Advances of reporter gene imaging monitoring stem cell therapy

International Nuclear Information System (INIS)

Pei Zhijun; Zhang Yongxue

2010-01-01

Stem cell transplantation in the treatment of various tissue damage or degenerative diseases are research hotspots both at home and abroad. However, ignorance of the homing, differentiation and functional expression of the stem cell in vivo influence the further development of stem cell therapy. As an important component of molecular imaging technology, reporter gene imaging dynamically monitors the change of stem cell in vivo via monitoring the expression of transfected reporter gene. This paper briefly describes the latest research progress and the future development trend of the monitoring of reporter gene imaging in stem cell therapy in vivo. (authors)

Cloning-free regulated monitoring of reporter and gene expression

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Demirkaya Omer

2009-03-01

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

Developmentally regulated expression of reporter gene in adult ...

Indian Academy of Sciences (India)

pression of reporter gene in adult brain specific GAL4 enhancer traps of. Drosophila ... genes based on their expression pattern, thus enabling us to overcome the ... order association and storage centres of olfactory learning and memory, and ...

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

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Gong Shiaochin

2009-03-01

Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

Science.gov (United States)

Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

2009-03-13

Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

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

Science.gov (United States)

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

2014-02-01

Ecdysone is a hormone known to play a pivotal role in crustaceans and insects. In order to evaluate the ecdysone activities in the environment and within the organism, we have developed a biomonitoring Daphnia strain by introducing a reporter gene. In this study, the ecdysone response element was inserted in the upstream region of a reporter gene, and the DNA construct was injected into Daphnia eggs. The expression of the reporter gene was detected during the early embryonic development stage. In addition, when the eggs expressing the reporter gene were exposed to ecdysone, there was enhanced expression of the reporter gene at detectable levels, while the presence of an antagonist led to its downregulation. These results suggested that this system could be potentially developed for monitoring ecdysone activities in media. Copyright © 2013 Elsevier Ltd. All rights reserved.

Advances in study of perpes simplex virus type 1-thymidine kinase reporter gene imaging

International Nuclear Information System (INIS)

Liu Ying; Lan Xiaoli; Zhang Yongxue

2007-01-01

Radionuclide reporter gene imaging is an effect way to provide qualitative and quantitative information for gene therapy. There are three systems of reporter gene including kinase reporter gene. perpes simplex virus type 1-thymidine kinase (HSV1-tk) has perfect physical and chemical characteristic which is suit for imaging as reporter gene. It has been widely investigated and intensively researched. Two substrates of HSV1-tk are purine nucleosite derivant and acyclovir derivant, which can also be used as reporter probes of HSV1-tk. (authors)

Advances of reporter gene monitoring stem cell therapy

International Nuclear Information System (INIS)

Zhou Xiang; Yin Hongyan; Zhang Yifan

2010-01-01

Stem cell therapy research has made great progress, demonstrating a broad application prospects. However, stem cell therapy as a new disease treatment, there are still many problems to be solved. Reporter gene imaging is a rapid development in recent years, a non-invasive, sensitive method of monitoring of stem cells, in particular radionuclide reporter gene imaging has high sensitivity and specificity of the advantages of strong and can carry out imaging of deep tissue and repeat imaging, is a tracer in vivo conditions, the most promising stem cell transplantation technique, showing good prospects for development. (authors)

Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma.

Science.gov (United States)

Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

2016-07-21

Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

Non-invasive imaging using reporter genes altering cellular water permeability

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

2016-12-01

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

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

Science.gov (United States)

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

2016-05-25

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

Repetitive Imaging of Reporter Gene Expression in the Lung

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Jean-Christophe Richard

2003-10-01

Full Text Available Positron emission tomographic imaging is emerging as a powerful technology to monitor reporter transgene expression in the lungs and other organs. However, little information is available about its usefulness for studying gene expression over time. Therefore, we infected 20 rats with a replication-deficient adenovirus containing a fusion gene encoding for a mutant Herpes simplex virus type-1 thymidine kinase and an enhanced green fluorescent protein. Five additional rats were infected with a control virus. Pulmonary gene transfer was performed via intratracheal administration of vector using a surfactant-based method. Imaging was performed 4–6 hr, and 4, 7, and 10 days after gene transfer, using 9-(4-[18F]-fluoro-3-hydroxymethylbutylguanine, an imaging substrate for the mutant kinase. Lung tracer uptake assessed with imaging was moderately but significantly increased 4–6 hr after gene transfer, was maximal after 4 days, and was no longer detectable by 10 days. The temporal pattern of transgene expression measured ex vivo with in vitro assays of thymidine kinase activity and green fluorescent protein was similar to imaging. In conclusion, positron emission tomography is a reliable new tool to evaluate the onset and duration of reporter gene expression noninvasively in the lungs of intact animals.

Molecular MR imaging of cancer gene therapy. Ferritin transgene reporter takes the stage

International Nuclear Information System (INIS)

Hasegawa, Sumitaka; Furukawa, Takako; Saga, Tsuneo

2010-01-01

Molecular imaging using magnetic resonance (MR) imaging has been actively investigated and made rapid progress in the past decade. Applied to cancer gene therapy, the technique's high spatial resolution allows evaluation of gene delivery into target tissues. Because noninvasive monitoring of the duration, location, and magnitude of transgene expression in tumor tissues or cells provides useful information for assessing therapeutic efficacy and optimizing protocols, molecular imaging is expected to become a critical step in the success of cancer gene therapy in the near future. We present a brief overview of the current status of molecular MR imaging, especially in vivo reporter gene imaging using ferritin and other reporters, discuss its application to cancer gene therapy, and present our research of MR imaging detection of electroporation-mediated cancer gene therapy using the ferritin reporter gene. (author)

Reporter gene bioassays in environmental analysis.

