to differentiate into a variety of cell types in residing tissues. BMSCs can stably maintain their plasticity and are an ideal cell population for delivery of therapeutic genes to non-hematopoietic tissues. Using lacZ as a reporter gene, we demonstrated that the lung-specific human surfactant protein B (hSP-B) 1.5-kb promoter is

C Yan; X Lian; Y Dai; X Wang; P Qu; A White; Y Qin; H Du



Analysis of Expression of the Alpha-Toxin Gene (hla )o f Staphylococcus aureus by Using a Chromosomally Encoded hla::lacZ Gene Fusion  

Microsoft Academic Search

The staphylococcal alpha-toxin (Hla) is a major virulence factor contributing to Staphylococcus aureus pathogenesis. To elucidate the conditions influencing hla expression, the determinant was fused to lacZ, the reporter gene coding for b-galactosidase. The hla::lacZ fusion was integrated into the chromosome of the wild-type S. aureus strain Wood 46, leading to the variant Wood 46-3. Alpha-toxin expression was found to




Sources of variability in data from a positive selection lacZ transgenic mouse mutation assay: an interlaboratory study.  


Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported. PMID:9057887

Piegorsch, W W; Lockhart, A C; Carr, G J; Margolin, B H; Brooks, T; Douglas, G R; Liegibel, U M; Suzuki, T; Thybaud, V; van Delft, J H; Gorelick, N J



Exploring sRNA-mediated gene silencing mechanisms using artificial small RNAs derived from a natural RNA scaffold in Escherichia coli  

PubMed Central

An artificial small RNA (afsRNA) scaffold was designed from an Escherichia coli sRNA, SibC. Using the lacZ reporter system, the gene silencing effects of afsRNAs were examined to explore the sRNA-mediated gene-silencing mechanisms in E. coli. Substitution of the original target recognition sequence with a new sequence recognizing lacZ mRNA led to effective reduction of lacZ gene expression. Single-strandedness of the target recognition sequences in the scaffold was essential for effective gene silencing. The target recognition sequence was shortened to 10 nt without significant loss of gene silencing, although this minimal length was limited to a specific target mRNA sequence. In cases where afsRNAs had mismatched (forming internal loops) or unmatched (forming bulges) regions in the middle of the target recognition sequence, internal loop-forming afsRNAs were more effective in gene silencing than those that formed bulges. Unexpectedly, gene silencing by afsRNA was not decreased but increased on hfq disruption in E. coli, particularly when interactions between afsRNA and mRNA were weak, suggesting that Hfq is possibly involved in destabilization of the RNA–RNA duplex, rather than enhancement of base pairing.

Park, Hongmarn; Bak, Geunu; Kim, Sun Chang; Lee, Younghoon



[Construction and characterization of a recombinant fowlpox virus co-expressing F, HN genes of Newcastle disease virus and gB gene of infectious laryngnotracheitis virus].  


The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2) while the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ were characterized by Western blot (F and gB proteins) and indirect immunofluorescence test (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10 kb gene fragment, could be expressed authentically and efficiently. Compared to the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in chicken embryo fibroblasts (CEF) cell culture. Overall, our work suggests that FPV can be a useful live virus vector for the expression of multi- foreign genes against multiple avian pathogens. PMID:17168315

Sun, Hui-Ling; Wang, Yun-Feng; Miao, De-Yuan; Zhang, Pei-Jun; Zhi, Hai-Dong; Xu, Ling-Long; Wang, Mei; Tong, Guang-Zhi; Wang, Ming



Structure and regulation of the salivary gland secretion protein gene Sgs-1 of Drosophila melanogaster.  

PubMed Central

The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae.

Roth, G E; Wattler, S; Bornschein, H; Lehmann, M; Korge, G



O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene  

Microsoft Academic Search

The authors previously reported increased expression of the Salmonella enterica serovar Typhi (S. typhi) rfaH gene when the bacterial cells reach stationary phase. In this study, using a lacZ fusion to the rfaH promoter region, they demonstrate that growth-dependent regulation of rfaH expression occurs at the level of transcription initiation. It was also observed that production of the lipopolysaccharide (LPS)

Mauricio Bittner; Soledad Saldi; Mercedes Zaldi; Cristina L. Marolda; Miguel A. Valvano


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



Adenovirus-mediated gene transfer to the ocular surface epithelium.  


Gene transfer to the ocular surface epithelium is of potential therapeutic value. It was determined whether a reporter gene can be introduced into the ocular surface epithelium in vitro (human cell lines), ex vivo (human tissues), and in vivo (rats) by treating with a recombinant, replication-deficient, adenovirus type 5. Human and conjunctival cell lines were cultured with various multiplicities of infection (MOI; 3.2x10(-5)-5x10(-1)) of adenovirus vector (Ad5:Adex1CAlacZ) containing the reporter gene lacZ (1.3-2.0x10(4) PFU ml-1). The ex vivo study used human corneal and conjunctival tissues obtained from an eye bank and during surgery. Non-specific upregulation of inflammatory cytokines of conjunctival epithelium infected by Ad5 was assayed and its suppression by steroids. For the in vivo study, Ad5 (5x10(5) PFU, 5-10 microliter) was applied to the eyes of 8-12-week-old cotton rats, which were enucleated 24 and 48 hr later. The maximum lacZ expression in vitro was demonstrated in the corneal epithelial cell line at 7 days (1x10(-1) MOI) and conjunctival epithelial cell line at 2 days (4x10(-4) MOI). Furthermore, lacZ was also expressed in the superficial corneal and conjunctival epithelium in the ex vivo study. IL-6, IL-8, and ICAM-1 expression from conjunctival epithelium by Ad5 was significantly inhibited by treatment with betamethasone (BM). For the in vivo study, only the conjunctival epithelium demonstrated beta-Gal activity at 24 and 48 hr after application. These data indicate that adenovirus vector is capable of directly delivering gene to the corneal and conjunctival epithelium, suggesting a variety of possible gene therapy uses. The concomitant application of steroid eye drops may avoid inflammation. PMID:9878215

Tsubota, K; Inoue, H; Ando, K; Ono, M; Yoshino, K; Saito, I



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



Image Processing and Analysis for Quantifying Gene Expression from Early Drosophila Embryos  

PubMed Central

Abstract Correlation of quantities of transcriptional activators and repressors with the mRNA output of target genes is a central issue for modeling gene regulation. In multicellular organisms, both spatial and temporal differences in gene expression must be taken into account; this can be achieved by use of in situ hybridization followed by confocal laser scanning microscopy (CLSM). Here we present a method to correlate the protein levels of the short-range repressor Giant with lacZ mRNA produced by reporter genes using images of Drosophila blastoderm embryos taken by CLSM. The image stacks from CLSM are processed using a semiautomatic algorithm to produce correlations between the repressor levels and lacZ mRNA reporter genes. We show that signals derived from CLSM are proportional to actual mRNA levels. Our analysis reveals that a suggested parabolic form of the background fluorescence in confocal images of early Drosophila embryos is evident most prominently in flattened specimens, with intact embryos exhibiting a more linear background. The data extraction described in this paper is primarily conceived for analysis of synthetic reporter genes that are designed to decipher cis-regulatory grammar, but the techniques are generalizable for quantitative analysis of other engineered or endogenous genes in embryos.

Ay, Ahmet; Fakhouri, Walid D.; Chiu, Chichia



Differential experimental micrometastasis to lung, liver, and bone with lacZ -tagged CWR22R prostate carcinoma cells  

Microsoft Academic Search

LacZ-tagged human prostate carcinoma CWR22Rv1 cells metastasize spontaneously to lung, liver, and bone from subcutaneous primary tumors in athymic nude mice; these organs are ‘natural’ targets of metastasis for the human disease. To evaluate the mechanism(s) of metastasis to these organs, an experimental metastasis model was used by taking advantage of the ultrasensitive detection of lacZ. Within 1 h after

Julianne L. Holleran; Carson J. Miller; Nancy L. Edgehouse; Theresa P. Pretlow; Lloyd A. Culp



Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression?  

PubMed Central

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

Ravin, Nikolai V.; Rech, Jerome; Lane, David



Establishment of lacZ marked strain of phosphate solubilizing bacterium in the rhizosphere and its effect on plant growth in mungbean.  


The establishment of lacZ marked strain of P-solubilizing bacterium Pseudomonas in the rhizosphere of mungbean (Vigna radiata) under pothouse conditions was studied. The lacZ marker was transferred to Pseudomonas P-36 on LB medium using donor strain of E. coli. The lacZ marked strain formed blue colonies on selective media and could be identified from soil on the basis of this character. The lacZ marked strain was able to survive in rhizosphere of mungbean under pothouse conditions and maintained a population of about 10(4) g(-1) of rhizosphere soils up to 60 days study period. Positive effect of inoculation with P-solubilizing bacterium on dry matter yield, P and N-uptake was observed using rock phosphate and single super phosphate as P sources with and without farmyard amendment. PMID:22815583

Sunita, S; Kapoor, K K; Goyal, S; Sharma, P K



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.

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



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

PubMed Central

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

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



Nucleotide sequence of Klebsiella pneumoniae lac genes.  

PubMed Central

The nucleotide sequences of the Klebsiella pneumoniae lacI and lacZ genes and part of the lacY gene were determined, and these genes were located and oriented relative to one another. The K. pneumoniae lac operon is divergent in that the lacI and lacZ genes are oriented head to head, and complementary strands are transcribed. Besides base substitutions, the lacZ genes of K. pneumoniae and Escherichia coli have suffered short distance shifts of reading frame caused by additions or deletions or both during evolutionary divergence from a common ancestral gene. Relative to corresponding E. coli sequences, the nucleotide sequences of the lacZ and lacY genes are 61 and 67% conserved, and the lacI genes are 49% conserved. A comparison of both nucleotide and amino acid sequences revealed that the K. pneumoniae and E. coli lacI genes and lac repressor proteins each are related to the galR gene and gal repressor of E. coli to about the same extent. In terms of evolutionary relationships, the divergence of the forerunner of the galR gene from an ancestral lac repressor gene preceded separation and differentiation of the K. pneumoniae and E. coli lac repressor genes.

Buvinger, W E; Riley, M



A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.  

PubMed Central

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

Selwood, T; Sinnott, M L



Yeast excision-repair gene is inducible by DNA damaging agents  

Microsoft Academic Search

Plasmids containing various RAD-lacZ gene fusions were integrated into the chromosome of haploid yeast cells. These integrant strains were tested for expression of Escherichia coli ..beta..-galactosidase after treatment with agents that damage DNA or interfere with normal DNA replication. The authors did not observe induction of single-copy RAD1-lacZ or RAD3-lacZ fusion genes under the experimental conditions used. However, exposure of

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



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


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

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



Recombinant Carcinoembryonic Antigen as a Reporter Gene for Molecular Imaging  

PubMed Central

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

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



Dicistronic Targeting Constructs: Reporters and Modifiers of Mammalian Gene Expression  

Microsoft Academic Search

To investigate the activity of candidate regulatory molecules in mammalian embryogenesis, we have developed a general strategy for modifying and reporting resident chromosomal gene expression. The picornaviral internal ribosome-entry site was incorporated into gene targeting constructs to provide cap-independent translation of a selectable marker from fusion transcripts generated following homologous recombination. These promoterless constructs were highly efficient and have been

Peter Mountford; Branko Zevnik; Annette Duwel; Jennifer Nichols; Meng Li; Christian Dani; Morag Robertson; Ian Chambers; Austin Smith



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

PubMed Central

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

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



Streptococcal Reporter Gene-Fusion Vector for Identification of in Vivo Expressed Genes  

Microsoft Academic Search

To study streptococcal genes that are specifically induced in the host during endocarditis, we have developed a novel plasmid for use in in vivo expression technology (IVET). This IVET uses an integration plasmid, pAK36, that carries dual (amy–cat) reporter genes. A gene-fusion strain library was constructed with the plasmid randomly inserted into the chromosome of Streptococcus gordonii V288 by insertion–duplication.

Ali O. Kiliç; Mark C. Herzberg; Maurice W. Meyer; Xuemei Zhao; Lin Tao



Pag-3, a Caenorhabditis Elegans Gene Involved in Touch Neuron Gene Expression and Coordinated Movement  

PubMed Central

Mutations in a newly identified gene, pag-3, cause ectopic expression of touch neuron genes mec-7, mec-7lacZ and mec-4lacZ in the lineal sisters of the ALM touch neurons, the BDU neurons. pag-3 mutants also show a reverse kinker uncoordinated phenotype. The first pag-3 allele was isolated in a screen for mutants with altered immunofluorescence staining patterns. Two additional pag-3 alleles were identified in a noncomplementation screen of 38,000 haploid genomes. All of the pag-3 alleles were recessive to wild type and cause the same phenotypes. Two-factor crosses, deficiency mapping and three-factor crosses located pag-3 to the right arm of the X chromosome between unc-3 and unc-7. Because recessive mutations in pag-3 result in expression of several touch cell specific genes in the BDU neurons, pag-3(+) must directly or indirectly suppress expression of these genes in the BDU neurons. Although pag-3 mutants did not show mec-3lacZ expression in their BDU neurons, expression of mec-7lacZ and mec-4lacZ in the BDU neurons of pag-3 mutants required mec-3(+).

Jia, Y.; Xie, G.; Aamodt, E.



In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism  

PubMed Central

Background The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p. Results In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain. Conclusions These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

De Mesquita, Joelma F; Panek, Anita D; de Araujo, Pedro S



Regulatory elements mediating expression of xylanase genes in Fusarium oxysporum.  


The role of DNA regulatory elements mediating activation of the xylanase-encoding gene xyl4 by the transcription factor XlnR in the fungal pathogen Fusarium oxysporum, was studied by in vitro and in vivo functional analysis of the xyl4 promoter. Recombinant XlnR protein specifically bound the sequence GGCTAA in electrophoretic mobility shift assays. Experiments with xyl4 promoter fusions with the lacZ reporter gene showed that the GGCTAA sequence is required for xylan-induced transcriptional activation of xyl4 in F. oxysporum. The results support a model in which the interaction between the transcriptional activator XlnR and an unknown constitutive repressor regulates xylanase gene expression in F. oxysporum. PMID:17664074

Calero-Nieto, Fernando; Hera, Concepción; Di Pietro, Antonio; Orejas, Margarita; Roncero, M Isabel G



Structure and regulation of the salivary gland secretion protein gene Sgs-1 of Drosophila melanogaster.  


The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae. PMID:10511555

Roth, G E; Wattler, S; Bornschein, H; Lehmann, M; Korge, G



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

NASA Technical Reports Server (NTRS)

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

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



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



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


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

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



Single plasmids expressing human steroid hormone receptors and a reporter gene for use in yeast signaling assays.  


Single plasmids designed to express the six human type I steroid hormone receptors and detect signaling activity are described in this report. These stably replicating plasmids reported ligand-induced transcriptional activation via lacZ assays in Baker's yeast (Saccharomyces cerevisiae). The ligand concentrations needed to activate signaling in yeast expressing these plasmids spanned five orders of magnitude as based on comparisons of EC(50) values. Radicicol, a direct inhibitor of heat shock protein 90 (Hsp90) and an indirect inhibitor of steroid hormone receptor signaling, was used to determine the functional utility of this yeast reporter system. The inhibitory effect of radicicol was similar on the signaling of all six steroid hormone receptors and was distinguishable from cytotoxic effects that occurred with higher concentrations. These yeast plasmids provide a high throughput system for comparative assessment of steroid hormone receptor signaling and may be useful in screening for pharmacological or xenobiotic activities. PMID:19962400

Miller, Charles A; Tan, Xiaobing; Wilson, Mark; Bhattacharyya, Sunanda; Ludwig, Sara



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



The Use of the L-Plastin Promoter for Adenoviral-mediated, Tumor-specific Gene Expression in Ovarian and Bladder Cancer Cell Lines1  

Microsoft Academic Search

A 2.4-kb truncated L-plastin promoter was inserted either 5* to the LacZ gene (Ad-Lp-LacZ) or 5* to the cytosine deaminase (CD) gene (Ad-Lp-CD) in a replication-incompetent adenoviral vector backbone. Infectivity and cytotoxicity experiments with the LacZ and CD vectors suggested that the L-plastin promoter-driven transcriptional units were expressed at much higher levels in explants of ovarian cancer cells from patients

Xue Yan Peng; Jong Ho Won; Thomas Rutherford; Takuma Fujii; Daniel Zelterman; Giuseppi Pizzorno; Eva Sapi; John Leavitt; Barry Kacinski; Ronald Crystal; Peter Schwartz; Albert Deisseroth



Parameters influencing ectopic gene expression in Aplysia neurons.  


DNA microinjection for expressing exogenous genes in Aplysia neurons has been very useful for analyzing not only functions of encoded proteins, but also regulations of gene expression in the nervous system. Some of the factors that affect expression of foreign genes microinjected into Aplysia neurons are described. The effect of the DNA form (supercoiled or linear) and promoter modification in the expression vectors are analyzed as well as buffer composition and DNA concentration in the microinjection solution. The time course of reporter gene expression was also monitored. Reporter gene expression was first detected as early as 1 h and maintained at a high level even until 7 days after microinjection. The presence of AP-1 enhancer in the promoter region of the expression vectors was essential in driving a high-level constitutive expression of reporter genes. Particularly, a pNEX derivative containing eight copies of AP-1 enhancer drove constitutive overexpression more effectively than ones harboring either four or 12 copies of AP-1 enhancer. We also found that a prokaryotic promoter/operator from E. coli lacZ gene placed upstream from an eukaryotic enhancer/promoter repressed the expression of the downstream reporter gene in Aplysia neurons. PMID:9014173

Kaang, B K



GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line  

PubMed Central

AIM: The GFAP was traditionally considered to be a biomarker for neural glia (mainly astrocytes and non-myelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other non-HSC cell types). The transgene expression specificity was determined by X-gal staining of the ?-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-?1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-?1 by upregulation in a dose- and time-dependent manner, similar to the endogenous GFAP. CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

Maubach, Gunter; Lim, Michelle Chin Chia; Zhang, Chun-Yan; Zhuo, Lang



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


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



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


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

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



Pleiotrophin Gene Therapy for Peripheral Ischemia: Evaluation of Full-Length and Truncated Gene Variants  

PubMed Central

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

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



Dual-modality gene reporter for in vivo imaging.  


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



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



Readministration of adenoviral gene delivery to dopamine neurons.  


An approach currently being explored as treatment for Parkinson's disease is gene therapy. An important question concerns the duration of transgene expression in dopamine neurons and the issues of vector persistence, neuronal damage and the feasibility of readministering vector to the same neuronal population. We show, using an adenoviral vector expressing the LacZ reporter gene, that transgene expression declined over time but with minimal loss of dopamine neurons or vector DNA. Readministration of vector resulted in low levels of transgene delivery to the neurons. Moreover, the neurons to which vector had already been delivered were unable to transport the retrograde tracer fluorogold. Our findings indicate that transgene expression declined in dopamine neurons despite the persistence of virus, and the capacity to readminister vector to these neurons was limited. PMID:17885611

Gonzalez, Sarah C; McMenamin, Margaret M; Charlton, Harry M; Goodman, James; Lantos, Tibor; Simpson, Christine; Wood, Matthew J A



Generation and analysis of Elf5-LacZ mouse: unique and dynamic expression of Elf5 (ESE2) in the inner root sheath of cycling hair follicles  

Microsoft Academic Search

The Elf5\\/ESE-2 transcription factor is a member of the Epithelium Specific Ets subfamily of Ets transcription factors. Expression\\u000a of Elf5 has been known to be restricted to organs and tissues rich in glandular or secretory epithelial cells such as kidney\\u000a and mammary gland as well as differentiated keratinocytes of the skin. We have engineered an Elf5-LacZ mouse strain in which

Yeon Sook Choi; Jun Cheng; Julie Segre; Satrajit Sinha



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.



Evaluating Reported Candidate Gene Associations with Polycystic Ovary Syndrome  

PubMed Central

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

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



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.



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


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

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



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


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

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



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


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

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



The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability.  


The yeast HPR1 gene plays an important role in genome stability, as indicated by the observation that hpr1 mutants have high frequencies of DNA repeat recombination and chromosome loss. Here we report that HPR1 is required for transcriptional elongation. Transcription driven from constitutive and regulated yeast promoters cannot elongate through the bacterial lacZ coding region in hpr1Delta cells, but progresses efficiently through other sequences such as yeast PHO5. We show that HPR1 is not required for transcription activation and that the previously reported effects of hpr1Delta on the activation of different promoters is a consequence of the incapacity of hpr1Delta cells to elongate transcription through lacZ, used as reporter. Transcriptional defects are also observed in yeast DNA sequences of hpr1Delta cells in the presence of the transcription elongation inhibitor 6-azauracil. In all cases, the blockage of transcription elongation in hpr1Delta is associated with both the high frequency of deletions and the increase in plasmid instability that we report here. Therefore, in addition to the identification of a new element involved in transcriptional elongation, our work provides evidence for a new source of genomic instability. PMID:9407037

Chávez, S; Aguilera, A



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

PubMed Central

Background Gene therapy has attracted attention for its potential to treat several cardiovascular diseases. The use of adeno-associated viral (AAV) vectors to facilitate therapeutic gene transfer to suppress intimal hyperplasia is a promising concept. The objective of this study was to analyze the in vivo transduction of a novel recombinant AAV-2/9 vector with SM22? promoter, containing ?-galactosidase gene (Lac Z) or green fluorescent protein (GFP) as reporter genes, to the medial layer smooth muscle cells (SMCs) of swine coronary and peripheral arteries. Methods The AAV2/9 vector containing SM22? (1×1013 pfu) were administered into carotid/femoral/coronary arteries of domestic swine using irrigating balloon catheter-based gene delivery. Following gene transfer, cryosections of arteries were processed for X-Gal and GFP analysis. Fluorescence microscopy and Western blotting were done to analyze the GFP expression in the SMCs. Results LacZ mRNA expression was visualized in the medial layer 7 days after vector administration. The GFP expression was detected at 7th day and lasted for at least 2 months showing the longer-lasting expression of the AAV2/9-vector. Control arteries did not show any expression of GFP or LacZ. There was no significant effect of AAV2/9 viral transduction on serum amylase, fibrinogen and serum CRP levels. Conclusion These finding support the use of AAV2/9 as a vector to effectively transduce a gene in SMCs of coronary and peripheral arteries without causing inflammation.

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



Identification of a class of Saccharomyces cerevisiae mutants defective in fatty acid repression of gene transcription and analysis of the frm2 gene.  


Exogenous fatty acids transcriptionally control the expression of a wide variety of eukaryotic genes, many of which encode proteins involved in lipid metabolism. To identify gene products involved in the lipid signalling pathway, a reporter plasmid containing the 5'-upstream region of a gene demonstrated to be repressed by unsaturated fatty acids (OLE1) was fused in frame to the Escherichia coli gene lacZ encoding beta-galactosidase. Saccharomyces cerevisiae mutants defective in transcriptional control by lipids were identified and this class of mutants has been named frm (fatty acid repression mutant). The mutants were organized into six complementation groups designated frm1-6. Mutants from two of the complementation groups, frm1 and frm3, were also defective in their ability to activate a reporter construct containing the 5'-upstream region of POX1. POX1 has been shown to be transcriptionally activated in the presence of unsaturated fatty acids. frm2 was rescued by a region of DNA localized to chromosome III. This region contained an open reading frame of 579 nucleotides predicted to encode a M(r) 21 116 polypeptide. The upstream region of FRM2 contained a number of potential response elements which have previously been identified as important in regulating gene expression in response to glucose and certain fatty acids. Consistent with this observation, lacZ activity driven by FRM2 or frm2 promoters was induced two- to three-fold dependent upon the carbon and fatty acid source utilized. The properties of FRM2 suggest that it functions in the fatty acid signalling pathway and that it is itself regulated by fatty acids. PMID:8701605

McHale, M W; Kroening, K D; Bernlohr, D A



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


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

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



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


Novel double and triple fusion reporter gene constructs harboring distinct 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) [Portola Valley, CA; Pritha, Ray (Mountain View, CA) [Mountain View, CA



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


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

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



Development of a LacZ-Based Transcriptional Reporter System for Use with Moraxella catarrhalis  

PubMed Central

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

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



Feasibility of sodium\\/iodide symporter gene as a new imaging reporter gene: comparison with HSV1-tk  

Microsoft Academic Search

Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D 2 receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium\\/iodide symporter ( NIS) gene can be easily monitored with radioiodines and technetium-99m using

JaeHoon Shin; June-Key Chung; JooHyun Kang; YongJin Lee; KwangIl Kim; ChulWoo Kim; JaeMin Jeong; DongSoo Lee; MyungChul Lee



Intravenous delivery of AAV9 vector mediates effective gene expression in ischemic stroke lesion and brain angiogenic foci  

PubMed Central

Background and Purpose Adeno-associated viral vector (AAV) is a powerful tool for delivering genes to treat brain diseases. Intravenous delivery of a self-complementary, but not single-stranded, AAV9 vector (ssAAV9) mediates robust gene expression in the adult brain. We tested if ssAAV9 effectively mediates gene expression in the ischemic stroke lesion and angiogenic foci. Methods Focal ischemic stroke was induced by permanent occlusion of the left middle cerebral artery (MCAO), and focal angiogenesis, by injecting an AAV vector expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have CMV promoter driving (AAV-CMVLacZ) or hypoxia response elements controlling (AAV-H9LacZ) LacZ expression were packaged in AAV9 or AAV1 capsid, and injected into mice through the jugular vein one hour after MCAO or four weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs five days post LacZ vector-injection. Results LacZ expression was detected in the peri-infarct region of AAV9-CMVLacZ and AAV9-H9LacZ-injected MCAO mice, and the brain angiogenic foci of AAV9-CMVLacZ-injected mice. Minimum LacZ expression was detected in the brain of AAV1-CMVLacZ-injected mice. Robust LacZ expression was found in the liver and heart of AAV-CMVLacZ-injected mice, but not AAV9-H9LacZ-injected mice. Conclusion ssAAV9 vector could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci.

Shen, Fanxia; Kuo, Robert; Milon-Camus, Marine; Han, Zhenying; Jiang, Lidan; Young, William L.; Su, Hua



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


The veA gene is one of the key genes in regulating sexual development of Aspergillus nidulans. During the study on the veA gene, it was observed that the veA expression level is slightly higher in a veA1 mutant than in a wild type at 37 degrees C, suggesting that the wild type veA gene is necessary for the negative regulation of the veA expression. In the veA1 mutant, the veA expression was higher than in a wild type grown at 42 degrees C but equal at 30 degrees C. Furthermore, in a veA deletion mutant having its own promoter and the N-terminus of the VeA ORF, expression of the N-terminus by the veA promoter was highly up-regulated, supporting the possibility that the veA gene is important for the negative regulation of the veA expression. Analyses of the lacZ transcript and the beta-galactosidase activity from the reporter strains in the veA1 background, which were constructed by transformation of the lacZ reporter plasmids containing the lacZ gene under the control of the intact or the truncated veA promoters from the -943 to +262 bp region, showed that the truncated promoters produced more veA transcript and higher beta-galactosidase activity than the intact one at 30 degrees C, but equal at 42 degrees C. In addition, the serial-deletion analysis of the veA promoter identified a crucial region in the promoter from -943 to -740 bp for this derepression of the veA expression. Taken together, these results indicated that the veA gene is necessary for the negative regulation of the veA expression. Moreover, the veA expression was derepressed in the light-illuminated condition, where the VeA protein is hardly transported into the nucleus. PMID:19479257

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



Gene overexpression as a tool for identifying new trans-acting factors involved in translation termination in Saccharomyces cerevisiae.  


In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process. PMID:12072456

Namy, Olivier; Hatin, Isabelle; Stahl, Guillaume; Liu, Hongmei; Barnay, Stephanie; Bidou, Laure; Rousset, Jean-Pierre



BCS1L gene mutation causing GRACILE syndrome: case report.  


Abstract 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, Ci?dem Seher; Tümer, Leyla; Ezgü, Fatih Suheyl; Küçükçongar, Aynur; Hasano?lu, Alev



A single secreted luciferase-based gene reporter assay.  


Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary. PMID:24583246

Barriscale, Kathy A; O'Sullivan, Sharon A; McCarthy, Tommie V



The Lon protease regulates swarming motility and virulence gene expression in Proteus mirabilis.  


A mini-Tn5lacZ1 transposon insertion in a gene encoding an orthologue of the Lon protease conferred a hyper-swarming phenotype on Proteus mirabilis. The lon mutation increased the accumulation of mRNA for representative class 1 (flhDC), class 2 (fliA) and class 3 (flaA) genes during swarmer cell differentiation. In addition, the stability of the FlhD protein was fourfold higher in the lon : mini-Tn5lacZ1 background. Expression of a single-copy lon : lacZ fusion increased during the swarming cycle and reached peak levels of expression at a point just after swarmer cell differentiation had initiated. In liquid media, a condition normally non-permissive for swarming, the lon : : mini-Tn5lacZ1 insertion resulted in motile, highly elongated cells that overexpressed flagellin. Finally, the lon : : mini-Tn5lacZ1 mutation was shown to result in increased expression of the hpmBA and zapA virulence genes during swarmer cell differentiation. PMID:18628491

Clemmer, Katy M; Rather, Philip N



Effect of L-ascorbic Acid on the hsp70 Expression and Tissue Damage in the Third Instar Larvae of Transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9).  


All living organisms respond to various physical or chemical stressors by the induction of heat shock protein (HSP). The present study was performed on transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9) in which the transformation vector is inserted with a P-element, the line contains wild-type hsp70 sequence up to the lacZ fusion point. The effect of L-ascorbic acid on the hsp70 expression and tissue damage was studied at the doses of 1, 2, 4, and 8 × 10(-4) g/ml in the third instar larvae of transgenic D. melanogaster (hsp70-lacZ) Bg(9). The larvae were exposed to different doses of L-ascorbic acid for 24 and 48 hours. A dose-dependent significant increase in the hsp70 expression was observed at 2, 4, and 8 × 10(-4) g/ml of L-ascorbic acid for both 24 and 48 hours. The tissue damage was observed only in the 48 hours of exposure and mostly only in the salivary glands of the third instar larvae of transgenic D. melanogaster (hsp70-lacZ) Bg(9). The present study also validates and supports the use of transgenic D. melanogaster (hsp70-lacZ) Bg(9) for the toxicological evaluations. PMID:23293470

Shakya, Barkha; Jyoti, Smita; Naz, Falaq; Khan, Safiya; Afzal, Rahul Mohammad; Siddique, Yasir Hasan



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

SciTech Connect

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

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



Genetic Engineering with a Gene Encoding a Soybean Storage Protein. Progress Report.  

National Technical Information Service (NTIS)

Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the alpha '-subunit of beta -conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ERA citation 10:021771)

R. N. Beachy



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

SciTech Connect

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

Beachy, R.N.



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

PubMed Central

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

Mukhopadhyay, S; Schellhorn, H E



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.

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



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.



Watch out for reporter gene assays with Renilla luciferase and paclitaxel.  


Luminescence-based reporter gene assays are widely used in biochemistry. Signals from reporter genes (e.g., firefly luminescence) are usually normalized to signals from constantly luminescing luciferases such as Renilla luciferase. This normalization step can be performed by modern luminometry devices automatically providing final results. Here we demonstrate paclitaxel to strikingly enhance Renilla luminescence, thereby potentially flawing results from reporter gene assays. In consequence, these data advocate for careful examination of raw data and militate against automatic data processing. PMID:23499973

Theile, Dirk; Spalwisz, Adriana; Weiss, Johanna



The peroxisomal transporter gene ANT1 is regulated by a deviant oleate response element (ORE): characterization of the signal for fatty acid induction.  

PubMed Central

Saccharomyces cerevisiae ANT1/YPR128c encodes the peroxisomal adenine nucleotide transporter that provides ATP for intra-peroxisomal activation of medium-chain fatty acids. A lacZ reporter construct comprising the ANT1 promoter was shown to be comparatively more highly expressed in a wild-type strain grown on oleic acid, a long-chain fatty acid, than in pip2Delta(oaf1)Delta mutant cells that are defective in fatty acid induction. The ANT1 promoter was demonstrated to contain a deviant oleate response element (ORE) that could bind the Pip2p-Oaf1p transcription factor and confer activation on a basal CYC1-lacZ reporter gene. Expression of Ant1p as well as other enzymes whose genes are known to be regulated by a canonical ORE was found to be increased in cells grown on lauric acid, a medium-chain fatty acid. We concluded that the signal for induction does not differentiate between long- and medium-chain fatty acids. This signal was independent of beta-oxidation or the biogenesis of the peroxisomal compartment where this process occurs, since a pox1Delta strain blocked in the first and rate-limiting step of beta-oxidation as well as various pex mutant cells devoid of intact peroxisomes produced sufficient amounts of Pip2p-Oaf1p for binding OREs in vitro and for expressing an ORE-driven reporter gene. The signal's durability was shown to be related to the concentration of fatty acids in the medium, since a pex6Delta strain expressed an ORE-driven reporter gene at high levels for a longer period than did isogenic wild-type cells. Generation of the signal was also independent of protein synthesis, as demonstrated by cycloheximide treatment.

Rottensteiner, Hanspeter; Palmieri, Luigi; Hartig, Andreas; Hamilton, Barbara; Ruis, Helmut; Erdmann, Ralf; Gurvitz, Aner



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



Translational Control of the Antibiotic Inducibility of the PA5471 Gene Required for mexXY Multidrug Efflux Gene Expression in Pseudomonas aeruginosa? †  

PubMed Central

The PA5471 gene required for induction of the MexXY multidrug efflux system in response to ribosome-targeting antimicrobials was itself shown to be inducible by ribosome-targeting antimicrobials (Y. Morita, M. L. Sobel, and K. Poole, J. Bacteriol. 188:1847-1855, 2006). Using a lacZ transcriptional reporter, drug inducibility of PA5471 was shown to require the entirety of the 367-bp PA5472-PA5471 intergenic region. A constitutive promoter activity was, however, localized to the first 75 bp of this region, within which a single PA5471 transcription initiation site was mapped. That 3? sequences of the intergenic region blocked PA5471 expression and made it antibiotic dependent was suggestive of an attenuation mechanism of control. A 13-amino-acid leader peptide (LP)-encoding open reading frame preceded by a Shine-Dalgarno sequence was identified ca. 250 bp upstream of the PA5471 coding sequence, and its expression and translation were confirmed using a lacZ translational reporter. Alteration of the initiation codon (M1T) or introduction of translational stop signals at codons 3 (Q3Am) and 8 (C8Op) of this LP sequence (PA5471.1) yielded high-level constitutive expression of PA5471, suggesting that interference with LP translation was linked to PA5471 gene expression. Consistent with this, a Q3K mutation in the LP sequence maintained the drug inducibility of PA5471 expression. Introduction of the LP Q3Am mutation into the chromosome of Pseudomonas aeruginosa yielded stronger expression of PA5471 than did antibiotic (chloramphenicol) exposure of wild-type P. aeruginosa, in agreement with lacZ transcriptional fusion data. Still, the Q3Am mutation yielded modest expression of mexXY, less than that seen for antibiotic-treated wild-type P. aeruginosa. These data suggest that PA5471 is not sufficient for MexXY recruitment in response to antibiotic exposure and that additional antibiotic-dependent effects are needed.

Morita, Yuji; Gilmour, Christie; Metcalf, Devon; Poole, Keith



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

PubMed Central

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



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



Feedback-Inducible Nuclear-Receptor-Driven Reporter Gene Expression in Transgenic Mice  

Microsoft Academic Search

Understanding nuclear receptor signaling in vivo would be facilitated by an efficient methodology to determine where a nuclear receptor is active. Herein, we present a feedback-inducible expression system in transgenic mice to detect activated nuclear receptor effector proteins by using an inducible reporter gene. With this approach, reporter gene induction is not limited to a particular tissue, and, thus, this

Alexander Mata de Urquiza; Ludmila Solomin; Thomas Perlmann



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


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

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



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


The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening. PMID:23385445

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



Yeast excision-repair gene is inducible by DNA damaging agents  

SciTech Connect

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

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



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


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

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



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

PubMed Central

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

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



Rational design of murine secreted alkaline phosphatase for enhanced performance as a reporter gene in mouse gene therapy preclinical studies.  