Science.gov (United States)

Köhler, S; Belkin, S; Schmid, R D

2000-01-01

In parallel to the continuous development of increasingly more sophisticated physical and chemical analytical technologies for the detection of environmental pollutants, there is a progressively more urgent need also for bioassays which report not only on the presence of a chemical but also on its bioavailability and its biological effects. As a partial fulfillment of that need, there has been a rapid development of biosensors based on genetically engineered bacteria. Such microorganisms typically combine a promoter-operator, which acts as the sensing element, with reporter gene(s) coding for easily detectable proteins. These sensors have the ability to detect global parameters such as stress conditions, toxicity or DNA-damaging agents as well as specific organic and inorganic compounds. The systems described in this review, designed to detect different groups of target chemicals, vary greatly in their detection limits, specificity, response times and more. These variations reflect on their potential applicability which, for most of the constructs described, is presently rather limited. Nevertheless, present trends promise that additional improvements will make microbial biosensors an important tool for future environmental analysis.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data

DEFF Research Database (Denmark)

Kannan, Soumya; Sams, Thomas; Maury, Jérôme

2018-01-01

activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system......Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds......, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter...

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

International Nuclear Information System (INIS)

Shin, Jae Hoon; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il; Kang, Joo Hyun; Jeong, Jae Min; Lee, Dong Soo; Kim, Chul Woo; Lee, Myung Chul

2004-01-01

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 a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([ 125 I]IVDU) and 125 I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [ 125 I]IVDU and 131 I, and 131 I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of 125 I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [ 125 I]IVDU than CT-26 and CTN. NIS gene expression and 125 I accumulation were found to be directly correlated (R 2 =0.923), as were HSV1-tk gene expression and [ 125 I]IVDU accumulation (R 2 =0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,240±3,755 cpm and 34,039±5,346 cpm, respectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of 131 I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [ 125 I]IVDU than CT-26 and CTN. Scintigraphy using 131 I easily visualised CTN and CMN tumours. In conclusion, the NIS gene may be viewed as an imaging

Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

Science.gov (United States)

Luke, Garry A; Ryan, Martin D

2018-01-01

To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

[Myocardial single photon emission tomography imaging of reporter gene expression in rabbits].

Science.gov (United States)

Liu, Ying; Lan, Xiao-li; Zhang, Liang; Wu, Tao; Jiang, Ri-feng; Zhang, Yong-xue

2009-06-01

To explore the feasibility of single photon emission computed tomography (SPECT) detection of heart reporter gene expression and observed the optimal transfecting titer and imaging time by using herpes simplex virus 1-thymidine kinase (HSV1-tk) as reporter gene and 131I-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (131I-FIAU) as reporter probe in rabbit myocardium. The recombinant Ad-tk carrying HSV1-tk gene and adenovirus (Ad) as vector was constructed and intramyocardially injected to rabbits at various concentrations (1 x 10(9) pfu, 5 x 10(8) pfu, 1 x 10(8) pfu, 5 x 10(7) pfu, 1 x 10(7) pfu). Two days later, rabbits were injected with 600 microCi 131I-FIAU in ear-margin vein and then underwent SPECT myocardium imaging for detection of HSV1-tk expression at 6 h, 24 h, 48 h and 72 h after injection, rabbits with 1 x 10(9) pfu Ad-tk injection were imaged at 96 h and 120 h. Rabbits were sacrificed after imaging and the total myocardial 131I-FIAU accumulation was quantified in percent of injected dose per gram myocardium (% ID/g). The myocardial Ad-tk expression was determined with RT-PCR. Reporter gene was detected by SPECT imaging in the injection site while not detected in the control myocardium and site remote from injection. RT-PCR results also evidenced HSV1-tk express in the injection site. The SPECT target/nontarget ratio was correlated with ex vivo gamma-counting (r2 = 0.933, Ppfu by SPECT imaging. The cardiac SPECT reporter gene imaging with HSV1-tk as reporter gene and 131I-FIAU as reporter probe is feasible.

Environmental Application of Reporter-Genes Based Biosensors for Chemical Contamination Screening

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Matejczyk Marzena

2014-12-01

Full Text Available The paper presents results of research concerning possibilities of applications of reporter-genes based microorganisms, including the selective presentation of defects and advantages of different new scientific achievements of methodical solutions in genetic system constructions of biosensing elements for environmental research. The most robust and popular genetic fusion and new trends in reporter genes technology – such as LacZ (β-galactosidase, xylE (catechol 2,3-dioxygenase, gfp (green fluorescent proteins and its mutated forms, lux (prokaryotic luciferase, luc (eukaryotic luciferase, phoA (alkaline phosphatase, gusA and gurA (β-glucuronidase, antibiotics and heavy metals resistance are described. Reporter-genes based biosensors with use of genetically modified bacteria and yeast successfully work for genotoxicity, bioavailability and oxidative stress assessment for detection and monitoring of toxic compounds in drinking water and different environmental samples, surface water, soil, sediments.

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

Directory of Open Access Journals (Sweden)

Shu-Ye Jiang

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

Biological Education of IVFRU and FIAU for HSV1-TK Reporter Gene Monitoring

Energy Technology Data Exchange (ETDEWEB)

Hong, Su Hee; Kim, Eun Jung; Lee, Eun Ah; Lee, Jong Chan; Choi, Tae Hyun; Lee, Kyo Chul; An, Gwang Il; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2006-07-01

The Herpes Simplex Virus Type1-thymidine kinase (HSV1-TK) system is a useful gene therapy monitoring method. HSV1-TK is one of the most widely used effector gene systems used for imaging gene expression, in association with its use as a reporter gene. It has resulted the development of a number of radiolabeled HSV1-TK substrates for the non-invasive detection of HSV1-TK expression. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. Prodrug activation or sucide gene therapy has been shown to be successful in potentiating the therapeutic index by sensitizing genetically modified tumor cells to various prodrugs or enhancing the action of commonly used chemotherapeutic agents. The most studied prodrug activation approaches involve transfection of tumors with HSV1-TK gene. (Z)-5-(2-iodovinyl)-2'-fluoro- 2'-deoxyuridine (IVFRU) possesses a 2'-fluoro substituent in the ribose configuration, is considered to protect IVFRU from enzyme mediated degradation in vivo. It is obviously potential substrates for HSV1-TK imaging. 2'-Fiuoro-2'-deoxy-1-{beta}-D-arabinofuranosyl- 5-iodo-uridine (FIAU), an anticancer drug widely used in clinical practice, is an analogue of thymidine. In a series of studies using adenovirus vector for gene transfer described the appropriate combination of exogenously introduced HSV1-TK as a 'marker/reporter gene' and radiolabelled FIAU as a 'marker substrate/reporter probe' for monitoring gene therapy and gene expression.