Many preclinical gene therapy studies use a reporter gene to evaluate vector design and performance in mouse models of human disease. Unfortunately, most commonly used reporter genes are immunogenic in mice, which confounds accurate evaluation of vector function. In previous studies, we showed that the murine secreted alkaline phosphatase (mSEAP) gene functions well as a simple and sensitive reporter gene in mice. In this study, we have used rational design to enhance mSEAP performance. The majority of native mSEAP remains attached to the outer surface of the cell through glycan phosphatidylinositol linkage; removal of the carboxy-terminal tail of mSEAP resulted in a dramatic enhancement of release of the protein into cell culture medium and into mouse plasma in whole animal experiments. We increased the heat stability of mSEAP through mutation of a key residue in the crown domain of the protein (H451E), thus allowing us to reduce endogenous, background AP activity through heat inactivation for enhanced sensitivity. We show that these alterations in mSEAP result in enhanced performance in tissue culture and mouse studies. Taken together, these data illustrate that mSEAP is a sensitive, nonimmunogenic reporter for preclinical mouse studies. PMID:21083426

Christou, Carin; Parks, Robin J



Microbubble-enhanced sonoporation: efficient gene transduction technique for chick embryos.  


The gene transduction technique is a useful method to study gene functions that underlie vertebrate embryogenesis. In this study, a new gene transduction technique is reported using microbubble-enhanced sonoporation (hereafter referred to as sonoporation) to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes. The technique proposed in this study has the advantages of 1) relatively simple gene transduction procedures, and 2) efficient exogenous gene transduction and expression with lower damages to embryos. Green fluorescent protein (GFP) or LacZ was misexpressed in limb bud mesenchymes by sonoporation, with the introduced expression transiently detected in the injected sites. Most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonoporation. To demonstrate its efficacy for assessing the effect of transient gene transduction, the Shh (sonic hedgehog) was transduced into the developing chick limb bud. The transduced limb bud displayed limb malformations including partial digit duplication. Advantages and possible future applications in relation to this method are discussed. PMID:14595845

Ohta, Sho; Suzuki, Kentaro; Tachibana, Katsuro; Yamada, Gen



A new lysis buffer for luciferase, CAT and beta- galactosidase reporter gene co-transfections  

Microsoft Academic Search

A new lysis buffer formulation allows assays of luciferase, CAT and beta-galactosidase reporter activity to be conveniently performed with the same cell extract. The new Reporter Lysis Buffer gives reporter enzyme activities comparable to or better than those obtained with the standard lysis methods for these reporter genes.

Elaine Schenborn; Virginia Goiffon


Organisation and expression of a cluster of yolk protein genes in the Australian sheep blowfly, Lucilia cuprina.  


The Australian sheep blowfly Lucilia cuprina is a major pest for the Australian and New Zealand sheep industries. With the long-term aim of making a strain of L. cuprina suitable for a genetic control program, we previously developed a tetracycline-repressible female lethal genetic system in Drosophila. A key part of this system is a female-specific promoter from a yolk protein (yp) gene controlling expression of the tetracycline-dependent transactivator (tTA). Here we report the sequence of a 14.2 kb genomic clone from L. cuprina that contains a cluster of three complete yp genes and one partial yp gene. The Lcyp genes are specifically expressed in females that have received a protein meal. A bioinformatic analysis of the promoter of one of the yp genes (LcypA) identified several putative binding sites for DSX, a known regulator of yp gene expression in other Diptera. A transgenic strain of L. cuprina was made that contained the LcypA promoter driving the expression of the Escherichia coli lacZ reporter gene. Transgenic females express high levels of ?-galactosidase after a protein meal. Thus the LcypA promoter could be used to obtain female-specific expression of tTA in transgenic L. cuprina. PMID:20844939

Scott, Maxwell J; Atapattu, Asela; Schiemann, Anja H; Concha, Carolina; Henry, Rebecca; Carey, Brandi-lee; Belikoff, Esther J; Heinrich, Jörg C; Sarkar, Abhimanyu



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.



Nomenclature report on rice WRKY's - Conflict regarding gene names and its solution  

PubMed Central

Background Since whole genome sequences of rice were made publically accessible, the number of articles on new rice genes has increased remarkably. The Committee on Gene Symbolization, Nomenclature and Linkage (CGSNL) of the Rice Genetics Cooperative published the gene nomenclature system for rice and encouraged researchers to follow the rules before publishing their results. The CGSNL provides an on-line registration system for newly identified rice genes to prevent conflicts and/or duplication of gene name in journal articles. Findings Recently, the CGSNL surveyed genes in the rice WRKY family in published journal articles and found several duplicated gene names. Conclusions To discuss and resolve inconsistencies in WRKY gene nomenclature, the rice WRKY working group was established and redefined the nomenclature. This report announces the conclusion.



Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast.  


We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro. It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin. In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid. We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S. cerevisiae. We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene. Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression. PMID:9161405

Maiorano, D; Cece, R; Badaracco, G



First Report of the Multiresistance Gene cfr in Streptococcus suis  

PubMed Central

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

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



Signal transduction pathways that regulate CAB gene expression. Progress report  

SciTech Connect

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

Chory, J.



A versatile element for gene addition in bacterial chromosomes  

PubMed Central

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

Sibley, Marion H.; Raleigh, Elisabeth A.



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


The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants. PMID:8336108

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



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.

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



Reporter gene technologies for imaging cell fates in hematopoiesis.  


Advances in noninvasive imaging technologies that allow for in vivo dynamic monitoring of cells and cellular function in living research subjects have revealed new insights into cell biology in the context of intact organs and their native environment. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Stem cells present unique challenges for imaging since after transplantation, they proliferate dramatically and differentiate. Therefore, the imaging modality used needs to have a large dynamic range, and the genetic regulatory elements used need to be stably expressed during differentiation. Multiple imaging technologies using different modalities are available, and each varies in sensitivity, ease of data acquisition, signal to noise ratios (SNR), substrate availability, and other parameters that affect utility for monitoring cell fates and function. For a given application, there may be several different approaches that can be used. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging (FLI), and all have been used to monitor bone marrow and stem cells after transplantation into mice. Photoacoustic imaging that utilizes the sound created by the thermal expansion of absorbed light to generate an image best represents hybrid technologies. Each modality requires that the cells of interest be marked with a genetic reporter that acts as a label making them uniquely visible using that technology. For each modality, there are several labels to choose from. Multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed protocols for gene delivery into hematopoietic cells for the purposes of applying these specific labels to cell trafficking. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice. PMID:24473775

Kusy, Sophie; Contag, Christopher H



A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis  

Microsoft Academic Search

We have found that the upstream region of the isocitrate lyase gene (UPR-ICL) from the n-alkane-utilizing yeast Candida tropicalis was functional in Saccharomyces cerevisiae as a novel promoter with non-fermentable carbon sources, such as oleic acid, acetate, ethanol, and glycerol\\/lactate. The expression of two foreign genes coding for #-galactosidase from Escherichia coli (LacZ) and glutamate decarboxylase from rat brain was

T. Kanai; H. Atomi; K. Umemura; H. Ueno; Y. Teranishi; M. Ueda; A. Tanaka



Inflammatory bowel disease gene discovery. CRADA final report  

SciTech Connect

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




Non-invasive imaging of cardiac transgene expression with PET: comparison of the human sodium\\/iodide symporter gene and HSV1-tk as the reporter gene  

Microsoft Academic Search

Purpose: Genes encoding for intracellular en- zymes or transmembrane proteins are suitable as reporters, but may differ in terms of applicability for cardiac im- aging. The aim of this study was to compare the human sodium iodide symporter gene (hNIS) with the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk )a s the reporter gene in non-invasive imaging of

Masao Miyagawa; Martina Anton; Bettina Wagner; Roland Haubner; Michael Souvatzoglou; Bernd Gansbacher; Markus Schwaiger; Frank M. Bengel



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

SciTech Connect

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

Wolstenholme, D.R.



Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice  

Microsoft Academic Search

Lentiviral-mediated gene delivery holds significant promise for sustained gene expression within living systems. Vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 1-based lentiviral vectors can be used to introduce transgenes in a broad spectrum of dividing as well as nondividing cells. In the current study, we construct a lentiviral vector carrying two reporter genes separated by an internal ribosomal entry

Abhijit De; Xiaoman Zhou Lewis; Sanjiv Sam Gambhir



Oxalate oxidase: a novel reporter gene for monocot and dicot transformations  

Microsoft Academic Search

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H2O2 generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay

John Simmonds; Leslie Cass; Elizabeth Routly; Keith Hubbard; Pauline Donaldson; Bonnie Bancroft; Andrea Davidson; Sheryl Hubbard; Daina Simmonds



[Construction and evaluation of efficient gene expression platforms in Synechocystis sp. strain PCC6803].  


For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a "Shine-Dalgarno -AUG" fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong beta-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA' gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future. PMID:24409696

Qi, Fengxia; Tan, Xiaoming; Lü, Xuefeng



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.



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))



Correlation of translation efficiency with the decay of lacZ mRNA in Escherichia coli.  


We isolated mutations in the leader of a ribosomal protein (r-protein)/lacZ fusion gene in Escherichia coli that caused the mRNA to be translated at efficiencies between < 1% and 62% of the rate of wild-type message. Using a subset of these mutants with translation efficiencies between 5% and 62%, we studied both physical and functional decay of the mRNA after rifampicin inhibition of transcription initiation. The decay of physically intact transcript was analyzed by gel electrophoresis of hybrid-selected messenger RNA segments. The output from the message was analyzed by measuring the synthesis rate of r-protein/lacZ fusion protein. Decay of physically intact message after rifampicin addition correlated with the translation efficiency, with the more active messengers being more stable. Different segments of the r-protein/lacZ fusion mRNA decayed with the same rate, indicating that there is no hyper-labile region in the transcript. The decay rate was also independent of the length of the segment probed, suggesting that the mRNA is not degraded by random attacks along the entire length of the molecule. Our results are consistent with an overall 5' to 3' degradation pathway. Surprisingly, the rate of fusion protein synthesis did not decrease immediately after rifampicin addition. Rather, a lag preceded the exponential decay phase; the length of this delay correlated with the translation efficiency, such that the lag increased with increasing efficiency of translation. We suggest that these lags indicate that mRNAs are normally competing for ribosomes during exponential growth and, after rifampicin addition, RNA molecules with longer physical half-lives are translated by ribosomes released from fast decaying messengers. PMID:8014986

McCormick, J R; Zengel, J M; Lindahl, L



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

PubMed Central

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

Juneja, Subhash C.; Schwarz, Edward M.; O'Keefe, Regis J.; Awad, Hani A.



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

Microsoft Academic Search

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

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



A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells  

Microsoft Academic Search

Hematopoietic stem cells are widely recognized as attractive targets for gene therapy but current protocols to transduce these cells using recombinant retroviral vectors are inefficient. To evaluate optimization of retroviral transduction of hematopoietic stem cells and stability of gene expression in their progeny, the green fluorescent protein (GFP) was explored as a reporter. We first improved sensitivity of detection >100-fold

L Cheng; C Du; D Murray; X Tong; YA Zhang; BP Chen; RG Hawley



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?.  


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 (Gnao(f/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




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, ...


Photoacoustic microscopy of tyrosinase reporter gene in vivo  

Microsoft Academic Search

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

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



Insulinoma-Induced Hypoglycemic Death in Mice is Prevented With Beta Cell-Specific Gene Therapy  

PubMed Central

Objective and Summary Background Data Tumor-specific gene therapy can be achieved if a tumor-specific promoter can be identified. In this study the authors investigated the use of the rat insulin promoter (RIP) for insulinoma-specific expression of a reporter gene. Insulinoma-specific cytotoxicity using the suicide gene thymidine kinase (tk) was studied both in vitro and in vivo. RIPtk gene therapy, delivered by a nontoxic, noninflammatory liposomal delivery system, was used in an insulinoma ICR/ SCID mouse model to prevent hypoglycemic death. Methods Rat insulin promoter (0.502 kb) was ligated to the reporter gene lacZ and ligated to the tk gene. These two genes were transfected into a mouse insulinoma (NIT) cell line to ascertain insulinoma-specific expression and insulinoma-specific cytotoxicity in vitro. Reverse transcriptase–polymerase chain reaction and electrophoretic mobility-shift assays were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse insulinoma model was created with NIT-1 cells. These mice were treated with the RIPtk gene, and both blood sugars and animal viability were monitored. Results Only NIT-1 cells stained blue after X-gal staining or had detectable levels of ?-galactosidase protein. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIPtk in vitro. Messenger RNA for both BETA2 and PDX-1 was found in NIT-1 cells, and a supershift was observed for both BETA2 and PDX-1. Experimental mice treated with the RIPtk gene, delivered by a liposomal gene delivery system, maintained their blood glucose levels, and the animals did not die of hypoglycemia. Conclusions The data suggest that the RIP is an insulinoma-specific promoter. An ICR/ SCID mouse insulinoma model was used to show that insulinoma-specific cytotoxicity can be accomplished by RIP coupled to a suicide gene in vivo, preventing hypoglycemic death.

Tirone, Thomas A.; Fagan, Shawn P.; Templeton, Nancy S.; Wang, Xaioping; Brunicardi, F. Charles



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))



Identification of a sporulation-specific promoter regulating divergent transcription of two novel sporulation genes in Saccharomyces cerevisiae  

Microsoft Academic Search

Promoters that control gene expression in Saccharomyces cerevisiae only in a sporulation-specific manner have previously been isolated from a genomic yeast DNA library fused to a promoterless Escherichia coli lacZ gene. Two novel sporulation-specific genes, SPS18 and SPS19, were isolated using this technique. These genes are divergently controlled by the same promoter but with SPS18 expressed at four times the

John G. S. Coe; Lois E. Murray; Ian W. Dawes



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


Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity. PMID:16188984

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



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

Microsoft Academic Search

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

Niren R Thanky; Jin H Son; Allan E Herbison



Low-energy (30 keV) carbon ion induced mutation spectrum in the LacZ? gene of M13mp18 double-stranded DNA  

Microsoft Academic Search

Double-stranded M13mp18 DNA was irradiated with 30keV carbon ions in dry state under vacuum to investigate the low-energy heavy ion induced mutation spectra. When the irradiated DNA was used to transfect Escherichia coli JM105, 3.6–5.7-fold increases in mutation frequency were observed, in contrast to the spontaneous group. Sequences of the 92 induced mutants showed that the carbon ions in this

Quan Wang; Gang Zhang; Yan-hua Du; Yong Zhao; Guan-ying Qiu



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

PubMed Central

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

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



Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness.  

PubMed Central

There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere. To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P. ultimum. The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P. fluorescens F113. By this screening procedure, five P. fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P. ultimum. The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type. Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil. With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type. This is the first reported case of a fungus repressing the expressing of bacterial genes.

Fedi, S; Tola, E; Moenne-Loccoz, Y; Dowling, D N; Smith, L M; O'Gara, F



Analysis of the expression of the Rhodobacter sphaeroides lexA gene  

Microsoft Academic Search

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN7GAACN7GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third\\u000a GAAC motif in this

A. Tapias; S. Campoy; J. Barbé



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.



Transgenic Mice Demonstrate Novel Promoter Regions for Tissue-Specific Expression of the Urokinase Receptor Gene  

PubMed Central

The urokinase-type plasminogen activator receptor (u-PAR) contributes to cell migration and proteolysis in normal and cancerous tissues. Currently, there are no reports on the regulatory regions directing tissue-specific expression. Consequently, we undertook a study to identify novel promoter regions required for expression of this gene in transgenic mice bearing a LacZ reporter regulated by varying amounts (0.4, 1.5, and 8.5 kb) of upstream sequence. The 0.4-kb u-PAR upstream sequence directed weak and strong LacZ expression in the placenta and epididymis, respectively, both of which are tissues that express endogenous u-PAR. Conversely, transgene expression in the apical cells of the colon positive for endogenous u-PAR protein required 1.5 kb of upstream sequence for optimal expression. Furthermore, chromatin accessibility assays coupled with real-time polymerase chain reaction suggested a putative regulatory region spanning ?1295/?1192 driving u-PAR expression in colonic cells. Interestingly, placental transgene expression was augmented with the 8.5-kb upstream fragment compared with the shorter 1.5-kb fragment indicating contributing element(s) between ?1.5 and ?8.5 kb. Thus, while 0.4 kb of upstream sequence directs u-PAR expression in the epididymis, sequences located between ?0.4 and ?1.5 kb and between ?1.5 and ?8.5 kb are required for optimal tissue-specific expression in the colon and the placenta, respectively.

Wang, Heng; Hicks, John; Khanbolooki, Parham; Kim, Sun-Jin; Yan, Chunhong; Wang, Yao; Boyd, Douglas



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



Novel SOST gene mutation in a sclerosteosis patient from Morocco: A case report.  


Sclerosteosis (OMIM 269500) is a rare autosomal recessive condition characterized by increased bone density associated with syndactyly. It is linked to a genetic defect in the SOST gene coding for sclerostin. So far, seven different loss-of-function mutations in SOST have been reported in patients with sclerosteosis. Recently, two mutations in LRP4 gene underlying sclerosteosis were identified, reflecting the genetic heterogeneity of this disease. We report here a 30-years-old Moroccan man presented with typical clinical and radiological features of sclerosteosis who carries a novel homozygous mutation in the SOST gene, characterized as a nonsense mutation (c.79C > T; p.Gln27?) in exon 1 of the SOST gene. This is to our knowledge the first case of sclerosteosis reported from Morocco and North Africa. PMID:24594238

Belkhribchia, Mohamed Reda; Collet, Corinne; Laplanche, Jean-Louis; Hassani, Redouane



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

NASA Astrophysics Data System (ADS)

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

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


Quantitative Analysis of Bacterial Gene Expression by Using the gusA Reporter Gene System  

PubMed Central

An Azospirillum brasilense Sp7 strain containing a plasmid-borne translational cytN-gusA fusion was grown in a continuous culture to quantitatively evaluate the influence of extracellular signals (such as O2) on expression of the cytNOQP operon. The dissolved oxygen concentration was shifted at regular time intervals before the steady state was reached. The measured ?-glucuronidase activity was used to monitor cytN gene expression. However, as the ?-glucuronidase activity in the experimental setup not only depended on altered transcription of the hybrid gene when the signal was varied but was also influenced by cellular accumulation, degradation, and dilution of the hybrid fusion protein, a mathematical method was developed to describe the intrinsic properties of the dynamic bioprocess. After identification and validation of the mathematical model, the apparent specific rate of expression of the fusion, which was independent of the experimental setup, could be deduced from the model and used to quantify gene expression regulated by extracellular environmental signals. In principle, this approach can be generalized to assess the effects of external signals on bacterial gene expression.

Sun, Jun; Smets, Ilse; Bernaerts, Kristel; Van Impe, Jan; Vanderleyden, Jos; Marchal, Kathleen



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.



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


Loss of hair and hair colour is associated with ageing, and when it involves the scalp hair, it can be distressing to both sexes. Hair loss resulting from cancer chemotherapy is particularly distressing. However, safe, effective therapies directed to hair have only just started to be developed. The hair follicle is a complex skin appendage composed of epidermal and dermal tissue, with specialized keratinocytes, the hair matrix cells, forming the hair shaft. Specific therapy of the hair follicle depends on selective targeting of specific cells of the hair follicle. We have developed the histoculture of intact hair-growing skin on sponge-gel matrices. We have recently found in histocultured skin that liposomes can selectively target hair follicles to deliver both small and large molecules. That liposomes can target the hair follicle for delivery has been confirmed independently. Two decades ago we introduced the technique of entrapping DNA in liposomes for use in gene therapy. In this report we describe the selective targeting of the lacZ reporter gene to the hair follicles in mice after topical application of the gene entrapped in liposomes. These results demonstrate that highly selective, safe gene therapy for the hair process is feasible. PMID:7585157

Li, L; Hoffman, R M



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


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

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



Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.  


We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells. PMID:9192666

Flannery, J G; Zolotukhin, S; Vaquero, M I; LaVail, M M; Muzyczka, N; Hauswirth, W W



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


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



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

PubMed Central

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

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



Molecular cloning of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum.  


A pLAFR1 cosmid genomic library of wild-type Bradyrhizobium japonicum J1063 was constructed. A cosmid clone designated pBjJ4, containing a 26-kilobase (kb) DNA insert, was identified as being able to confer the ability to convert alpha-naphthaleneacetamide acid on B. japonicum J1B7 Rifr, which cannot perform this conversion. The gene coding for the enzyme that converts alpha-naphthaleneacetamide to alpha-naphthaleneacetic acid was localized in the 3.5-kb region of pBjJ4 by recloning in plasmid pSUP202. The gene coding for the enzyme was also mapped by Tn5 insertion mutagenesis to a region of ca. 2.3 kb. When the gene was placed behind the lacZ promoter and used to transform Escherichia coli, a high level of expression of indole-3-acetamide hydrolase activity was found. Since there have been no reports of this activity in E. coli, we have thus confirmed that the gene cloned here is a structural gene for indole-3-acetamide hydrolase and have designated it as the bam (Bradyrhizobium amidehydrolase) gene. Southern hybridization with the central region of the bam gene indicated that a high degree of similarity exists among the bam gene, the iaaH gene from Pseudomonas savastonoi, and the tms-2 gene from Agrobacterium tumefaciens. The result suggests that there is a common origin for the gene that encodes the enzyme that catalyzes the biosynthesis of indoleacetic acid. PMID:2646294

Sekine, M; Watanabe, K; Syono, K



Report of a chimeric origin of transposable elements in a bovine-coding gene.  


Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is approximately 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage. PMID:18273826

Almeida, L M; Amaral, M E J; Silva, I T; Silva, W A; Riggs, P K; Carareto, C M



Pre-existing herpes simplex virus 1 (HSV1) immunity decreases, but does not abolish, gene transfer to experimental brain tumors by a HSV1 vector  

Microsoft Academic Search

The influence of pre-existing anti-herpes simplex type 1 (HSV-1) immunity on HSV-1 vector-mediated gene transfer to glioma cells was analyzed in this gene marking study using intracranial D74 gliomas in syngeneic Fischer rats. The HSV-1 mutant virus used, hrR3, is defective in ribonucleotide reductase and bears the marker genes E. coli lacZ and HSV-1 thymidine kinase (HSVtk). Initial marker gene

U Herrlinger; CM Kramm; KS Aboody-Guterman; JS Silver; K Ikeda; KM Johnston; PA Pechan; RF Barth; D Finkelstein; EA Chiocca; DN Louis; XO Breakefield



Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation  

PubMed Central

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

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



The mkaC virulence gene of the Salmonella serovar Typhimurium 96 kb plasmid encodes a transcriptional activator  

Microsoft Academic Search

Summary. The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four Mka proteins indicated that mkaC enhances the expression of \\/3-galactosidase from

Suvi Taira; Petri Riikonen; Hannu Saarilahti; Soila Sukupolvi; Mikael Rhen



Reporter gene approaches for mapping cell fate decisions by MRI: promises and pitfalls.  


The central dogma of molecular biology, namely the process by which information encoded in the DNA serves as the template for transcriptional activation of specific mRNA resulting in temporal and spatial control of the translation of specific proteins, stands at the basis of normal and pathological cellular processes. Serving as the primary mechanism linking genotype to phenotype, it is clearly of significant interest for in vivo imaging. While classically, imaging revolutionized the ability to phenotype the anatomical and physiological impact of induction of changes in gene expression, the preceding molecular events remained invisible. Reporter gene-based imaging techniques provide a window for in vivo visualization of such transcriptional activation events. In addition to the widespread use of fluorescent and bioluminescent reporter genes and development of a number of reporter genes for positron emission tomography (PET) imaging, there has been significant progress in the development of reporter genes for MRI. With the development of strategies for cellular based therapies, such imaging tools could become central components for personalized patient monitoring. PMID:24375898

Vande Velde, Greetje; Himmelreich, Uwe; Neeman, Michal



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

NASA Astrophysics Data System (ADS)

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

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



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

PubMed Central

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



Adenylyl Cyclase A Expression Is Tip-Specific in Dictyostelium Slugs and Directs StatA Nuclear Translocation and CudA Gene Expression  

Microsoft Academic Search

cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organizer function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except

Irene Verkerke-van Wijk; Masashi Fukuzawa; Peter N. Devreotes; Pauline Schaap



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


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

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



Gene transcription and electromagnetic fields. Final progress report  

SciTech Connect

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

Henderson, A.S.



Rubinstein-Taybi syndrome with normal FISH result and CREBBP gene analysis: a case report.  


We report on a six-year-old boy with typical Rubinstein-Taybi syndrome (RSTS) phenotype. Clinical findings included mental and motor retardation, patent ductus arteriosus (PDA), undescended testes, hirsutism, broad thumbs with radial angulation and broad toes, and inguinal hernia. His karyotype was normal (46, XY) and fluorescence in situ hybridization (FISH) showed no deletion of the CREBBP [cAMP response element-binding (CREB) binding protein] gene on chromosome 16p13.3. CREBBP gene sequencing also revealed normal results. We wish to present this case because this patient had typical RSTS phenotype, but normal FISH and CREBBP gene sequencing results. It could be possible that genetic heterogeneity is related with novel mutations in other genes. With the publication of such cases, their significance will be brought to the attention of researchers in this field. PMID:18773673

Balci, Sevim; Ergün, Mehmet Ali; Yüksel-Konuk, E Berrin; Bartsch, Oliver



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))



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

PubMed Central

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

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



Oral contrast enhances the resolution of in-life NIS reporter gene imaging  

PubMed Central

NIS reporter gene imaging is an excellent technology for noninvasive cell fate determination in living animals unless the NIS-transduced cells reside in perigastric organs such as spleen, liver, diaphragm, omentum, pancreas, perigastric lymph nodes or perigastric tumor deposits. Here we report that orally administered barium sulfate enhances CT definition of the stomach, masks background gamma ray emissions from the stomach, and enhances signal detection from radiotracer uptake in NIS-transduced organs.

Suksanpaisan, Lukkana; Pham, Linh; McIvor, Scott; Russell, Stephen J; Peng, Kah-Whye



Oral contrast enhances the resolution of in-life NIS reporter gene imaging.  


Sodium iodide symporter (NIS) reporter gene imaging is an excellent technology for noninvasive cell fate determination in living animals unless the NIS-transduced cells reside in perigastric organs such as the spleen, liver, diaphragm, omentum, pancreas, perigastric lymph nodes or perigastric tumor deposits. Here we report that orally administered barium sulfate enhances CT definition of the stomach, masks background gamma ray emissions from the stomach and enhances signal detection from radiotracer uptake in NIS-transduced organs. PMID:24030210

Suksanpaisan, L; Pham, L; McIvor, S; Russell, S J; Peng, K-W



Imaging Adenoviral-Directed Reporter Gene Expression in Living Animals with Positron Emission Tomography  

Microsoft Academic Search

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the

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



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

PubMed Central

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

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



Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes  

PubMed Central

Advances in genetic engineering methods have allowed the development of an increasing number of practical and scientific applications for bioluminescence with lux genes cloned from a variety of organisms. Bioluminescence derived from the shortened lux operon (luxAB genes) is a complex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this report, we describe a two-phase kinetic behavior of the light emission which must be properly taken into account in any quantitative measurements of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and interpreted in terms of the biochemistry underlying the bacterial luciferase mechanism. We show that the intensity profile of each of the two phases of the luminescence signal is responsive (and exhibits different sensitivities) to the concentration of added decanal and other components of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol design, and specific recommendations for using the luxAB system as a molecular reporter are presented, along with versatile assay protocols that yield meaningful and reproducible signals.

Blouin, K.; Walker, S. G.; Smit, J.; Turner, R.



Long term non-invasive imaging of embryonic stem cells using reporter genes  

Microsoft Academic Search

Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and

Ning Sun; Andrew Lee; Joseph C Wu



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



Multiple insulin degrading enzyme variants alter in vitro reporter gene expression.  


The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma ?-amyloid (A?), decreased risk for Alzheimer's disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p?=?0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5' to the promoter (1.18 fold-change, p?=?0.006) and 3' to the reporter gene (1.29 fold change, p?=?0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates A? degradation, variants that regulate IDE expression could represent good therapeutic targets for AD. PMID:21731745

Belbin, Olivia; Crump, Michael; Bisceglio, Gina D; Carrasquillo, Minerva M; Morgan, Kevin; Younkin, Steven G



Multiple Insulin Degrading Enzyme Variants Alter In Vitro Reporter Gene Expression  

PubMed Central

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma ?-amyloid (A?), decreased risk for Alzheimer's disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p?=?0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5? to the promoter (1.18 fold-change, p?=?0.006) and 3? to the reporter gene (1.29 fold change, p?=?0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates A? degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.

Belbin, Olivia; Crump, Michael; Bisceglio, Gina D.; Carrasquillo, Minerva M.



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

SciTech Connect

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

Not Available



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

PubMed Central

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

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



Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system.  


Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn5-based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42 degrees C) is applied to a bacterial culture carrying the transposon delivery plasmid, approximately 0.1% of the population is recovered with Tn5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains. PMID:18344342

Buchan, Blake W; McLendon, Molly K; Jones, Bradley D



In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells  

PubMed Central

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

Wilson, Kitchener; Yu, Jin; Lee, Andrew; Wu, Joseph C.



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

SciTech Connect

The Drosophilia homeotic gene Sex combs reduced (Scr) controls the segmental identity of the labial and prothoracic segments in the embryo and adult. It encodes a sequence-specific transcription factor that controls, in concert with other gene products, differentiative pathways of tissues in which Scr is expressed. During embryogenesis, Scr accumulation is observed in a discrete spatiotemporal pattern that includes the labial and prothoracic ectoderm, the subesophageal ganglion of the ventral nerve cord and the visceral mesoderm of the anterior and posterior midgut. Previous analyses have demonstrated that breakpoint mutations located in a 75-kb interval, including the Scr transcription unit and 50 kb of upstream DNA, cause Scr misexpression during development, presumably because these mutations remove Scr cis-regulatory sequences from the proximity of the Scr promoter. To gain a better understanding of the regulatory interactions necessary for the control of Scr transcription during embryogenesis, we have begun a molecular analysis of the Scr regulatory interval. DNA fragments from this 75-kb region were subcloned into P-element vectors containing either an Scr-lacZ or hsp70-lacZ fusion gene, and patterns of reporter gene expression were assayed in transgenic embryos. Several fragments appear to contain Scr regulatory sequences, as they direct reporter gene expression in patterns similar to those normally observed for Scr, whereas other DNA fragments direct Scr reporter gene expression in developmentally interesting but non-Scr-like patterns during embryogenesis. Scr expression in some tissues appears to be controlled by multiple regulatory elements that are separated, in some cases, by more than 20 kb of intervening DNA. This analysis provides an entry point for the study of how Scr transcription is regulated at the molecular level. 60 refs., 7 figs., 1 tab.

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



Inducible gene manipulations in brain serotonergic neurons of transgenic rats.  


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

Weber, Tillmann; Schönig, Kai; Tews, Björn; Bartsch, Dusan



Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli : Use of AUA as a restart codon  

Microsoft Academic Search

An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the ß-galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active ß-galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result

Ad A. C. M. Peijnenburg; Gerard Venema; Sierd Bron



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



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

PubMed Central

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

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



Optical Bioluminescence and Positron Emission Tomography Imaging of a Novel Fusion Reporter Gene in Tumor Xenografts of Living Mice  

Microsoft Academic Search

Noninvasive imaging of reporter gene expression using various imaging modalities is playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. In this study, a novel reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk )( tk), a positron

Pritha Ray; Anna M. Wu; Sanjiv S. Gambhir



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


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

Sergi, Consolato; Gekas, Jean; Kamnasaran, Deepak



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.



Transcriptional analysis of the bglP gene from Streptococcus mutans  

PubMed Central

Background An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. Results To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. Conclusion The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript.

Cote, Christopher K; Honeyman, Allen L



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.

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



Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles.  


Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting. PMID:16525478

Fink, T L; Klepcyk, P J; Oette, S M; Gedeon, C R; Hyatt, S L; Kowalczyk, T H; Moen, R C; Cooper, M J



Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria.  

PubMed Central

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 the Vibrio harveyi luciferase structural genes (luxAB) was introduced downstream of the promoter for the Synechococcus psbAI gene, which encodes a photosystem II protein. This reporter construction was recombined into the Synechococcus chromosome, and bioluminescence was monitored under conditions of constant illumination following entrainment to light and dark cycles. The reporter strain, AMC149, expressed a rhythm of bioluminescence which satisfies the criteria of circadian rhythms: persistence in constant conditions, phase resetting by light/dark signals, and temperature compensation of the period. Rhythmic changes in levels of the native psbAI message following light/dark entrainment supported the reporter data. The behavior of this prokaryote disproves the dogma that circadian mechanisms must be based on eukaryotic cellular organization. Moreover, the cyanobacterial strain described here provides an efficient experimental system for molecular analysis of the circadian clock. Images Fig. 2

Kondo, T; Strayer, C A; Kulkarni, R D; Taylor, W; Ishiura, M; Golden, S S; Johnson, C H



A tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice.  

PubMed Central

Uroplakins are a group of integral membrane proteins that are synthesized as the major differentiation products of urothelium. The luminal portions of these proteins form 12-nm protein particles arranged in a two-dimensional crystalline array. The expression of uroplakin genes is bladder specific and differentiation dependent; little is known, however, about their molecular regulation. Here we describe the cloning of mouse uroplakin II gene and demonstrate, in transgenic mouse experiments, that a 3.6-kb 5'-flanking sequence of this gene can drive a bacterial lacZ (reporter) gene to express in the suprabasal cell layers of the urothelium. The transgene was not expressed in any tested (nonurothelial) epithelial and other tissues (except hypothalamus). These results suggest that most of the cis elements that confer the bladder-specific and differentiation-dependent expression of mouse uroplakin II gene must reside in the 3.6-kb sequence. The availability of a promoter capable of delivering a foreign molecule to the differentiated cell layers of bladder epithelium opens avenues for studying normal and pathological urothelial differentiation in transgenic mice. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5

Lin, J H; Zhao, H; Sun, T T



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

Microsoft Academic Search

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




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

PubMed Central

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

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



Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection  

SciTech Connect

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

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



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


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

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



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

PubMed Central

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

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



SDHB Gene Mutation in a Carotid Body Paraganglioma: Case Report and Review of the Paraganglioma Syndromes.  


Carotid body tumors represent the most common of head and neck tumors. They account for <0.03% of all human tumors. The underlying physiology and pathogenesis of this tumor type are not well understood. Several different genetic abnormalities have been associated with the development of carotid body paragangliomas. We present a case report with an unusual genetic mutation in the SDHB gene and a review of the paraganglioma syndromes. PMID:24509376

Peterson, Laura A; Litzendorf, Maria; Ringel, Matthew D; Vaccaro, Patrick S



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



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

Microsoft Academic Search

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

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



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.

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



Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging  

PubMed Central

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.

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



Temporal and spatial regulation of gene expression mediated by the promoter for the human tissue inhibitor of metalloproteinases-3 (TIMP-3)-encoding gene.  


A complex interplay between enzymes involved in extracellular matrix formation and their inhibitors is thought to control organogenesis during mammalian development. Disturbance of this balance may result in a wide range of diseases, including macular degeneration, arthritis, and tumor metastases. In order to define elements which may be involved in regulating human tissue inhibitor of metalloproteinase 3 (TIMP3) expression, we isolated and sequenced a clone containing 1315 bp of the 5'-upstream region of the human TIMP-3-encoding gene. A 1.2 kb fragment of this clone, which contains multiple motifs which are binding sites for known transcription factors, was used to drive expression of the lacZ reporter gene in multiple lines of transgenic mice. TIMP3 promoter activity, detected through beta-galactosidase histochemical assay, was observed at high levels in selected tissues, the identity of which varied according to developmental stage. TIMP3 promoter activity was detected at embryonic and early postnatal stages in tissues undergoing extensive remodeling, such as developing somites, bones and joints, choroid plexus, webs between the digits, and the spongiotrophoblastic portion of the placenta. In adulthood, TIMP3 promoter activity was restricted to a few tissues which exhibit high metabolic activity or rapid turnover. These include the retinal pigment epithelium (RPE), cells of the kidney cortex, hair follicles, gingiva, ovarian follicles, and testis. The results suggest that TIMP3 expression plays an active role in developmental patterning and in the maintenance of specific differentiated tissues. PMID:9520110

Zeng, Y; Rosborough, R C; Li, Y; Gupta, A R; Bennett, J



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

PubMed Central

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

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



Minimal phenotype of mice homozygous for a null mutation in the forkhead/winged helix gene, Mf2.  


Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2(lacZ)) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes. PMID:10648626

Kume, T; Deng, K; Hogan, B L



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

Microsoft Academic Search

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

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



Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice.  


Noninvasive imaging of reporter gene expression using various imaging modalitiesis playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. In this study, a novel reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) (tk), a positron emission tomography (PET) reporter gene, and renilla luciferase (rl), a bioluminescence optical reporter gene joined by a 20 amino acid long spacer sequence. We validated the activity of the two enzymes encoded by the fusion protein (tk(20)rl) in cell culture. Then, tumors stably expressing the tk(20)rl fusion gene were imaged both by microPET and optically using a cooled charge coupled device camera in xenograft-bearing living mice. Using a single fusion reporter (PET/optical) gene should accelerate the validation of reporter gene approaches developed in cell culture for translation into preclinical and clinical models. PMID:12649169

Ray, Pritha; Wu, Anna M; Gambhir, Sanjiv S



Evaluating the microRNA targeting sites by luciferase reporter gene assay.  