Biological Education of IVFRU and FIAU for HSV1-TK Reporter Gene Monitoring

International Nuclear Information System (INIS)

Hong, Su Hee; Kim, Eun Jung; Lee, Eun Ah; Lee, Jong Chan; Choi, Tae Hyun; Lee, Kyo Chul; An, Gwang Il; Cheon, Gi Jeong

2006-01-01

The Herpes Simplex Virus Type1-thymidine kinase (HSV1-TK) system is a useful gene therapy monitoring method. HSV1-TK is one of the most widely used effector gene systems used for imaging gene expression, in association with its use as a reporter gene. It has resulted the development of a number of radiolabeled HSV1-TK substrates for the non-invasive detection of HSV1-TK expression. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. Prodrug activation or sucide gene therapy has been shown to be successful in potentiating the therapeutic index by sensitizing genetically modified tumor cells to various prodrugs or enhancing the action of commonly used chemotherapeutic agents. The most studied prodrug activation approaches involve transfection of tumors with HSV1-TK gene. (Z)-5-(2-iodovinyl)-2'-fluoro- 2'-deoxyuridine (IVFRU) possesses a 2'-fluoro substituent in the ribose configuration, is considered to protect IVFRU from enzyme mediated degradation in vivo. It is obviously potential substrates for HSV1-TK imaging. 2'-Fiuoro-2'-deoxy-1-β-D-arabinofuranosyl- 5-iodo-uridine (FIAU), an anticancer drug widely used in clinical practice, is an analogue of thymidine. In a series of studies using adenovirus vector for gene transfer described the appropriate combination of exogenously introduced HSV1-TK as a 'marker/reporter gene' and radiolabelled FIAU as a 'marker substrate/reporter probe' for monitoring gene therapy and gene expression

hNIS-IRES-eGFP Dual Reporter Gene Imaging

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Jiantu Che

2005-04-01

Full Text Available The human and rodent sodium iodide symporters (NIS have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES-linked human NIS (hNIS-enhanced green fluorescent protein (eGFP hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.

SPECT imaging of cardiac reporter gene expression in living rabbits

International Nuclear Information System (INIS)

Liu Ying; Lan Xiaoli; Zhang Liang; Wu Tao; Jiang Rifeng; Zhang Yongxue

2009-01-01

This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine kinase (HSVI-tk) reporter gene in rabbits myocardium by using the reporter probe 131 I-2'-fluoro-2'-deoxy-l-β-D-arabinofuranosyl-5-iodouracil ( 131 I-FIAU) and SPECT. Rabbits of the study group received intramyocardial injection of Ad5-tk and control group received aseptic saline injection. Two sets of experiments were performed on the study group. Rabbits of the 1st set were injected with 131 I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1xl0 9 , 5x10 8 , 1x10 8 , 5x10 7 and 1x10 7 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1x10 9 , 5x10 8 , 1x10 8 , 5x10 7 , 1x10 7 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131 I-FIAU was injected and heart SPECT imaging was performed at 6, 24 and 48 h, before killing them for gamma counting of the hearts. Reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSVI-tk gene expression. Semi-quantitative analysis derived of region of interest (ROI) of SPECT images and RT-PCR images was performed and the relationship of SPECT images with ex vivo gamma counting and mRNA level were evaluated. SPECT images conformed 131 I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images quality was obtained at 24-48 h for different viral titers. The highest radioactivity in the focal myocardium was seen at 6 h, and then declined with time. The threshold was 5x10 7 pfu of virus titer. The result could be set better in 1-5x10 8 pfu by SPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECT images correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The HSVI-tk/ 131 I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter

Early Infantile Leigh-like Gene Defects Have a Poor Prognosis: Report and Review

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Majid Alfadhel

2017-10-01

Full Text Available Solute carrier family 19 (thiamine transporter, member 3 ( SCL19A3 gene defect produces an autosomal recessive neurodegenerative disorder associated with different phenotypes and acronyms. One of the common presentations is early infantile lethal Leigh-like syndrome. We report a case of early infantile Leigh-like SLC19A3 gene defects of patients who died at 4 months of age with no response to a high dose of biotin and thiamine. In addition, we report a novel mutation that was not reported previously. Finally, we review the literature regarding early infantile Leigh-like SLC19A3 gene defects and compare the literature with our patient.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

Science.gov (United States)

Kannan, Soumya; Sams, Thomas; Maury, Jérôme; Workman, Christopher T

2018-03-16

Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils.

Science.gov (United States)

Cheng, Hsiao-Ying; Masiello, Caroline A; Del Valle, Ilenne; Gao, Xiaodong; Bennett, George N; Silberg, Jonathan J

2018-03-16

Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, C 2 H 4 is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CH 3 Br is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.

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

Science.gov (United States)

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

2009-04-01

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

Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter.

Science.gov (United States)

Kathiria, Palak; Sidler, Corinne; Woycicki, Rafal; Yao, Youli; Kovalchuk, Igor

2013-07-01

The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding β-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA.

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

Science.gov (United States)

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

2014-08-01

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. © 2014. Published by The Company of Biologists Ltd.

GAL4 enhancer trap strains with reporter gene expression during ...