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

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



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


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

Sperandio, V; Mellies, J L; Nguyen, W; Shin, S; Kaper, J B



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.

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



Organization of the Saccharomyces cerevisiae actin gene UAS: functional significance of reiterated REB1 binding sites and AT-rich elements.  


The upstream activation sequence (UAS) in the Saccharomyces cerevisiae actin gene promoter contains three different motifs, specifically two AT-rich tracts, two binding sites for the yeast protein REB1, and an Mlul site. Synthetic UAS elements containing individual motifs, or combinations of them, were inserted in place of the natural UAS, and assayed using a lacZ reporter gene. The REB1 binding sites were found to be essential for, and sufficient to restore partial, UAS activity. AT-rich tracts alone were inactive. Multimerization of a REB1 binding site created a UAS that in galactose is more active, but in glucose less active, than a UAS having a single REB1 site with one AT-rich tract. In general, transcription during growth in galactose or glycerol/lactate responds more to multimerization of motifs. The results suggest that the natural actin promoter UAS retains activity on these alternative carbon sources because of reiteration of sequence elements within it; the additional elements appear to be redundant when cells are grown on glucose. The Mlul site, which is present upstream of a number of yeast genes involved in DNA synthesis and confers cell cycle periodicity to those genes, contributes to the activity of the synthetic UAS elements, but not in a cell-cycle-dependent manner. PMID:8817483

McLean, M; Hubberstey, A V; Bouman, D J; Pece, N; Mastrangelo, P; Wildeman, A G



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


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

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



New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria.  


Three types of new variants of the broad-host-range transposon Tn5 are described. (i) Tn5-mob derivatives with the new selective resistance (R) markers GmR, SpR and TcR facilitate the efficient mobilization of replicons within a wide range of Gram-negative bacteria. (ii) Promoter probe transposons carry the promoterless reporter genes lacZ, nptII, or luc, and NmR, GmR or TcR as selective markers. These transposons can be used to generate transcriptional fusions upon insertion, thus facilitating accurate determinations of gene expression. (iii) Tn5-P-out derivatives carry the npt- or tac-promoter reading out from the transposon, and TcR, NmR or GmR genes. These variants allow the constitutive expression of downstream genes. The new Tn5 variants are available on mobilizable Escherichia coli vectors suitable as suicidal carriers for transposon mutagenesis of non-E. coli recipients and some on a phage lambda mutant to be used for transposon mutagenesis in E. coli. PMID:2551782

Simon, R; Quandt, J; Klipp, W



The Escherichia coli stpA gene is transiently expressed during growth in rich medium and is induced in minimal medium and by stress conditions.  

PubMed Central

The transcriptional regulation of the stpA gene, encoding the Escherichia coli H-NS-like protein StpA, has been studied as a function of a variety of environmental conditions, and its response to trans-acting factors has been characterized. Chromosomally located stpA is expressed primarily from a promoter immediately upstream of the gene which is severely repressed by the homologous nucleoid-associated protein H-NS. However, we show here that even in a strain containing functional H-NS, stpA is transiently induced during growth of a batch culture in rich medium. It can also be induced strongly by osmotic shock and, to a lesser extent, by an increase in growth temperature. Moreover, when cells are grown in minimal medium, we observe a more sustained induction of stpA which is dependent on the leucine-responsive regulatory protein (Lrp). This enhanced level of stpA transcription is virtually abolished in an H-NS-independent manner when the culture undergoes carbon starvation. A sensitivity of the stpA promoter to DNA topology may contribute to some of these responses. Results reported here show that cloned fragments of the stpA promoter region can confer H-NS and Lrp responsiveness upon a lacZ reporter gene and suggest that several hundred base pairs of DNA upstream of the transcriptional start may be required for regulation by these two proteins.

Free, A; Dorman, C J



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

PubMed Central

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

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



Chromosome microarray analysis: a case report of infertile brothers with CATSPER gene deletion.  


We present the case of two brothers who were referred to a male infertility clinic for infertility workup. Conventional chromosome analysis and Y chromosome microdeletions did not reveal any genetic alterations. We utilized the chromosome microarray analysis (CMA) to identify novel and common variations associated with this severely impaired spermatogenesis cases. CMA specific results showed a common deletion in the 15q15.3 region that harbors genes like CATSPER2, STRC and PPIP5K1 in both cases (M18 and M19). In addition we identified small duplication in X and 11 chromosomes of M19. This is the first familial case report from India on occurrence of CATSPER gene deletion in human male infertility. PMID:24690399

Jaiswal, Deepika; Singh, Vertika; Dwivedi, U S; Trivedi, Sameer; Singh, Kiran



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


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



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

SciTech Connect

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




Sex-dependent regulation of hypothalamic neuropeptide Y-Y1 receptor gene expression in moderate\\/high fat, high-energy diet-fed mice  

Microsoft Academic Search

In this study we investigated whether long-term consumption of a moderate\\/high fat (MHF), high-energy diet can affect the gene expression of the Y1 receptor (Y1R) for neuropeptide Y (NPY) in the dorsomedial (DMH), ventromedial (VMH), arcuate (ARC) and paraventricular (PVN) hypothalamic nuclei of male and female Y 1R\\/LacZ transgenic mice, carrying the murine Y1R promoter linked to the LacZ gene.

Francesca Zammaretti; Giancarlo Panzica; Carola Eva



Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon  

Microsoft Academic Search

We have constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding ß-galactosidase) from E. coli, mro (encoding

Thomas Rygus; Wolfgang Hillen



Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast Saccharomyces cerevisiae  

PubMed Central

Saccharomyces cerevisiae transcribes two genes, ARE1 and ARE2, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 and ARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from the ARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded by ARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1 promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2 are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold while ARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in a rox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2 transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

Jensen-Pergakes, Kristen; Guo, Zhongmin; Giattina, Mara; Sturley, Stephen L.; Bard, Martin



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.



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

PubMed Central

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

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



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

PubMed Central

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



HaloTag: a novel reporter gene for positron emission tomography.  


Among the many molecular imaging techniques, reporter gene imaging has been a dynamic area of research. The HaloTag protein is a modified haloalkane dehalogenase which was designed to covalently bind to synthetic ligands (i.e. the HaloTag ligands [HTL]). Covalent bond formation between the HaloTag protein and the chloroal-kane within the HTL occurs rapidly under physiological conditions, which is highly specific and essentially irreversible. Over the years, HaloTag technology has been investigated for various applications such as in vitro/in vivo imaging, protein purification/trafficking, high-throughput assays, among others. The goal of this study is to explore the use of the HaloTag protein as a novel reporter gene for positron emission tomography (PET) imaging. By attaching a HaloTag -reactive chloroalkane to 1, 4, 7-triazacyclononane-N, N', N"-triacetic acid (NOTA) through hydrophilic linkers, the resulting NOTA-conjugated HTLs were labeled with (64)Cu and tested for PET imaging in living mice bearing 4T1-HaloTag-ECS tumors, which stably express the HaloTag protein on the cell surface. Significantly higher uptake of (64)Cu-NOTA-HTL-S (which contains a short hydrophilic linker) in the 4T1-HaloTag-ECS than the non-HaloTag-expressing 4T1 tumors was observed, which demonstrated the HaloTag specificity of (64)Cu-NOTA-HTL-S and warranted future investigation of the HaloTag protein as a PET reporter gene. PMID:21904659

Hong, Hao; Benink, Hélčne A; Zhang, Yin; Yang, Yunan; Uyeda, H Tetsuo; Engle, Jonathan W; Severin, Gregory W; McDougall, Mark G; Barnhart, Todd E; Klaubert, Dieter H; Nickles, Robert J; Fan, Frank; Cai, Weibo



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

ERIC Educational Resources Information Center

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

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



Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.  


A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 ?M but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection. PMID:20833789

Uchiyama, Taku; Miyazaki, Kentaro



Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium.  


Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum-sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5' to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR-regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine-responsive transcriptional regulator. PMID:10361309

Piper, K R; Beck Von Bodman, S; Hwang, I; Farrand, S K



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


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

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



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

PubMed Central

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

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



A genetic system that reports transient activation of genes in Bacillus.  


Site-specific recombination is a powerful tool for precise excision of DNA fragments. We used this characteristic to construct a genetic system to report the transient activation of a promoter by promoting the stable acquisition of an antibiotic resistance marker by the bacterium. The system is composed of two compatible plasmid derivatives from Gram-positive bacteria. One of the plasmids allows the insertion of promoters upstream from tnpI, which encodes the site-specific recombinase of Tn4430. The second plasmid carries two selectable resistance genes: one is flanked by two site-specific recombination sequences and is lost following recombination; in contrast, the other resistance gene becomes functional after the site-specific recombination event. By inserting conditionally controlled promoters (the xylose-inducible xylA promoter or the plcA promoter whose expression is dependent on the growth medium) upstream of tnpI, we demonstrated that our genetic system responds to signals inducing transcription by conferring a new resistance phenotype to the host bacteria. Thus, this system can be used to identify genes which are transiently or conditionally expressed. PMID:9427554

Salamitou, S; Agaisse, H; Lereclus, D



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

PubMed Central

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

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



Identification of a novel missense GLRA1 gene mutation in hyperekplexia: a case report  

PubMed Central

Introduction Hereditary hyperekplexia is a neurological disorder characterized by excessive startle responses with violent jerking to noise or touch, stiffening of the trunk and limbs, clenching of the fists and attacks of a high-frequency trembling. Hyperekplexia has a heterogeneous genetic background with several identified causative genes and demonstrates both dominant and recessive inheritance. Mutations in the glycine receptor alpha 1 subunit gene occur in about 30 percent of hyperekplexia cases. Case presentation In this study, we report the case of a Hungarian boy whose abnormal movements, muscle stiffness and convulsions were first noted when he was 4 days old. Neurological and electrophysiological investigation suggested the clinical diagnosis of hyperekplexia. Conclusions Direct sequencing of the coding regions and the flanking introns of the glycine receptor alpha 1 subunit gene revealed a novel heterozygous missense mutation (c.211A/T, p.Ile71Phe). Genetic screening of our patient’s family revealed that the clinically unaffected parents and sister do not carry the mutation, suggesting that the identified sequence change is a de novo mutation. Since hyperekplexia can have severe consequences, including sudden infant death due to laryngospasm and cardiorespiratory failure, identification of the causative genetic alteration(s) of the disease is high priority. Such knowledge is necessary for prenatal diagnosis, which would allow informed family planning and greater parental sensitivity to hyperekplexia 1-associated risks.



Inhibition of reporter gene expression in mammalian cells. Effects of distinct carcinogen lesions in DNA.  


The effect of UV photoproducts or benzo[a]pyrene-diol-epoxide-I (BPDE-I) adducts in DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of cell extracts used in the chloramphenicol acetyl transferase (CAT) assays to contain equal beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid DNA treated with Micrococcus luteus UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by BPDE-I adducts and pyrimidine dimers in DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar threshold densities of BPDE-I adducts and pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (xeroderma pigmentosum group A); calculated D0 values were 1.2 pyrimidine dimers of 2.1 BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that BPDE-I adducts and UV-induced cyclobutane pyrimidine dimers in template DNA block transcription with similar efficacy. PMID:8200075

Sorscher, D H; Cordeiro-Stone, M



Transient reporter gene expression in oocysts and sporozoites of Cryptosporidium parvum controlled by endogenous promoters.  


The apicomplexan protozoan Cryptosporidium parvum is an enteric parasite that affects a variety of mammal hosts including humans, and causes serious diarrheal disease in immunocompromised individuals, notably AIDS patients. Despite many advances in the development of transgenic techniques in many protozoan parasites over the past two decades, rare reports have been documented on the genetic manipulation on C. parvum. Achievement of the DNA-based transfection chiefly depends on the selection of an effective parasite genus-specific promoter. This report described the successful yellow (YFP-YFP) or red (RFP) fluorescent protein expression in oocysts and sporozoites of C. parvum controlled by the endogenous promoters of actin, alpha tubulin, and myosin genes using the restricted enzyme-mediated integration technique. One expression cassette in pBluescript backbone, YFP-YFP or RFP fused between 5' and 3' untranslated regions of actin gene, displayed the highest transfection efficiency with fluorescence rate around 50%. The established DNA-based transient transfection assay may contribute to a better understanding of the biology of Cryptosporidium species and their relationship with hosts and may also result in the development of more efficient molecule-based vaccines and drugs. PMID:24768672

Li, Wei; Diao, Yumei; Gong, Pengtao; Suo, Xun; Li, Jianhua; Zhang, Xichen



Phenotyping of cytomegalovirus drug resistance mutations by using recombinant viruses incorporating a reporter gene.  


A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate. PMID:15980340

Chou, Sunwen; Van Wechel, Laura C; Lichy, Heather M; Marousek, Gail I



Use of reporter-gene based bacteria to quantify phenanthrene biodegradation and toxicity in soil.  


A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed. PMID:21093134

Shin, Doyun; Moon, Hee Sun; Lin, Chu-Ching; Barkay, Tamar; Nam, Kyoungphile



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


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

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



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

NASA Astrophysics Data System (ADS)

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

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



Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection.  


We describe a highly efficient calcium phosphate transfection protocol capable of achieving 100% transfection efficiency of reporter genes transiently expressed in the human hepatoma cell lines HuH7 and HepG2. This procedure, a modification of that described by Chen and Okayama, is reliable, reproducible, and eliminates the requirement for the inclusion of cotransfected internal control plasmids. While Chen and Okayama described the pH of the 2x BBS (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid-buffered saline) and DNA concentration as being critical factors for optimal transfection efficiency, we show that a reduced and strictly monitored standing time of the DNA/CaCl2/2x BBS cocktail prior to addition to cultured cells is essential for a particular combination of pH and DNA concentration. We also show that the inclusion of internal control plasmids can inhibit reporter gene activity in a promoter- and dose-dependent manner. The method so described is also applicable for the transfection of other mammalian cell lines including COS and HeLa, and conceivably for the generation of stable transfectants at high frequency. PMID:7811389

O'Mahoney, J V; Adams, T E



Transcription of the muscle regulatory gene Myf4 is regulated by serum components, peptide growth factors and signaling pathways involving G proteins  

PubMed Central

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell- surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle- specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.



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:



The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication  

PubMed Central

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between ?54 and ?43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

Gariano, Grazia Rosaria; Dell'Oste, Valentina; Bronzini, Matteo; Gatti, Deborah; Luganini, Anna; De Andrea, Marco; Gribaudo, Giorgio; Gariglio, Marisa; Landolfo, Santo



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

SciTech Connect

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

Higgins, D.R.



Long-Term Gene Delivery into the Livers of Immunocompetent Mice with E1\\/E4Defective Adenoviruses  

Microsoft Academic Search

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1\\/E4-defective adenovi- ruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that




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

Microsoft Academic Search

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

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



Effect of chelating agents and respiratory inhibitors on regulation of the cadA gene in Escherichia coli  

Microsoft Academic Search

The cadA gene that encodes lysine decarboxylase in Escherichia coli is induced by low pH and – during anaerobic growth – by the substrate, lysine. We used operon fusions of cadA to lacZ to investigate the effects of aeration on cadA regulation. When an insertion mutation in osmZ (= hns) was introduced, a cadA-lacZ fusion was derepressed in the presence

S. G. Reams; Norizan Lee; F. Mat-Jan; D. P. Clark



Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells  

PubMed Central

Background Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. Results A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene. Conclusion IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.

D'Aiuto, Leonardo; Robison, Clinton S; Gigante, Margherita; Nwanegbo, Edward; Shaffer, Benjamin; Sukhwani, Meena; Castro, Carlos A; Chaillet, J Richard



Genetically Marked Male Sterile Genes and Hybrid Seed Production. Phase 1 Report.  

National Technical Information Service (NTIS)

The objective of the research was to engineer gene fusions which would allow the expression of the naphthalene dioxygenase enzyme in plants. This was done by joining plant gene expression signals to the coding sequence of the naphthalene dioxygenase genes...

R. Jorgensen



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


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

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



?-Globin Gene Sequencing of Hemoglobin Austin Revises the Historically Reported Electrophoretic Migration Pattern.  


Context.-Hemoglobin (Hb) Austin was defined in 1977, using amino acid sequencing of samples from 3 unrelated Mexican-Americans, as a substitution of serine for arginine at position 40 of the ?-globin chain (Arg40Ser). Its electrophoretic migration on both cellulose acetate (pH 8.4) and citrate agar (pH 6.2) was reported between Hb F and Hb A, and this description persists in reference literature. Objectives.-To review the clinical features and redefine the diagnostic characteristics of Hb Austin. Design.-Eight samples from 6 unrelated individuals and 2 siblings, all with Hispanic surnames, were submitted for abnormal Hb identification between June 2010 and September 2011. High-performance liquid chromatography, isoelectric focusing (IEF), citrate agar electrophoresis, and bidirectional DNA sequencing of the entire ?-globin gene were performed. Results.-DNA sequencing confirmed all 8 individuals to be heterozygous for Hb Austin (Arg40Ser). Retention time on high-performance liquid chromatography and migration on citrate agar electrophoresis were consistent with that identification. Migration on IEF, however, was not between Hb F and Hb A, as predicted from the report of cellulose acetate electrophoresis. By IEF, Hb Austin migrated anodal to ("faster than") Hb A. Conclusions.-Hemoglobin Austin (Arg40Ser) appears on IEF as a "fast," anodally migrating, Hb variant, just as would be expected from its amino acid substitution. The cited historic report is, at best, not applicable to IEF and is probably erroneous. Our observation of 8 cases in 16 months suggests that this variant may be relatively common in some Hispanic populations, making its recognition important. Furthermore, gene sequencing is proving itself a powerful and reliable tool for definitive identification of Hb variants. PMID:24878022

Racsa, Lori D; Luu, Hung S; Park, Jason Y; Mitui, Midori; Timmons, Charles F



Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay  

PubMed Central

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.

Parekh, Bhavin S.; Berger, Elaine; Sibley, Sharon; Cahya, Suntara; Xiao, Liqun; LaCerte, Melinda Ann; Vaillancourt, Peter; Wooden, Scott; Gately, Dennis



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


The authors constructed recombinant adenoviral vectors to investigate their potential for gene therapy treatment of leptomeningeal metastases. Several human cell lines that were derived from tumors occurring as leptomeningeal metastases and that were infected in vitro with major late promoter recombinant adenovirus containing the luciferase (luc) gene (IG.Ad.MLP.luc) showed high levels of expression. When these human tumor cell lines were infected in vitro with recombinant adenovirus harboring the herpes simplex virus-thymidine kinase (HSV-tk) gene (IG.Ad.MLP.TK), they were highly sensitive to the killing effects of ganciclovir (GCV). Transduction efficiency of leptomeningeal tumor cells in vivo was assessed by injecting 9-L rat brain tumor cells into the cerebrospinal fluid of Fischer rats via the cisterna magna. After 3 days, recombinant adenovirus containing the lacZ reporter gene (IG.Ad.MLP.lacZ) was injected via the same route. Six days after tumor cell injection, expression of the reporter gene was observed in tumor cells along the total neural axis. Subsequently, rats with leptomeningeal metastases were treated 3 days after tumor cell injection with HSV-tk. Beginning on the next day, GCV was injected intraperitoneally for 10 days. The rats that developed neurological symptoms were killed immediately. The symptom-free latency of every rat was determined. The rats treated with HSV-tk and subsequent GCV had significantly longer (p < 0.01) symptom-free latency than all control groups. This study demonstrates the feasibility and efficacy of this therapeutic approach in a rat model. Clinically, it should be used in the palliative treatment of patients with leptomeningeal metastases. PMID:8814169

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



Expression of the Distalless-B gene in Ciona is regulated by a pan-ectodermal enhancer module  

PubMed Central

The Ci-Dll-B gene is an early regulator of ectodermal development in the ascidian Ciona intestinalis (Imai et al., 2006). Ci-Dll-B is located in a convergently transcribed bigene cluster with a tandem duplicate, Ci-Dll-A. This clustered genomic arrangement is the same as those of the homologous vertebrate Dlx genes, which are also arranged in convergently transcribed bigene clusters. Sequence analysis of the C. intestinalis Dll-A-B cluster reveals a 378 bp region upstream of Ci-Dll-B, termed B1, which is highly conserved with the corresponding region from the congener Ciona savignyi. The B1 element is necessary and sufficient to drive expression of a lacZ reporter gene in a pattern mimicking the endogenous expression of Ci-Dll-B at gastrula stages. This expression pattern which is specific to the entire animal hemisphere is activated preferentially in posterior, or b-lineage, cells by a central portion of B1. Expression in anterior, or a-lineage cells, can be activated by this central portion in combination with the distal part of B1. Anterior expression can also be activated by the central part of B1 plus both the proximal part of B1 and non-conserved sequence upstream of B1. Thus, cis-regulation of early Ci-Dll-B expression is activated by a required submodule in the center of B1, driving posterior expression, which works in combination with redundant submodules that respond to differentially localized anterior factors to produce the total animal hemisphere expression pattern. Interestingly, the intergenic region of the cluster, which is important for expression of the Dlx genes in vertebrates, does not have a specific activating function in the reporter genes tested, but acts as an attenuator in combination with upstream sequences.

Irvine, Steven Q.; Vierra, David A.; Millette, Brad J.; Blanchette, Matthew D.; Holbert, Rachel E.



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

PubMed Central

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

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



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

PubMed Central

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

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



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

NASA Astrophysics Data System (ADS)

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

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



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.

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



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

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



Combined effect of BMP-2 gene transfer and bioactive glass microspheres on enhancement of new bone formation.  


Adenovirus-mediated recombinant human BMP-2 (RAdBMP-2) gene transfer has been found to have significant osteoinductive properties. The hypothesis of the current study was that bioactive glass surface could provide favorable osteoconductive conditions for cellular action of osteoinductive RAdBMP-2 gene transfer. In the rat proximal tibia, a portion of the medullary cavity was evacuated and filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (BG/RAdBMP-2). Control defects filled with BG microspheres were injected with adenovirus carrying the LacZ reporter gene (BG/RAdLacZ) or saline (BG). Empty control defects were also used. Bone healing response was analyzed at 4 days, and at 2 and 8 weeks by radiography, peripheral quantitative computed tomography (pQCT), histomorphometry, and backscattered electron imaging of scanning electron microscopy (BEI-SEM) equipped with energy dispersive X-ray analysis (EDXA). In empty controls, the amount of intramedullary new bone peaked at 2 weeks, whereas defects filled with bioactive glass with and without RAdBMP-2 gene transfer showed a constant time-related increase of intramedullary new bone. At 8 weeks, there was significantly more new bone in defects treated with BG and RAdBMP-2 than in defects left to heal without filling (p < 0.001). Compared with the other controls (BG only or BG/RAdLacZ), the difference was not significant. In the current model, the osteopromotive effect of bioactive glass microspheres appears synergistic with the osteoinductive action of BMP-2 gene transfer, or one overshadows the other, as no additive effect was observed. PMID:16116592

Välimäki, V-V; Yrjans, J J; Vuorio, E; Aro, H T



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


Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local treatment for malignant mesothelioma. PMID:9176512

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



Measurement of Firefly Luciferase Reporter Gene Activity from Cells and Lysates Using Escherichia coli Arsenite and Mercury Sensors  

Microsoft Academic Search

The structural gene encoding firefly luciferase from Photinus pyralis is a widely used reporter both in traditional monitoring of gene expression and in bacterial sensors. Its activity can be detected from living cells (in vivo) without disruption or from cell-free lysate (in vitro). We compared the two measurement methods by using an overall toxicity detecting strain Escherichia coli MC1061(pCSS810), a

Sisko Tauriainen; Marko Virta; Wei Chang; Matti Karp



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


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

PubMed Central

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

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



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

National Technical Information Service (NTIS)

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

M. Karin



Development of gusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service  

Microsoft Academic Search

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzymeß-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend

David McElroy; Douglas A. Chamberlain; Eunpyo Moon; Kate J. Wilson



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

NASA Astrophysics Data System (ADS)

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

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



Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening†  

PubMed Central

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

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



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.



Suppression of experimental crescentic glomerulonephritis by interleukin-10 gene transfer  

Microsoft Academic Search

Suppression of experimental crescentic glomerulonephritis by interleukin-10 gene transfer.BackgroundInvestigated were effects of overexpression of interleukin-10 (IL-10) on the outcome and progression of crescentic glomerulonephritis in Wistar-Kyoto (WKY) rats.MethodsRats were singly or simultaneously injected with antiglomerular basement membrane (a-GBM) antibody and adenoviral vector encoding rat IL-10 (Ad-rIL-10) or LacZ (Ad-LacZ) (3 × 1010 pfu\\/rat) intravenously, and were sacrificed at day 7.




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.

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



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.  


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 vttR(A) (locus tag A33_1664) and vttR(B) (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 vttR(A) or vttR(B) 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, VttR(A) and VttR(B) 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 vttR(B) reporter fusion activity was observed in a DeltavttR(B) strain background, suggesting that VttR(B) 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



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.

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



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

NASA Astrophysics Data System (ADS)

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

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



287. Enhancement of Therapeutic Efficacy of G207 for the Treatment of Malignant Glioma Through Musashi1 Promotor Retargeting of ICP 34.5 Gene-Mediated Virulence  

Microsoft Academic Search

G207 is a conditionally replicating derivative of herpes simplex virus type1 (HSV-1) engineered with deletions of both ICP34.5 loci and a lacZ insertion disabling the ICP6 gene. G207 exhibits an efficient oncolytic activity in vitro and in vivo studies, yet minimal toxicity in normal tissue, and is now in clinical trial for malignant glioma. Accoding to the results of clinical

Takahito Yazaki; Ryuichi Kanai; Hideyuki Tomita; Atsuo Shinoda; Hideyuki Okano; Takeshi Kawase



Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery  

Microsoft Academic Search

The development of accurate, safe, and efficient gene delivery remains a major challenge towards the realization of gene therapeutic prevention and treatment of cardiovascular diseases. In this study, we investigated the ability of high-intensity focused ultrasound (HIFU), a form of mechanical wave transmission, to act as a noninvasive tool for the enhancement of in vivo gene transfer into rabbit carotid

P E Huber; M J Mann; L G Melo; A Ehsan; D Kong; L Zhang; M Rezvani; P Peschke; F Jolesz; V J Dzau; K Hynynen



Nodulation gene factors and plant response in the Rhizobium-legume symbiosis. Final report.  

National Technical Information Service (NTIS)

Our original application aimed to identify genes outside the common nod region involved in nodulation and host range of alfalfa. This has been revised by adding other studies on nodulation gene action and removing molecular studies of gene action. Our res...

S. R. Long



Genetically Marked Male Sterile Genes and Hybrid Seed Production. Final Report on Phase 2.  

National Technical Information Service (NTIS)

The project was intended to make practical the use of nuclear male sterile (ms) genes in hybrid seed production by linking a marker gene to the male fertile allele of the ms gene and selecting for the segregating ms seed by discarding segregants showing t...

A. Morgan



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.

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



A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging  

Microsoft Academic Search

Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (?45HSV1-tk) and with firefly luciferase at

Vladimir Ponomarev; Michael Doubrovin; Inna Serganova; Jelena Vider; Aleksander Shavrin; Tatiana Beresten; Anna Ivanova; Ludmila Ageyeva; Vilia Tourkova; Julius Balatoni; William Bornmann; Ronald Blasberg; Juri Gelovani Tjuvajev



The Association Between Serotonin Transporter Gene Promoter Polymorphism (5-HTTLPR), Self-Reported Symptoms, and Dental Mercury Exposure  

Microsoft Academic Search

The associations between a polymorphism of the serotonin transporter gene (5-HTTLPR), dental mercury exposure, and self-reported symptoms were evaluated among 157 male dentists and 84 female dental assistants. Self-reported symptoms and detailed work histories were obtained by computerized questionnaire. Spot urine samples were collected and analyzed for mercury concentrations to evaluate recent exposures, whereas a chronic mercury exposure index was

Nicholas J. Heyer; Diana Echeverria; Federico M. Farin; James S. Woods



Effect of bisphenol A, tetrachlorobisphenol A and pentachlorophenol on the transcriptional activities of androgen receptor-mediated reporter gene  

Microsoft Academic Search

Previously, we developed an androgen receptor (AR)-mediated reporter gene assay system, and verified the antiandrogenic activity of bisphenol A (BPA) based on this assay. There were some similar phenol-ring structure chemicals which were widely used while little had been done to evaluate their effect to androgen. In the present study, we applied this assay to evaluate the androgenic and antiandrogenic

Hong Sun; Li-Chun Xu; Jian-Feng Chen; Ling Song; Xin-Ru Wang



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



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


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



Validating parameters of a luciferase reporter gene assay to measure neutralizing antibodies to IFN? in multiple sclerosis patients  

Microsoft Academic Search

Neutralizing antibodies (NAbs) can occur in some multiple sclerosis (MS) patients receiving interferon ? (IFN?) therapy. NAbs reduce drug bioavailabity and high NAb titers reduce drug efficacy. We describe the validation of the R. Farrell and G. Giovannoni luciferase reporter gene assay to measure NAbs to INF?. We assayed 163 sera from IFN? treated MS patients with an optimized luciferase

Regina Lam; Rachel Farrell; Tariq Aziz; Ebrima Gibbs; Gavin Giovannoni; Sidney Grossberg; Joel Oger



[24] Green fluorescent protein as a reporter for promoter analysis of testis-specific genes in transgenic mice  

Microsoft Academic Search

The application of green fluorescent protein (GFP) as a reporter in transgenic mice is particularly useful for the study of testis-specific gene promoters. A major advantage is that GFP can be detected directly in freshly isolated spermatogenic cells without the need for cytochemical or immunohistochemical reactions. When combined with the transillumination-assisted microdissection technique, expression of GFP in the testis permits

P. Prabhakara Reddi; Marko Kallio; John C. Herr



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


Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria. PMID:23002229

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



Temporal and spatial regulation of fliP, an early flagellar gene of Caulobacter crescentus that is required for motility and normal cell division.  

PubMed Central

In Caulobacter crescentus, the genes encoding a single polar flagellum are expressed under cell cycle control. In this report, we describe the characterization of two early class II flagellar genes contained in the orfX-fliP locus. Strains containing mutations in this locus exhibit a filamentous growth phenotype and fail to express class III and IV flagellar genes. A complementing DNA fragment was sequenced and found to contain two potential open reading frames. The first, orfX, is predicted to encode a 105-amino-acid polypeptide that is similar to MopB, a protein which is required for both motility and virulence in Erwinia carotovora. The deduced amino acid sequence of the second open reading frame, fliP, is 264 amino acids in length and shows significant sequence identity with the FliP protein of Escherichia coli as well as virulence proteins of several plant and mammalian pathogens. The FliP homolog in pathogenic organisms has been implicated in the secretion of virulence factors, suggesting that the export of virulence proteins and some flagellar proteins share a common mechanism. The 5' end of orfX-fliP mRNA was determined and revealed an upstream promoter sequence that shares few conserved features with that of other early Caulobacter flagellar genes, suggesting that transcription of orfX-fliP may require a different complement of trans-acting factors. In C. crescentus, orfX-fliP is transcribed under cell cycle control, with a peak of transcriptional activity in the middle portion of the cell cycle. Later in the cell cycle, orfX-fliP expression occurs in both poles of the predivisional cell. Protein fusions to a lacZ reporter gene indicate that FliP is specifically targeted to the swarmer compartment of the predivisional cell.

Gober, J W; Boyd, C H; Jarvis, M; Mangan, E K; Rizzo, M F; Wingrove, J A



Recommendations for Analyzing and Reporting TP53 Gene Variants in the High-Throughput Sequencing Era.  


The architecture of TP53, the most frequently mutated gene in human cancer, is more complex than previously thought. Using TP53 variants as clinical biomarkers to predict response to treatment or patient outcome requires an unequivocal and standardized procedure toward a definitive strategy for the clinical evaluation of variants to provide maximum diagnostic sensitivity and specificity. An intronic promoter and two novel exons have been identified resulting in the expression of multiple transcripts and protein isoforms. These regions are additional targets for mutation events impairing the tumor suppressive activity of TP53. Reassessment of variants located in these regions is needed to refine their prognostic value in many malignancies. We recommend using the stable Locus Reference Genomic reference sequence for detailed and unequivocal reports and annotations of germ line and somatic alterations on all TP53 transcripts and protein isoforms according to the recommendations of the Human Genome Variation Society. This novel and comprehensive description framework will generate standardized data that are easy to understand, analyze, and exchange across various cancer variant databases. Based on the statistical analysis of more than 45,000 variants in the latest version of the UMD TP53 database, we also provide a classification of their functional effects ("pathogenicity"). PMID:24729566

Soussi, Thierry; Leroy, Bernard; Taschner, Peter E M



Genotoxic evaluation of the insecticide endosulfan based on the induced GADD153-GFP reporter gene expression.  


Endosulfan is one of the few organochlorine insecticides still in use in China for protecting crops from a variety of insects. Endosulfan is toxic in fishes and rodents in the in vivo assays, but its genotoxicity in mammalian cells has not been well tested. In this work, a genotoxic testing system has been developed based on the induction of a HepG2/GADD153-GFP reporter gene expression in response to the DNA-damaging agents. Methyl methanesulfonate, a known carcinogenic and genotoxic agent, was used to test the effects of damage dose and post-treatment incubation time on GADD153-GFP expression. Subsequently, the system was applied to the genotoxicity evaluation of endosulfan. Endosulfan was able to cause the increase of GADD153-GFP expression at a sublethal dose (0.02-20 mg/L). In particular, it induced a maximum green fluorescent protein expression at the tested concentration of 0.2 mg/L, with 4.07-fold inflorescence relative to untreated cells. The results suggest that endosulfan has the potential genotoxicity for HepG2 cell line by inducing DNA damage. The study also confirms that the induced GADD153-GFP expression system is an appropriate and sensitive method for the assessment of genotoxicity from a broad range of pesticides with the DNA-damaging potential. PMID:20625822

Li, Dahui; Liu, Jianzhang; Li, Jianzhong



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


The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA). This system utilizes human ovarian carcinoma cells (BG1) stably transfected with an estrogen-responsive luciferase reporter gene plasmid. It was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity. Several pesticides are often present in agricultural products. Therefore the estrogenicity of the mixtures of two kinds of pesticides was evaluated. The activity of diazinon/tolclofos-methyl, pyriproxyfen/prothiofos and TBZ/o-phenylphenol (OPP) was increased up to 1.2-5.3 fold. On the other hand, chlorfluazuron, imazalil and chlorfenapyr had anti-estrogenic activity. Further, to evaluate the change in the estrogenic activity of pesticide metabolites, an experimental system was established using a rat S9 mixture. Metabolites of permethrin and OPP had no estrogenic activity, but they had weak activity after the metabolism. On the other hand, the metabolites of TBZ exhibited less estrogenic activity than the original compounds. PMID:16175743

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



Feline immunodeficiency virus as a gene transfer vector in the rat nucleus tractus solitarii.  


Gene transfer has been used to examine the role of putative neurotransmitters in the nucleus tractus solitarii (NTS). Most such studies used adenovirus vector-mediated gene transfer although adenovirus vector transfects both neuronal and non-neuronal cells. Successful transfection in the NTS has also been reported with lentivirus as the vector. Feline immunodeficiency virus (FIV), a lentivirus, may preferentially transfect neurons and could be a powerful tool to delineate physiological effects produced by altered synthesis of transmitters in neurons. However, it has not been studied in NTS. Therefore, we sought to determine whether FIV transfects rat NTS cells and to define the type of cell transfected. We found that injection of FIV encoding LacZ gene (FIVLacZ) into the NTS led to transfection of numerous NTS cells. Injection of FIVLacZ did not alter immunoreactivity (IR) for neuronal nitric oxide synthase, which we have shown resides in NTS neurons. A majority (91.7 +/- 3.9%) of transfected cells contained IR for neuronal nuclear antigen, a neuronal marker; 2.1 +/- 3.8% of transfected cells contained IR for glial fibrillary acidic protein, a glial marker. No transfected neurons or fibers were observed in the nodose ganglion, which sends afferents to the NTS. We conclude that FIV almost exclusively transfects neurons in the rat NTS from which it is not retrogradely transported. The cell-type specificity of FIV in the NTS may provide a molecular method to study local physiological functions mediated by potential neurotransmitters in the NTS. PMID:19777342

Lin, L H; Langasek, J E; Talman, L S; Taktakishvili, O M; Talman, W T



Feline Immunodeficiency Virus as a Gene Transfer Vector in the Rat Nucleus Tractus Solitarii  

PubMed Central

Gene transfer has been used to examine the role of putative neurotransmitters in the nucleus tractus solitarii (NTS). Most such studies used adenovirus vector-mediated gene transfer although adenovirus vector transfects both neuronal and non-neuronal cells. Successful transfection in the NTS has also been reported with lentivirus as the vector. Feline immunodeficiency virus (FIV), a lentivirus, may preferentially transfect neurons and could be a powerful tool to delineate physiological effects produced by altered synthesis of transmitters in neurons. However, it has not been studied in NTS. Therefore, we sought to determine whether FIV transfects rat NTS cells and to define the type of cell transfected. We found that injection of FIV encoding LacZ gene (FIVLacZ) into the NTS led to transfection of numerous NTS cells. Injection of FIVLacZ did not alter immunoreactivity (IR) for neuronal nitric oxide synthase, which we have shown resides in NTS neurons. A majority (91.7 ± 3.9%) of transfected cells contained IR for neuronal nuclear antigen, a neuronal marker; 2.1 ± 3.8% of transfected cells contained IR for glial fibrillary acidic protein, a glial marker. No transfected neurons or fibers were observed in the nodose ganglion, which sends afferents to the NTS. We conclude that FIV almost exclusively transfects neurons in the rat NTS from which it is not retrogradely transported. The cell-type specificity of FIV in the NTS may provide a molecular method to study local physiological functions mediated by potential neurotransmitters in the NTS.