Indian Academy of Sciences (India)

the development of adult brain in Drosophila melanogaster. C. R. VENKATESH ... vous system (CNS), at different time points during the pupal stage—a critical .... in frontal view, with further reduced reporter gene expression. Orthodenticle and ...

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

Science.gov (United States)

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

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

Science.gov (United States)

Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

2011-06-07

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

Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

International Nuclear Information System (INIS)

Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung; Kim, Yeon Soo

2004-01-01

For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells

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

Science.gov (United States)

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

2011-01-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

Effects of Epigenetic Modulation on Reporter Gene Expression: Implications for Stem Cell Imaging

Science.gov (United States)

Krishnan, Manickam; Park, Jinha M.; Cao, Feng; Wang, Dongxu; Paulmurugan, Ramasay; Tseng, Jeffrey R.; Gonzalgo, Mark L.; Gambhir, Sanjiv S.; Wu, Joseph C.

2013-01-01

Tracking stem cell localization, survival, differentiation, and proliferation following transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3–8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage-1 (843±28) to passage-20 (250±10) to passage-40 (44±3) to passage-60 (3±1 RLU/µg) (P<0.05 vs. passage-1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared to trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator) (P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 weeks compared to 1 week for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation. PMID:16246867

Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans

DEFF Research Database (Denmark)

Schalén, Martin; Anyaogu, Diana Chinyere; Hoof, Jakob Blæsbjerg

2016-01-01

roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans....... The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. Results: Fourteen protein secretion...... pathway related genes were overexpressed with a tet-ON promoter in the RFP-secreting reporter strain and macromorphology, physiology and protein secretion were monitored when the secretory genes were induced. Overexpression of several of the chosen genes was shown to cause anomalies on growth, micro...

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Science.gov (United States)

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-03-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

Directory of Open Access Journals (Sweden)

Dashuai Mu

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

Volatile Gas Production by Methyl Halide Transferase: An In Situ Reporter Of Microbial Gene Expression In Soil.

Science.gov (United States)

Cheng, Hsiao-Ying; Masiello, Caroline A; Bennett, George N; Silberg, Jonathan J

2016-08-16

Traditional visual reporters of gene expression have only very limited use in soils because their outputs are challenging to detect through the soil matrix. This severely restricts our ability to study time-dependent microbial gene expression in one of the Earth's largest, most complex habitats. Here we describe an approach to report on dynamic gene expression within a microbial population in a soil under natural water levels (at and below water holding capacity) via production of methyl halides using a methyl halide transferase. As a proof-of-concept application, we couple the expression of this gas reporter to the conjugative transfer of a bacterial plasmid in a soil matrix and show that gas released from the matrix displays a strong correlation with the number of transconjugant bacteria that formed. Gas reporting of gene expression will make possible dynamic studies of natural and engineered microbes within many hard-to-image environmental matrices (soils, sediments, sludge, and biomass) at sample scales exceeding those used for traditional visual reporting.

Splicing defect in FKBP10 gene causes autosomal recessive osteogenesis imperfecta disease: a case report.

Science.gov (United States)

Maghami, Fatemeh; Tabei, Seyed Mohammad Bagher; Moravej, Hossein; Dastsooz, Hassan; Modarresi, Farzaneh; Silawi, Mohammad; Faghihi, Mohammad Ali

2018-05-25

Osteogenesis imperfecta (OI) is a group of connective tissue disorder caused by mutations of genes involved in the production of collagen and its supporting proteins. Although the majority of reported OI variants are in COL1A1 and COL1A2 genes, recent reports have shown problems in other non-collagenous genes involved in the post translational modifications, folding and transport, transcription and proliferation of osteoblasts, bone mineralization, and cell signaling. Up to now, 17 types of OI have been reported in which types I to IV are the most frequent cases with autosomal dominant pattern of inheritance. Here we report an 8- year- old boy with OI who has had multiple fractures since birth and now he is wheelchair-dependent. To identify genetic cause of OI in our patient, whole exome sequencing (WES) was carried out and it revealed a novel deleterious homozygote splice acceptor site mutation (c.1257-2A > G, IVS7-2A > G) in FKBP10 gene in the patient. Then, the identified mutation was confirmed using Sanger sequencing in the proband as homozygous and in his parents as heterozygous, indicating its autosomal recessive pattern of inheritance. In addition, we performed RT-PCR on RNA transcripts originated from skin fibroblast of the proband to analyze the functional effect of the mutation on splicing pattern of FKBP10 gene and it showed skipping of the exon 8 of this gene. Moreover, Real-Time PCR was carried out to quantify the expression level of FKBP10 in the proband and his family members in which it revealed nearly the full decrease in the level of FKBP10 expression in the proband and around 75% decrease in its level in the carriers of the mutation, strongly suggesting the pathogenicity of the mutation. Our study identified, for the first time, a private pathogenic splice site mutation in FKBP10 gene and further prove the involvement of this gene in the rare cases of autosomal recessive OI type XI with distinguished clinical manifestations.

WellReader: a MATLAB program for the analysis of fluorescence and luminescence reporter gene data.

Science.gov (United States)

Boyer, Frédéric; Besson, Bruno; Baptist, Guillaume; Izard, Jérôme; Pinel, Corinne; Ropers, Delphine; Geiselmann, Johannes; de Jong, Hidde

2010-05-01

Fluorescent and luminescent reporter gene systems in combination with automated microplate readers allow real-time monitoring of gene expression on the population level at high precision and sampling density. This generates large amounts of data for the analysis of which computer tools are missing to date. We have developed WellReader, a MATLAB program for the analysis of fluorescent and luminescent reporter gene data. WellReader allows the user to load the output files of microplate readers, remove outliers, correct for background effects and smooth and fit the data. Moreover, it computes biologically relevant quantities from the measured signals, notably promoter activities and protein concentrations, and compares the resulting expression profiles of different genes under different conditions. WellReader is available under a LGPL licence at http://prabi1.inrialpes.fr/trac/wellreader.

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

Science.gov (United States)

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

2017-01-01

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

High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.