Langasek, J. E.; Talman, L. S.; Taktakishvili, O. M.; Talman, W. T.



Targeted Disruption of Peptide Transporter Pept1 Gene in Mice Significantly Reduces Dipeptide Absorption in Intestine  

PubMed Central

PEPT1 is a high-capacity, low-affinity peptide transporter that mediates the uptake of di- and tripeptides in the intestine and kidney. PEPT1 also has significance in its ability to transport therapeutic agents and because of its potential as a target for anti-inflammatory therapies. To further understand the relevance of specific peptide transporters in intestinal physiology, pharmacology and pathophysiology, we have generated Pept1 null mice by targeted gene disruption. The Pept1 gene was disrupted by insertion of a lacZ reporter gene under the control of the endogenous Pept1 promoter. Phenotypic profiling of wild-type and Pept1 null mice was then performed, along with in vitro intestinal uptake, in situ intestinal perfusion and in vivo pharmacokinetic studies of glycylsarcosine (GlySar). Pept1 null mice lacked expression of PEPT1 protein in the intestine and kidney, tissues in which this peptide transporter is normally expressed. Pept1-deficient mice were found to be viable, fertile, grew to normal size and weight, and were without any obvious abnormalities. Nevertheless, Pept1 deletion dramatically reduced the intestinal uptake and effective permeability of the model dipeptide GlySar (i.e., by at least 80%), and its oral absorption following gastric gavage (i.e., by about 50%). In contrast, the plasma profiles of GlySar were almost superimposable between wild-type and Pept1 null animals after intravenous dosing. These novel findings provide strong evidence that PEPT1 has a major role in the in vivo oral absorption of dipeptides.

Hu, Yongun; Smith, David E.; Ma, Ke; Jappar, Dilara; Thomas, Winston; Hillgren, Kathleen M.



Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers  

PubMed Central

BACKGROUND—When recombinant adenoviruses are infused directly into the circulation, transgene expression is almost completely restricted to the liver.?AIMS—Efficiency and safety of adenovirus mediated gene transfer into damaged livers were examined in mice with liver cirrhosis or fulminant hepatitis.?METHODS—Liver cirrhosis and fulminant hepatitis were induced by intraperitoneal administration of thioacetamide and D-galactosamine followed by lipopolysaccharide, respectively. Mice were infused with adenoviruses carrying the Escherichia coli ?-galactosidase gene, lacZ gene, into the tail vein. Transduction efficiency of the lacZ gene was estimated histochemically by X-gal staining and quantitatively using a chemiluminescent assay. Activation of adenovirus specific T cells and development of neutralising antibodies against adenovirus were also examined.?RESULTS—Histochemical evaluation revealed that approximately 40%, 80%, and 40% of cells in normal, cirrhotic, and fulminant hepatitis livers, respectively, were stained blue using X-gal staining. Quantitative analyses revealed that levels of lacZ expression in cirrhotic livers were approximately 2.5-fold and sixfold greater than those in normal and fulminant hepatitis livers, respectively. Although transgene expression in fulminant hepatitis livers was significantly lower than that in normal livers, marked levels of transgene expression were achieved even in fulminant hepatitis livers. Significant adverse effects of adenoviruses were not observed in damaged livers. There were no significant differences in cellular or humoral immune responses to adenoviruses among animals with normal, cirrhotic, and fulminant hepatitis livers.?CONCLUSIONS—Our results suggest that gene therapy with adenoviruses may be used efficiently and safely, even in patients with severe liver disease.???Keywords: adenovirus; transduction efficiency; safety; liver cirrhosis; fulminant hepatitis

Nakatani, T; Kuriyama, S; Tominaga, K; Tsujimoto, T; Mitoro, A; Yamazaki, M; Tsujinoue, H; Yoshiji, H; Nagao, S; Fukui, H



Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses.  

PubMed Central

The group B entomopoxvirus (EPV) from Amsacta moorei (AmEPV) productively infects only insect cells. A series of AmEPV-lacZ recombinants was constructed in which the lacZ gene was regulated by either late (the AmEPV spheroidin or the cowpox virus A-type inclusion [ATI]) or early (the AmEPV esp [early strong promoter; derived from a 42-kDa AmEPV protein] or the Melolontha melolontha EPV fusolin, fus) virus promoters. When the AmEPV recombinants were used to infect vertebrate cells, beta-galactosidase expression occurred (in >30% of the cells) when lacZ was regulated by either the fus or esp early promoters but not when lacZ was regulated by the late promoters (spheroidin or ATI). Therefore, AmEPV enters vertebrate cells and undergoes at least a partial uncoating and early, but not late, viral genes are expressed. Neither viral DNA synthesis nor cytopathic effects were observed under any infection conditions. When an AmEPV recombinant virus containing the Aequorea victoria green fluorescent protein gene (gfp) under the control of the esp promoter was used to infect vertebrate cells at a low multiplicity of infection, single fluorescent cells resulted, which continued to divide over a period of several days, ultimately forming fluorescent cell clusters, suggesting that vertebrate cells survive the infection and continue to grow. Therefore, AmEPV may prove to be a highly efficient, nontoxic method of gene delivery into vertebrate cells for transient gene expression.

Li, Y; Hall, R L; Moyer, R W



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


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

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



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

SciTech Connect

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

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



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

ERIC Educational Resources Information Center

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

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



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


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

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



Green fluorescent protein: an in vivo reporter of plant gene expression  

Microsoft Academic Search

Summary  Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under

Randall P. Niedz; Michael R. Sussman; John S. Satterlee



Expression of a reporter gene is reduced by a ribozyme in transgenic plants  

Microsoft Academic Search

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene

Dorothee Wegener; Peter Steinecke; Thomas Herget; Iris Petereit; Christina Philipp; Peter H. Schreier



The neural restrictive silencer element can act as both a repressor and enhancer of L1 cell adhesion molecule gene expression during postnatal development  

PubMed Central

The cell adhesion molecule L1 mediates axonal guidance during neural development and mutations in its gene result in severe neurological defects. In previous studies, we identified the promoter for the L1 gene and showed that a neural restrictive silencer element (NRSE) was critical for preventing ectopic expression of L1 during early embryonic development. In the present study, we have investigated the role of the NRSE in the regulation of L1 expression during postnatal development. In gel mobility shift experiments, the NRSE formed DNA–protein complexes with nuclear extracts prepared from the brains of postnatal mice. To examine the influence of the NRSE on postnatal patterns of L1 expression in vivo, we compared the expression of two lacZ transgene constructs, one containing the native L1 gene regulatory sequences (L1lacZ) and another (L1lacZ?N) lacking the NRSE. Newborn mice carrying the L1lacZ?N showed enhanced ?-galactosidase expression relative to L1lacZ in the brain and ectopic expression in nonneural tissues. In contrast to L1lacZ mice, however, L1lacZ?N mice showed an unexpected loss, during postnatal development and in the adult, of ?-galactosidase expression in several neural structures, including the neural retina, cerebellum, cortex, striatum, and hippocampus. These data support the conclusion that the NRSE not only plays a role in the silencing of L1 expression in nonneural tissues during early development but also can function as a silencer and an enhancer of L1 expression in the nervous system of postnatal and adult animals.

Kallunki, Pekka; Edelman, Gerald M.; Jones, Frederick S.



Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters.  

PubMed Central

Translational lacZ fusions to forespore genes of Bacillus subtilis were not expressed in spoIIAC (sigma F) or spoIIIE mutants when the lacZ fusions were integrated at the loci of the same genes or at the SP beta locus. However, some of these genes, including gerA, gpr, spoIIIG (sigma G), and sspE, were expressed in spoIIIE mutants and spoIIIE spoIIIG double mutants (but not in spoIIAC mutants) when the lacZ fusions were integrated at the amyE locus. When tested, the beta-galactosidase made in these mutants was found only in the forespore, and the 5' ends of the mRNAs produced in these mutants were identical to those in a Spo+ background. Analysis of the in vitro transcription of forespore genes by RNA polymerase containing sigma F (E sigma F) revealed a direct correlation between good in vitro transcription by E sigma F and expression at the amyE locus in spoIIIE mutants. This result suggests that forespore genes are transcribed by E sigma F in spoIIIE and spoIIIE spoIIIG mutants. Comparison of the promoter regions of genes transcribed well and poorly by E sigma F in vivo and in vitro showed that good transcription by E sigma F was correlated with G residues at positions -15 and -16, a purine residue at position -13, and a T residue at position -7 relative to the start site of transcription. The importance of these residues in sigma F recognition was confirmed by analysis of the E sigma F-dependent transcription in vivo and in vitro of mutant ssp genes. Images FIG. 2

Sun, D; Fajardo-Cavazos, P; Sussman, M D; Tovar-Rojo, F; Cabrera-Martinez, R M; Setlow, P



Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products.  


Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (beta-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3' end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities. PMID:11790749

Vaillancourt, Katy; Moineau, Sylvain; Frenette, Michel; Lessard, Christian; Vadeboncoeur, Christian



Noninvasive Visualization of MicroRNA-16 in the Chemoresistance of Gastric Cancer Using a Dual Reporter Gene Imaging System  

PubMed Central

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

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



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


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

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



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

PubMed Central

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

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



Gene expression noise in spatial patterning: hunchback promoter structure affects noise amplitude and distribution in Drosophila segmentation.  


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 hb(14F), 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; Travençolo, Bruno A N; Golyandina, Nina; Usevich, Konstantin; Spirov, Alexander V



Cell-based reporter gene assay for therapy-induced neutralizing antibodies to interferon-beta in multiple sclerosis.  


Patients with therapy-induced neutralizing antibodies (NAbs) to interferon-beta (IFN-?) have reduced responses to IFN-? treatment, resulting in higher relapse rates, increased magnetic resonance imaging activity, and a higher risk of disease progression. A functional assay was employed for both screening and titering of IFN-? NAbs utilizing a human cell line transfected with a luciferase reporter gene responsive to IFN-?. This assay demonstrated 100% sensitivity and specificity compared with the traditional cytopathic effect (CPE) assay and normal donor specimens. Additionally, 183 patients with multiple sclerosis (MS) undergoing therapy with IFN-? were tested in the reporter gene assay. Percent positivity for NAbs to the IFN-? was as follows: Avonex (1?) 26.5%, Rebif (1?) 34.1%, and Betaseron (1?) 31.8%. The IFN-? reporter gene assay showed excellent correlation with the well-established CPE assay offering clear advantages. The 50% false-positivity rate typically seen in enzyme-linked immunosorbent assays could be eliminated by using a functional assay for both screening and titering. Results can be reported within 20 h, and the cell line is cryopreserved, eliminating the need to maintain live viral and cell cultures. The use of this functional assay should be a valuable tool for detecting and monitoring the presence of NAbs in IFN-?-treated patients with MS. PMID:23153300

Martins, Thomas B; Rose, John W; Gardiner, Gareth L; Kusukawa, Noriko; Husebye, Dee; Hill, Harry R



Recombinase-based reporter system and antisense technology to study gene expression and essentiality in hypoxic nonreplicating mycobacteria.  


The study of the mechanisms used by Mycobacterium tuberculosis to survive in the absence of growth is hampered by the absence of appropriate genetic tools. Here, we report two strategies, a recombinase-based reporter system and an antisense technology, to study gene expression and essentiality in hypoxic nonreplicating mycobacteria. The recombinase-based reporter system relies on the resolution of an antibiotic marker flanked by the gammadelta-res sites. This system was developed to identify M. tuberculosis promoters, which are specifically expressed under anaerobic conditions. The antisense strategy was designed to study the role of a gene candidate during anaerobic survival. To validate this approach, the dosR, narK2 and rv2466c promoters were selected to drive dosR antisense mRNA expression in quiescent mycobacteria. The conditional knockout strains were found to be attenuated to adapt and survive under anaerobic conditions, as observed for the dosR knockout strain. Together, our work demonstrates that the recombinase-based reporter system and antisense technology represent two genetic tools useful for the identification and characterization of genes essential for the survival of hypoxic nonreplicating M. tuberculosis. PMID:18544099

Rao, Srinivasa P S; Camacho, Luis; Huat Tan, Bee; Boon, Calvin; Russel, David G; Dick, Thomas; Pethe, Kevin



Efficacy of all-trans-beta-carotene, canthaxanthin, and all-trans-, 9-cis-, and 4-oxoretinoic acids in inducing differentiation of an F9 embryonal carcinoma RAR beta-lacZ reporter cell line.  


A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation. PMID:7864621

Nikawa, T; Schulz, W A; van den Brink, C E; Hanusch, M; van der Saag, P; Stahl, W; Sies, H



8-(18F)Fluoropenciclovir: An Improved Reporter Probe for Imaging HSV1-tk Reporter Gene Expression In Vivo Using PET  

Microsoft Academic Search

We have synthesized and evaluated 8-(18F)fluoropenciclovir (FPCV) and compared it with 8-(18F)fluoroganciclovir (FGCV) for monitoring the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene in cell culture and in vivo. Methods: C6 rat glioma cells stably transfected with HSV1-tk (C6-stb-tk1) and control C6 cells were evaluated for their ability to accumulate FGCV versus FPCV. For in

Meera Iyer; Jorge R. Barrio; Mohammad Namavari; Eileen Bauer; Nagichettiar Satyamurthy; Khoi Nguyen; Tatsushi Toyokuni; Michael E. Phelps; Harvey R. Herschman; Sanjiv S. Gambhir


Homozygous G320V mutation in the HJV gene causing juvenile hereditary haemochromatosis type A. A case report.  


While classical hereditary haemochromatosis, usually associated with mutations in the HFE gene, has an adult age onset and a long, progressive evolution, juvenile haemochromatosis, most often associated with mutations in the HJV gene, is a more severe, rapidly progressive condition and has an onset before the age of 30. We report a 26-year old woman with a severe iron overload, affected by hypogonadotropic hypogonadism and moderate dilative cardiomyopathy, in whom the molecular analysis revealed a homozygous genotype for G320V mutation in the HJV gene. As juvenile haemochromatosis is a severe disease, death usually occurring from cardiac involvement, an efficient iron removal from the body strategy should be started as soon as possible, in order to prevent irreversible damage. PMID:20593054

Militaru, Mariela S; Popp, Radu A; Trifa, Adrian P



Paratesticular myxoid/round cell liposarcoma harboring type 3 DDIT3-FUS fusion gene: report of a very rare case.  


Myxoid/round cell liposarcomas are rare mesenchymal neoplasms. They preferentially occur in the lower extremity, and most of them have type 1 or type 2 DDIT3-FUS fusion gene. We report here a very rare case of myxoid/round cell liposarcoma of the paratesticular region with type 3 DDIT3-FUS fusion gene. A 46-year-old Japanese man noticed a gradually enlarged intrascrotal mass without pain. Surgical resection of 3.4 cm × 2.1 cm oval mass was carried out, and it was located in the right paratesticular region apart from the spermatic cord and epididymis. Histological examination of the tumor revealed ovoid cell proliferation with anastomosing vascular network and scattered lipoblasts. Genetic analysis elucidated that the tumor had a chromosomal translocation, type 3 DDIT3-FUS chimeric gene. The tumor was definitely diagnosed as myxoid/round cell liposarcoma of the paratesticular region. PMID:23276404

Zozumi, Masataka; Nakai, Mayumi; Matsuda, Ikuo; Hao, Hiroyuki; Tsukamoto, Yoshitane; Shiraishi, Yusuke; Nojima, Michio; Yamamoto, Shingo; Hirota, Seiichi




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


Effect of CaCl2 concentration on the rate of foreign gene transfer and expression by in vivo electroporation in the mouse ovary.  


We tested the effect of electroporation (EP) medium composition on the rate of gene transfer and expression in the mouse ovary in vivo. FITC labeled oligonucleotides were dissolved in a medium with varying levels of CaCl2 concentration from 0 to 250 mM, and transferred by in vivo EP. Gene transfer efficiency was assessed by examining fluorescence signal intensity with a fluorescent microscope at 3 h after in vivo EP. The results indicated that CaCl2 concentration at 50 mM gave the highest transfer efficiency of the oligonucleotides only in the presence of phosphate buffered saline (PBS). Without PBS or CaCl2, the oligonucleotide transfer was negligible. A further increase in CaCl2 from 50 to 250 mM lowered the transfer efficiency. Little fluorescence signal was attained by substituting CaCl2 for MgCl2, NaCl or KCl. Addition of glycerol to the EP medium with 50 mM CaCl2 did not improve the transfer efficiency in the presence of PBS, although a marginal increase was observed in the absence of PBS. The stimulating effect of increased CaCl2 concentration from 0 to 50 mM was further evaluated by examining the intensity of reporter protein expression after transferring the bacterial lacZ gene. The results of X-gal staining demonstrated that CaCl2 with a range of 20 to 100 mM, showed enhanced gene expression in comparison with 0 mM. However, no remarkable difference was observed between the different CaCl2 concentrations, suggesting that the stimulating effect of CaCl2 on gene transfer and expression in the mouse ovary in vivo may not necessarily parallel in terms of the optimal concentration. PMID:12883653

Suzuki, Takayuki; Tsunekawa, Jun; Murai, Atsushi; Muramatsu, Tatsuo



Identification and characterization of spdR mutations that bypass the BsgA protease-dependent regulation of developmental gene expression in Myxococcus xanthus.  


The BsgA protease of Myxococcus xanthus is an intracellular protease closely related to the Lon protease of Escherichia coli. BsgA is required for normal levels of developmentally induced gene expression. In this report, we describe the identification of mutations that suppress the developmental defect of bsgA mutants. These mutations localized to the spdR gene (suppressor protease deficiency regulator) that appears to play a role in the regulation of early developmental gene expression. Mutations in spdR fully restored the ability of a bsgA mutant to form fruiting bodies and spores and, with one exception, restored the expression of several development-specific lacZ fusions. spdR mutants exhibited characteristic phenotypic properties including increased expression of the development-specific tps gene during vegetative growth, formation of fruiting bodies and spores on semi-rich nutrient medium and completion of starvation-induced development in a shorter time period than wild-type strains. The spdR locus was cloned and sequenced and found to encode a member of the NtrC family of two-component transcriptional regulators. One interpretation of these data is that SpdR acts, directly or otherwise, to regulate developmental gene expression negatively and that the BsgA protease is required to relieve this inhibitory effect at the onset of development. However, Western immunoblot analysis indicated that SpdR is present at a relatively constant level during growth and early development in both wild-type and BsgA protease-deficient cells. This finding suggests that BsgA does not function to degrade SpdR at the onset of development. PMID:11169116

Hager, E; Tse, H; Gill, R E



Carbon Limitation Induces ?S-Dependent Gene Expression in Pseudomonas fluorescens in Soil  

PubMed Central

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the ?S-dependent Escherichia coli promoter Pfic and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of ?-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing ?-galactosidase from Pfic. Changes in cell size and expression of ?-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of ?-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil ?-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.

Koch, Birgit; Worm, Jakob; Jensen, Linda E.; H?jberg, Ole; Nybroe, Ole



The murine MHC class I genes, H-2Dq and H-2Lq, are strikingly homologous to each other, H-2Ld, and two genes reported to encode tumor- specific antigens  

PubMed Central

Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.



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

PubMed Central

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.

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



Endocrine-related effects of perfluorooctanoic acid (PFOA) in zebrafish, H295R steroidogenesis and receptor reporter gene assays.  


Perfluorooctanoic acid (PFOA), a persistent perfluorinated compound, is distributed widely in wildlife and humans. Recent studies showed that PFOA is a suspected endocrine disruptor. But the results are somewhat contradictory and the mechanisms are unclear. In this study, we investigated the endocrine-related effects of PFOA using a series of assays. The lower dose effect of PFOA on development and endocrine-related gene expression were assessed in a short-term zebrafish assay in vivo. To clarify the mechanism of PFOA, in vitro assays were performed. We tested the hormone receptor activities of ER, AR, and TR against PFOA using reporter gene assays. The hormone levels of estradiol (E2) and testosterone (T), the expression of major steroidogenic genes and the key steroidogenic gene regulator steroidogenic factors 1 (SF-1) were measured after PFOA exposure in H295R steroidogenesis assay. Exposure of zebrafish embryo to PFOA resulted in higher expression of esr1, hhex and pax. PFOA is able to interfere with hormone receptor ER and TR. In H295R cells, PFOA could increase the E2 production and decrease the T production, altered the expression of major steroidogenic genes and regulator SF-1. The current findings indicated the potential endocrine-related effects of PFOA and provided novel information for human risk assessment. PMID:23399300

Du, Guizhen; Huang, Hongyu; Hu, Jialei; Qin, Yufeng; Wu, Di; Song, Ling; Xia, Yankai; Wang, Xinru



Transcriptional reporters for genes activated by cell wall stress through a non-catalytic mechanism involving Mpk1 and SBF  

PubMed Central

The Mpk1 MAP kinase of the Cell Wall Integrity (CWI) signaling pathway induces transcription of the FKS2 gene in response to cell wall stress through a non-catalytic mechanism that involves stable association of Mpk1 with the Swi4 transcription factor. This dimeric complex binds to a Swi4 recognition site in the FKS2 promoter. The Swi6 transcription factor is also required to bind this ternary complex for transcription initiation to ensue. In this context, the Mlp1 pseudokinase serves a redundant function with Mpk1. We have identified three additional genes, CHA1, YLR042c, and YKR013w that are induced by cell wall stress through the same mechanism. We report on the behavior of several promoter-lacZ reporter plasmids designed to detect cell wall stress transcription through this pathway.

Kim, Ki-Young; Levin, David E.



Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model  

PubMed Central

Intramyocardial injection of genes encoding angiogenic factors could provide a useful approach for the treatment of ischemic heart disease. However, uncontrolled expression of angiogenic factors in vivo may cause some unwanted side effects, such as hemangioma formation, retinopathy, and arthritis. It may also induce occult tumor growth and artherosclerotic plaque progression. Because hypoxia-inducible factor 1 is up-regulated in a variety of hypoxic conditions and it regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we propose to use HRE, found in the 3? end of the erythropoietin gene to control gene expression in ischemic myocardium. A concatemer of nine copies of the consensus sequence of HRE isolated from the erythropoietin enhancer was used to mediate hypoxia induction. We constructed two adeno-associated viral vectors in which LacZ and vascular endothelial growth factor (VEGF) expressions were controlled by this HRE concatemer and a minimal simian virus 40 promoter. Both LacZ and VEGF expression were induced by hypoxia and/or anoxia in several cell lines transduced with these vectors. The functions of these vectors in ischemic myocardium were tested by injecting them into normal and ischemic mouse myocardium created by occlusion of the left anterior descending coronary artery. The expression of LacZ gene was induced eight times and of VEGF 20 times in ischemic myocardium compared with normal myocardium after the viral vector transduction. Hence, HRE is a good candidate for the control of angiogenic factor gene expression in ischemic myocardium.

Su, Hua; Arakawa-Hoyt, Janice; Kan, Yuet Wai



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

PubMed Central

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

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



First report of carbapenem-resistant Acinetobacter nosocomialis isolates harboring ISAba1-blaOXA-23 genes in Latin America.  


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

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



Androgen receptor activities of p, p?DDE, fenvalerate and phoxim detected by androgen receptor reporter gene assay  

Microsoft Academic Search

In this study, we have developed a transient human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1. The assay displayed appropriate response to the known androgen receptor (AR) agonist 5?-dihydrotestosterone (DHT) and AR antagonist nilutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC50) value of 3.90×10?10M. Nilutamide exhibited potent

Li-Chun Xu; Hong Sun; Jian-Feng Chen; Qian Bian; Ling Song; Xin-Ru Wang



Electrochemical single-cell gene-expression assay combining dielectrophoretic manipulation with secreted alkaline phosphatase reporter system  

Microsoft Academic Search

Scanning electrochemical microscopy (SECM) was used for the analysis of single-cell gene-expression signals on the basis of a reporter system. We microfabricated a single-cell array on an Indium tin oxide (ITO) electrode comprising 4×4 SU-8 microwells with a diameter of 30?m and a depth of 25?m. HeLa cells transfected with plasmid vectors encoding the secreted alkaline phosphatase (SEAP) were seeded

Tatsuya Murata; Tomoyuki Yasukawa; Hitoshi Shiku; Tomokazu Matsue



Context-dependent Pax5 repression of a PU.1\\/NF-?B regulated reporter gene in B lineage cells  

Microsoft Academic Search

Enhancers located in the 3? end of the locus in part regulate immunoglobulin heavy chain (IgH) gene expression. One of these enhancers, HS 1,2, is developmentally regulated by DNA binding proteins like NF-?B, Pax-5 and the protein complex NF-?P in B lineage cells. Here we report that NF-?P is the ets protein PU.1. A glutathione-S-transferase (GST)-pulldown assay demonstrated that PU.1

Ylva Linderson; Neil S. French; Markus F. Neurath; Sven Pettersson



Molecular characterization of a maize regulatory gene. Annual progress report, November 1991--October 1992.  

National Technical Information Service (NTIS)

All aspects of this year's work have converged on the central theme of post-transcriptional control of R gene expression. Unlike transcriptional control, relatively little is known about post-transcriptional regulation, especially in plants. We believe th...

S. R. Wessler



Mapping Our Genes. Federal Genome Projects: How Vast. How Fast. Contractor Reports. Volume 1.  

National Technical Information Service (NTIS)

Contents: Bibliometric analysis of work on human gene mapping; Medical implications of extensive physical and sequence characterization of the human genome; Mapping the human genome: Some implications; Mapping and sequencing the human genome: Consideratio...

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



Analysis of novel nonviral gene transfer systems for gene delivery to cells of the musculoskeletal system.  


The aim of the present study was to evaluate the efficacy of novel nonviral gene delivery systems in cells of musculoskeletal origin. Primary cultures of lapine skeletal muscle cells, lapine articular chondrocytes, human cells from fibrous dysplasia and cell lines established from human osteosarcoma (SAOS-2), chondrosarcoma (CS-1), murine skeletal myoblasts (L8) and fibroblasts (NIH 3T3) were transfected with the P. pyralis luc or the E. coli lacZ genes using Nanofectin 1 and 2, Superfect, JetPEI, GeneJammer, Effectene, TransPass D2, FuGENE 6, Lipofectamine 2000, Dreamfect, Metafectene, Escort III, and calcium phosphate. Maximal transfection efficiency in lapine skeletal muscle cells was of 60.8 +/- 21.2% using Dreamfect, 38.9 +/- 5.0% in articular chondrocytes using Gene Jammer, 5.2 +/- 8.0% in human cells from fibrous dysplasia using Lipofectamine 2000, 12.7 +/- 16.2% in SAOS-2 cells using FuGENE 6, 29.9 +/- 3.5% in CS-1 cells using Lipofectamine 2000, 70.7 +/- 8.6% in L8 cells using FuGENE 6, and 48.9 +/- 13.0% in NIH 3T3 cells using Metafectene. When the cells were transfected with a human IGF-I gene, significant amounts of the IGF-I protein were secreted. These results indicate that relatively high levels of transfection can be achieved using novel nonviral gene transfer methods. PMID:18219593

Orth, Patrick; Weimer, Anja; Kaul, Gunter; Kohn, Dieter; Cucchiarini, Magali; Madry, Henning



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

SciTech Connect

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

Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (United States)); Schild, D. (Lawrence Berkeley Lab., CA (United States))



Novel transgenic mice for inducible gene overexpression in pancreatic cells define glucocorticoid receptor-mediated regulations of beta cells.  


Conditional gene deletion in specific cell populations has helped the understanding of pancreas development. Using this approach, we have shown that deleting the glucocorticoid receptor (GR) gene in pancreatic precursor cells leads to a doubled beta-cell mass. Here, we provide genetic tools that permit a temporally and spatially controlled expression of target genes in pancreatic cells using the Tetracycline inducible system. To efficiently target the Tetracycline transactivator (tTA) in specific cell populations, we generated Bacterial Artificial Chromosomes (BAC) transgenic mice expressing the improved Tetracycline transactivator (itTA) either in pancreatic progenitor cells expressing the transcription factor Pdx1 (BAC-Pdx1-itTA), or in beta cells expressing the insulin1 gene (BAC-Ins1-itTA). In the two transgenic models, itTA-mediated activation of reporter genes was efficient and subject to regulation by Doxycycline (Dox). The analysis of a tetracycline-regulated LacZ reporter gene shows that in BAC-Pdx1-itTA mice, itTA is expressed from embryonic (E) day 11.5 in all pancreatic precursor cells. In the adult pancreas, itTA is active in mature beta, delta cells and in few acinar cells. In BAC-Ins1-itTA mice tTA is active from E13.5 and is restricted to beta cells in fetal and adult pancreas. In both lines, tTA activity was suppressed by Dox treatment and re-induced after Dox removal. Using these transgenic lines, we overexpressed the GR in selective pancreatic cell populations and found that overexpression in precursor cells altered adult beta-cell fraction but not glucose tolerance. In contrast, GR overexpression in mature beta cells did not alter beta-cell fraction but impaired glucose tolerance with insufficient insulin secretion. In conclusion, these new itTA mouse models will allow fine-tuning of gene expression to investigate gene function in pancreatic biology and help us understand how glucocorticoid signaling affects on the long-term distinct aspects of beta-cell biology. PMID:22363422

Blondeau, Bertrand; Sahly, Iman; Massouridčs, Emmanuelle; Singh-Estivalet, Amrit; Valtat, Bérengčre; Dorchene, Delphine; Jaisser, Frédéric; Bréant, Bernadette; Tronche, Francois



Novel Transgenic Mice for Inducible Gene Overexpression in Pancreatic Cells Define Glucocorticoid Receptor-Mediated Regulations of Beta Cells  

PubMed Central

Conditional gene deletion in specific cell populations has helped the understanding of pancreas development. Using this approach, we have shown that deleting the glucocorticoid receptor (GR) gene in pancreatic precursor cells leads to a doubled beta-cell mass. Here, we provide genetic tools that permit a temporally and spatially controlled expression of target genes in pancreatic cells using the Tetracycline inducible system. To efficiently target the Tetracycline transactivator (tTA) in specific cell populations, we generated Bacterial Artificial Chromosomes (BAC) transgenic mice expressing the improved Tetracycline transactivator (itTA) either in pancreatic progenitor cells expressing the transcription factor Pdx1 (BAC-Pdx1-itTA), or in beta cells expressing the insulin1 gene (BAC-Ins1-itTA). In the two transgenic models, itTA-mediated activation of reporter genes was efficient and subject to regulation by Doxycycline (Dox). The analysis of a tetracycline-regulated LacZ reporter gene shows that in BAC-Pdx1-itTA mice, itTA is expressed from embryonic (E) day 11.5 in all pancreatic precursor cells. In the adult pancreas, itTA is active in mature beta, delta cells and in few acinar cells. In BAC-Ins1-itTA mice tTA is active from E13.5 and is restricted to beta cells in fetal and adult pancreas. In both lines, tTA activity was suppressed by Dox treatment and re-induced after Dox removal. Using these transgenic lines, we overexpressed the GR in selective pancreatic cell populations and found that overexpression in precursor cells altered adult beta-cell fraction but not glucose tolerance. In contrast, GR overexpression in mature beta cells did not alter beta-cell fraction but impaired glucose tolerance with insufficient insulin secretion. In conclusion, these new itTA mouse models will allow fine-tuning of gene expression to investigate gene function in pancreatic biology and help us understand how glucocorticoid signaling affects on the long-term distinct aspects of beta-cell biology.

Massourides, Emmanuelle; Singh-Estivalet, Amrit; Valtat, Berengere; Dorchene, Delphine; Jaisser, Frederic; Breant, Bernadette; Tronche, Francois



Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assay  

PubMed Central

Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences. We also observed enrichment for putative promoter regions of genes predicted in at least two annotation databases and for clones overlapping with CpG islands. Functional validation of randomly selected clones enriched by this method showed that a large fraction of these putative promoters can drive the expression of a reporter gene in transient transfection experiments. This method promises to be a useful genome-wide function-based approach that can complement existing methods to look for promoters.

Khambata-Ford, Shirin; Liu, Yueyi; Gleason, Christopher; Dickson, Mark; Altman, Russ B.; Batzoglou, Serafim; Myers, Richard M.



Combined use of two transcriptional reporters improves signalling assays for G protein-coupled receptors in fission yeast.  


The biochemical and genetic tractability of yeasts make them ideal hosts for the analysis of signalling from G protein-coupled receptors (GPCRs). Selected modifications to the strains allow the introduction of non-yeast components, while signal-dependent expression of reporter genes provides growth selection or enzyme read-out as assays for signalling. One issue with such systems is reporter expression in the absence of stimulation, usually because of spontaneous activation of intracellular signalling components and/or incomplete repression of the signal-dependent promoter. This limits the difference between reporter activity in the presence and absence of stimulation, often referred to as the signal:background ratio. In an effort to extend the applicability of the yeast system, we generated a Schizosaccharomyces pombe strain containing pheromone-dependent reporters for both growth selection and beta-galactosidase production. Simultaneous use of the two reporters provided several advantages over strains expressing only one reporter, particularly when coupled to the use of a competitive inhibitor of the nutritional reporter. For example, the beta-galactosidase signal:background ratio following stimulation with 10(-6) M P-factor increased from 35 for a strain containing a single lacZ reporter to almost 2500 for the double reporter. The sensitivity of the system was also improved, with higher signal:background ratios allowing detection of lower concentrations of P-factor. Although we have used Sz. pombe and focused on GPCR-based induction of beta-galactosidase, the principles described can be applied to other yeasts, different signalling pathways and alternative reporters. PMID:17001618

Das, Anamika; Forfar, Rachel; Ladds, Graham; Davey, John



In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes  

PubMed Central

Background Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. Results The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. Conclusion We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.

Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan MW



Craniofacial Dysmorphogenesis Including Cleft Palate in Mice with an Insertional Mutation in the discs large Gene  

PubMed Central

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.

Caruana, Georgina; Bernstein, Alan



Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome  

PubMed Central

The endogenous human telomerase reverse transcriptase (hTERT) gene is repressed in somatic cells. To study the mechanisms of its repression, we developed a strategy of retrovirus-directed Cre recombinase-mediated BAC targeting, or RMBT, to generate single-copy integrations of BAC at pre-engineered chromosomal sites. This technique involved retroviral transduction of acceptor loci, containing an HSV thymidine kinase marker, and subsequent integration of BAC constructs into the acceptor sites, utilizing the loxP and lox511 sites present in the vector backbones. The BAC reporter, with a Renilla luciferase cassette inserted downstream of the hTERT promoter, was retrofitted with a puromycin marker. Through puromycin selection and ganciclovir counter-selection, a targeting efficiency of over 50% was achieved. We demonstrated that the activity and chromatin structures of the hTERT promoter in chromosomally integrated BAC reporter recapitulated its endogenous counterpart of the host cells. Therefore, we have established a genetically amendable platform to study chromatin and epigenetic regulation of the hTERT gene. The highly efficient and versatile RMBT technique has general applicability for studying largely unexplored chromatin-dependent mechanisms of promoter regulation of various genes.

Wang, Shuwen; Zhao, Yuanjun; Leiby, Melanie A.; Zhu, Jiyue



Green fluorescent protein: an in vivo reporter of plant gene expression.  


Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under the control of the CaMV 35S promoter. Protoplasts were viewed by incident-light fluorescence microscopy twentyfour h after electroporation. 20-60% of the protoplasts emitted an intense green light when illuminated with bl