Science.gov (United States)

Keegan, Alissa; Plath, Kathrin; Damoiseaux, Robert

2018-01-01

Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.

Gene polymorphisms in association with self-reported stroke in US adults

Directory of Open Access Journals (Sweden)

Amy Z Fan

2010-03-01

Full Text Available Amy Z Fan1, Jing Fang1, Ajay Yesupriya2, Man-huei Chang2, Greta Kilmer1, Meaghan House3, Donald Hayes1, Renée M Ned2, Nicole F Dowling2, Ali H Mokdad1 1National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 2Office of Public Health Genomics, Coordinating Center for Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 3School of Public Health, Emory University, Atlanta, GA, USAPurpose: Epidemiologic studies suggest that several gene variants increase the risk of stroke, and population-based studies help provide further evidence. We identified polymorphisms associated with the prevalence of self-reported stroke in US populations using a representative sample.Methods: Our sample comprised US adults in the Third National Health and Nutrition Examination (NHANES III DNA bank. We examined nine candidate gene variants within ACE, F2, F5, ITGA2, MTHFR, and NOS3 for associations with self-reported stroke. We used multivariate regression and Cox proportional hazards models to test the association between these variants and history of stroke.Results: In regression models, the rs4646994 variant of ACE (I/I and I/D genotypes was associated with higher prevalence adjusted prevalence odds ratio [APOR] = 2.66 [1.28, 5.55] and 2.23 [1.30, 3.85], respectively compared with the D/D genotype. The heterozygous genotype of MTHFR rs1801131 (A/C was associated with lower prevalence of stroke (APOR = 0.48 [0.25, 0.92] compared with A/A and C/C genotypes. For rs2070744 of NOS3, both the C/T genotype (APOR = 1.91 [1.12, 3.27] and C/C genotype (APOR = 3.31 [1.66, 6.60] were associated with higher prevalence of stroke compared with the T/T genotype.Conclusion: Our findings suggest an association between the prevalence of self-reported stroke and polymorphisms in ACE, MTHFR, and NOS3 in a population-based sample. Keywords: stroke, gene, polymorphisms, NHANES III, gene

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

Energy Technology Data Exchange (ETDEWEB)

Shin, Doyun [Department of Civil and Environmental Engineering, Seoul National University, Gwanakno 599, Seoul 151-742 (Korea, Republic of); Moon, Hee Sun [School of Earth and Environmental Science, Seoul National University, Gwanakno 599, Seoul 151-742 (Korea, Republic of); Lin, Chu-Ching; Barkay, Tamar [Department of Biochemistry and Microbiology, Rutgers University, 76 Lipman Drive, New Brunswick, NJ 08901 (United States); Nam, Kyoungphile, E-mail: kpnam@snu.ac.k [Department of Civil and Environmental Engineering, Seoul National University, Gwanakno 599, Seoul 151-742 (Korea, Republic of)

2011-02-15

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. - Research highlights: A novel reporter bacterial strain has been developed. The bacterium can quantitatively determine the change in fluorescence intensity. The intensity can represent the bioavailable phenanthrene in solid matrix. - A cell-killing gene harboring reporter bacterium shows phenanthrene toxicity.

MR molecular imaging of tumours using ferritin heavy chain reporter gene expression mediated by the hTERT promoter

Energy Technology Data Exchange (ETDEWEB)

Yang, Yan [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China); The First Affiliated Hospital of ChengDu Medical College, Department of Radiology, ChengDu (China); Gong, Ming-fu; Yang, Hua; Zhang, Song; Wang, Guang-xian; Su, Tong-sheng; Wen, Li; Zhang, Dong [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China)

2016-11-15

Using the human telomerase reverse transcriptase (hTERT) promoter and the modified ferritin heavy chain (Fth) reporter gene, reporter gene expression for MRI was examined in telomerase positive and negative tumour cells and xenografts. Activity of the reporter gene expression vector Lenti-hTERT-Fth1-3FLAG-Puro was compared to constitutive CMV-driven expression and to the untransfected parental control in five tumour cell lines: A549, SKOV3, 293T, U2OS and HPDLF. In vitro, transfected cells were evaluated for FLAG-tagged protein expression, iron accumulation and transverse relaxation. In vivo, tumours transduced by lentiviral vector injection were imaged using T2*WI. Changes in tumour signal intensity were validated by histology. Only telomerase positive tumour cells expressed FLAG-tagged Fth and displayed an increase in R2* above the parental control, with a corresponding change in T2*WI. In addition, only telomerase positive tumours, transduced by injection of the reporter gene expression construct, exhibited a change in signal intensity on T2*WI. Tumour histology verified the expression of FLAG-tagged Fth and iron accumulation in telomerase positive tissue. Reporter gene expression for MRI, using the Fth reporter and the hTERT promoter, may be a useful strategy for the non-invasive diagnosis of many types of cancer. (orig.)

Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees.

Science.gov (United States)

Chabot, Adrien; Shrit, Ralla A; Blekhman, Ran; Gilad, Yoav

2007-08-01

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

Science.gov (United States)

Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

2006-11-15

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

Science.gov (United States)

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Gene expression and gene therapy imaging

International Nuclear Information System (INIS)

Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

2007-01-01

The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

The first report of prion-related protein gene (PRNT) polymorphisms in goat.

Science.gov (United States)

Kim, Yong-Chan; Jeong, Byung-Hoon

2017-06-01

Prion protein is encoded by the prion protein gene (PRNP). Polymorphisms of several members of the prion gene family have shown association with prion diseases in several species. Recent studies on a novel member of the prion gene family in rams have shown that prion-related protein gene (PRNT) has a linkage with codon 26 of prion-like protein (PRND). In a previous study, codon 26 polymorphism of PRND has shown connection with PRNP haplotype which is strongly associated with scrapie vulnerability. In addition, the genotype of a single nucleotide polymorphism (SNP) at codon 26 of PRND is related to fertilisation capacity. These findings necessitate studies on the SNP of PRNT gene which is connected with PRND. In goat, several polymorphism studies have been performed for PRNP, PRND, and shadow of prion protein gene (SPRN). However, polymorphism on PRNT has not been reported. Hence, the objective of this study was to determine the genotype and allelic distribution of SNPs of PRNT in 238 Korean native goats and compare PRNT DNA sequences between Korean native goats and several ruminant species. A total of five SNPs, including PRNT c.-114G > T, PRNT c.-58A > G in the upstream of PRNT gene, PRNT c.71C > T (p.Ala24Val) and PRNT c.102G > A in the open reading frame (ORF) and c.321C > T in the downstream of PRNT gene, were found in this study. All five SNPs of caprine PRNT gene in Korean native goat are in complete linkage disequilibrium (LD) with a D' value of 1.0. Interestingly, comparative sequence analysis of the PRNT gene revealed five mismatches between DNA sequences of Korean native goats and those of goats deposited in the GenBank. Korean native black goats also showed 5 mismatches in PRNT ORF with cattle. To the best of our knowledge, this is the first genetic research of the PRNT gene in goat.

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

Science.gov (United States)

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

2014-10-01

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

Science.gov (United States)

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

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

Directory of Open Access Journals (Sweden)

Mingyue Sun

2014-09-01

Full Text Available The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Establishment of a hepatocellular carcinoma cell line expressing dual reporter genes: sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP)

International Nuclear Information System (INIS)

Kwak, Won Jung; Koo, Bon Chul; Kwon, Mo Sun

2007-01-01

Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, T 1/2 of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene

Imaging gene expression in gene therapy

International Nuclear Information System (INIS)

Wiebe, Leonard I.

1997-01-01

Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

Gene-gene, gene-environment, gene-nutrient interactions and single nucleotide polymorphisms of inflammatory cytokines.

Science.gov (United States)

Nadeem, Amina; Mumtaz, Sadaf; Naveed, Abdul Khaliq; Aslam, Muhammad; Siddiqui, Arif; Lodhi, Ghulam Mustafa; Ahmad, Tausif

2015-05-15

Inflammation plays a significant role in the etiology of type 2 diabetes mellitus (T2DM). The rise in the pro-inflammatory cytokines is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress and beta cell apoptosis in T2DM. Among the recognized markers are interleukin (IL)-6, IL-1, IL-10, IL-18, tissue necrosis factor-alpha (TNF-α), C-reactive protein, resistin, adiponectin, tissue plasminogen activator, fibrinogen and heptoglobins. Diabetes mellitus has firm genetic and very strong environmental influence; exhibiting a polygenic mode of inheritance. Many single nucleotide polymorphisms (SNPs) in various genes including those of pro and anti-inflammatory cytokines have been reported as a risk for T2DM. Not all the SNPs have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups. The inter-ethnic variations can be explained by the fact that gene expression may be regulated by gene-gene, gene-environment and gene-nutrient interactions. This review highlights the impact of these interactions on determining the role of single nucleotide polymorphism of IL-6, TNF-α, resistin and adiponectin in pathogenesis of T2DM.

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

Science.gov (United States)

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

2016-05-01

Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

Imaging gene expression in gene therapy

Energy Technology Data Exchange (ETDEWEB)

Wiebe, Leonard I. [Alberta Univ., Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

1997-12-31

Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on `suicide gene therapy` of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k{sup +}) has been use for `suicide` in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k{sup +} gene expression where the H S V-1 t k{sup +} gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([{sup 18} F]F H P G; [{sup 18} F]-A C V), and pyrimidine- ([{sup 123}/{sup 131} I]I V R F U; [{sup 124}/{sup 131I}]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [{sup 123}/{sup 131I}]I V R F U imaging with the H S V-1 t k{sup +} reporter gene will be presented

A novel mutation of WFS1 gene in a Chinese patient with Wolfram syndrome: a case report.

Science.gov (United States)

Li, Min; Liu, Jia; Yi, Huan; Xu, Li; Zhong, Xiufeng; Peng, Fuhua

2018-03-17

Wolfram syndrome (WS), caused by mutations of the Wolfram syndrome 1 (WFS1) gene on chromosome 4p16.1, is an autosomal recessive disorder characterized by diabetes insipidus (DI), neuro-psychiatric disorders, hearing deficit, and urinary tract anomalies. Here we report a 11-year-old Chinese boy who presented with visual loss, was suspected with optic neuritis (ON) or neuromyelitis optica (NMO) and referred to our department for further diagnosis. Finally he was diagnosed with WS because of diabetes mellitus (DM) and optic atrophy (OA). Eight exons and flanking introns of WFS1 gene were analyzed by sequencing. A novel mutation c.1760G > A in WFS1 gene of exon 8 was identified. This report reviews a case of WS associated with a novel mutation, c.1760G > A in WFS1 gene of exon 8, and emphasizes that WS should be taken into account for juveniles with visual loss and diabetes mellitus.

Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.

Directory of Open Access Journals (Sweden)

Gabriel Rinaldi

2008-07-01

Full Text Available The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC. We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP, and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth

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

Science.gov (United States)

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

2015-05-15

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

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

Energy Technology Data Exchange (ETDEWEB)

Marton, L.

1994-12-31

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

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

NARCIS (Netherlands)

Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

2002-01-01

Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The

Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart

Energy Technology Data Exchange (ETDEWEB)

Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: LXL730724@hotmail.com; Liu Ying; He Yong; Wu Tao; Zhang Binqing; Gao Zairong; An Rui [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: zhyx1229@163.com

2010-04-15

Aim: To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with {sup 131}I-2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil ({sup 131}I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models. Methods: HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with {sup 131}I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1x10{sup 8} pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5x10{sup 8}, 1x10{sup 8}, 5x10{sup 7} and 1x10{sup 7} pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models. Results: From the ARG images, rats injected with Ad5-tk showed significant {sup 131}I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of {sup 131}I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5x10{sup 7} pfu, and the optimal Ad titer was 1x10{sup 8} pfu

Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

Directory of Open Access Journals (Sweden)

Hackett Perry B

2006-06-01

Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

The experimental study of reporter probe 131I-FIAU in neonatal cardiac myocytes after transfer of herpes simplex virus type 1 thymidine kinase reporter gene by different vectors

International Nuclear Information System (INIS)

Yin Xiaohua; Lan Xiaoli; Wang Ruihua; Liu Ying; Zhang Yongxue

2009-01-01

Objective: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In the present study, the recombinant plasmid and adenoviral vector carrying reporter gene. herpes simplex virus type 1 thymidine kinase (HSV1-tk), were constructed and transferred into nee-natal cardiac myocytes, and a series of in vitro studies were carried out on the cells transferred to evaluate the uptake of radiolabeled reporter probe and to compare both vectors for cardiac reporter gene imaging. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSVI-tk. chosen as the reporter gene.was inserted into adenovirus vector (Ad5-tk) and plasmid (pDC316-tk), thus it could be transferred into neonatal cardiac myocytes. Recombinant adenovirus containing enhanced green fluorescent protein (Ad5-EGFP) was used as control. Recombinant plasmid was coated with lipofectamine TM 2000 (pDC316-tk/lipoplex). The specific reporter probe of HSV1-tk, 2'-fluoro-2'-deoxy-l-β-D-arabinofuranosyl-uracil (FAU), was labeled with 131 I by solid phase oxidation with lodogen. Product wag purified on a reverse. phase Sep-Pak C18 column and the radiochemical purity wag then assessed. The accumulation of it in the transferred cardiac myocytes wag detected as uptake rate. Furthermore, mRNA expression of HSV1-tk was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), while its protein expression wag located by immunocytochemistry. Results: FAU could be labeled with 131 I and the labeling efficiency was (53.82 ±2.05)%. The radiochemical purity was (94.85 ± 1.76)% after purification, and it kept stable in vitro for at least 24h. Time-dependent increase of the ac- cumulation of 131 I-FIAU was observed in both Ad5-tk group and pDC316-tk/lipoplex group. and the highest uptake rate occurred at 5h, with peak values of (12.55 ± 0.37)% and (2.09 ± 0.34)% respectively. However, it also indicated that greater

Late onset bipolar disorder and frontotemporal dementia with mutation in progranulin gene: a case report.

Science.gov (United States)

Rubino, Elisa; Vacca, Alessandro; Gallone, Salvatore; Govone, Flora; Zucca, Milena; Gai, Annalisa; Ferrero, Patrizia; Fenoglio, Pierpaola; Giordana, Maria Teresa; Rainero, Innocenzo

2017-11-01

Bipolar disorder is a chronic psychiatric illness characterised by fluctuation in mood state, with a relapsing and remitting course. Frontotemporal dementia (FTD) is a clinically and genetically heterogeneous syndrome, with the most frequent phenotype being behavioural variant frontotemporal dementia (bvFTD). Here, we report the case of an Italian male presenting with late-onset bipolar disorder that developed into bvFTD over time, carrying a mutation in the GRN gene. Interestingly, the patient carried the c.1639 C > T variant in the GRN gene, resulting in a R547C substitution. Our case report further corroborates the notion that, in addition to FTD, progranulin may be involved in the neurobiology of bipolar disorder type 1, and suggests to screen patients with late-onset bipolar disorder for GRN mutations.

Case report of novel CACNA1A gene mutation causing episodic ataxia type 2

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David Alan Isaacs

2017-05-01

Full Text Available Background: Episodic ataxia type 2 (OMIM 108500 is an autosomal dominant channelopathy characterized by paroxysms of ataxia, vertigo, nausea, and other neurologic symptoms. More than 50 mutations of the CACNA1A gene have been discovered in families with episodic ataxia type 2, although 30%–50% of all patients with typical episodic ataxia type 2 phenotype have no detectable mutation of the CACNA1A gene. Case: A 46-year-old Caucasian man, with a long history of bouts of imbalance, vertigo, and nausea, presented to our hospital with 2 weeks of ataxia and headache. Subsequent evaluation revealed a novel mutation in the CACNA1A gene: c.1364 G > A Arg455Gln. Acetazolamide was initiated with symptomatic improvement. Conclusion: This case report expands the list of known CACNA1A mutations associated with episodic ataxia type 2.

Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports.

Science.gov (United States)

Yoshimura, Hidekane; Oshikawa, Chie; Nakayama, Jun; Moteki, Hideaki; Usami, Shin-Ichi

2015-05-01

This study examines the CLRN1 gene mutation analysis in Japanese patients who were diagnosed with Usher syndrome type 3 (USH3) on the basis of clinical findings. Genetic analysis using massively parallel DNA sequencing (MPS) was conducted to search for 9 causative USH genes in 2 USH3 patients. We identified the novel pathogenic mutation in the CLRN1 gene in 2 patients. The missense mutation was confirmed by functional prediction software and segregation analysis. Both patients were diagnosed as having USH3 caused by the CLRN1 gene mutation. This is the first report of USH3 with a CLRN1 gene mutation in Asian populations. Validating the presence of clinical findings is imperative for properly differentiating among USH subtypes. In addition, mutation screening using MPS enables the identification of causative mutations in USH. The clinical diagnosis of this phenotypically variable disease can then be confirmed. © The Author(s) 2015.

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

Science.gov (United States)

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

2013-05-01

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

Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

Energy Technology Data Exchange (ETDEWEB)

Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

2007-07-01

To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter

Energy Technology Data Exchange (ETDEWEB)

Zhou Xiang; Li Biao; Wang Jun; Yin Hongyan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Zhang Yifan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)], E-mail: zhangyifan1992@yahoo.com.cn

2010-04-15

Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene. Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a {gamma} counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells. Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with

Heterologous expression of mannanase and developing a new Reporter gene system in Lactobacillus casei and Escherichia coli

DEFF Research Database (Denmark)

Lin, Jinzhong; Zou, Yexia; Ma, Chengjie

2015-01-01

Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were clone....... casei and E.coli....

Endogenous Locus Reporter Assays.

Science.gov (United States)

Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

2018-01-01

Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

Evaluation of transcriptional activity of the oestrogen receptor with sodium iodide symporter as an imaging reporter gene.

Science.gov (United States)

Kang, Joo Hyun; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul

2006-10-01

Oestrogen receptors are ligand-dependent transcription factors whose activity is modulated either by oestrogens or by an alternative signalling pathway. Oestrogen receptors interact via a specific DNA-binding domain, the oestrogen responsive element (ERE), in the promoter region of sensitive genes. This binding leads to an initiation of gene expression and hormonal effects. To determine the transcriptional activity of the oestrogen receptor, we developed a molecular imaging system using sodium iodide symporter (NIS) as a reporter gene. The NIS reporter gene was placed under the control of an artificial ERE derived from pERE-TA-SEAP and named as pERE-NIS. pERE-NIS was transferred to MCF-7, human breast cancer cells, which highly expressed oestrogen receptor-alpha with lipofectamine. Stably expressing cells were generated by selection with G418 for 14 days. After treatment of 17beta-oestradiol and tamoxifen with serial doses, the (125)I uptake was measured for the determination of NIS expression. The inhibition of NIS activity was performed with 50 micromol x l(-1) potassium perchlorate. The MCF7/pERE-NIS treated with 17beta-oestradiol accumulated (125)I up to 70-80% higher than did non-treated cells. NIS expression was increased according to increasing doses of 17beta-oestradiol. MCF7/pERE-NIS treated with tamoxifen also accumulated (125)I up to 50% higher than did non-treated cells. Potassium perchlorate completely inhibited (125)I uptake. When MDA-MB231 cells, the oestrogen receptor-negative breast cancer cells, were transfected with pERE-NIS, (125)I uptake of MDA-MB-231/pERE-NIS did not increase. This pERE-NIS reporter system is sufficiently sensitive for monitoring transcriptional activity of the oestrogen receptor. Therefore, cis-enhancer reporter systems with ERE will be applicable to the development of a novel selective oestrogen receptor modulator with low toxicity and high efficacy.

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

Science.gov (United States)

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

2015-10-01

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

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

Directory of Open Access Journals (Sweden)

Yu Shi-Feng

2006-12-01

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

Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12.

Science.gov (United States)

Sellars, Laura E; Bryant, Jack A; Sánchez-Romero, María-Antonia; Sánchez-Morán, Eugenio; Busby, Stephen J W; Lee, David J

2017-08-03

In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

Developmental status of bioassays in genetic toxicology: a report of Phase II of the US Environmental Protection Agency Gene-Tox program

Energy Technology Data Exchange (ETDEWEB)

Brusick, D; Auletta, A

1985-01-01

The Gene-Tox Program was structured around two phases of genetic test data evaluation. The first phase consisted of 36 Work Group reports, each evaluating the results and performance of a specific bioassay. The second phase consisted of a plan to summarize the information provided by the Work Groups. The Gene-Tox Coordinating Committee was to be responsible for Phase II, and several subgroups were assigned specific goals in implementing this analysis. This report deals with Goal I which is to identify the developmental status of the individual bioassays reviewed by the Gene-Tox Work Groups in the first phase of the Program. 5 references, 6 tables.

Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

Energy Technology Data Exchange (ETDEWEB)

Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

2007-07-01

As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

DEFF Research Database (Denmark)

Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper

2011-01-01

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...... relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means...

Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

Energy Technology Data Exchange (ETDEWEB)

Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

2009-02-15

Aim: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyluracil) was labeled with {sup 131}I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [{sup 131}I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry. Results: FAU could be labeled with {sup 131}I, and the labeling efficiency and radiochemical purity rates were 53.82{+-}2.05% and 94.85{+-}1.76%, respectively. Time-dependent increase of the accumulation of [{sup 131}I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55{+-}0.37% and 2.09{+-}0.34%, respectively. Greater uptakes of [{sup 131}I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene

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

Directory of Open Access Journals (Sweden)

Chao-Hsun Yang

2013-01-01

Full Text Available 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.

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

Science.gov (United States)

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

2017-01-01

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

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

Czech Academy of Sciences Publication Activity Database

Svobodová, Kateřina; Cajthaml, Tomáš

2010-01-01

Roč. 88, č. 4 (2010), s. 839-847 ISSN 0175-7598 R&D Projects: GA ČR GAP503/10/0408 Institutional research plan: CEZ:AV0Z50200510 Keywords : endocrine disruptors * in vitro bioassays * reporter gene assays Subject RIV: EE - Microbiology, Virology Impact factor: 3.280, year: 2010

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

Science.gov (United States)

Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

2010-07-01

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Directory of Open Access Journals (Sweden)

Emanuela Chiarella

Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Science.gov (United States)

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

DEFF Research Database (Denmark)

Larashati, Sekar; Schyth, Brian Dall; Lorenzen, Niels

2011-01-01

be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human...... coinjection and the assay is important in order to detect knock down by siRNA. Our experiment reveal in vivo knock down at 72 hours post injection of reporter gene and siRNA, but further dose-response experiments are required to confirm specifity....

Positron emission tomography imaging of gene expression

International Nuclear Information System (INIS)

Tang Ganghua

2001-01-01

The merging of molecular biology and nuclear medicine is developed into molecular nuclear medicine. Positron emission tomography (PET) of gene expression in molecular nuclear medicine has become an attractive area. Positron emission tomography imaging gene expression includes the antisense PET imaging and the reporter gene PET imaging. It is likely that the antisense PET imaging will lag behind the reporter gene PET imaging because of the numerous issues that have not yet to be resolved with this approach. The reporter gene PET imaging has wide application into animal experimental research and human applications of this approach will likely be reported soon

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

Science.gov (United States)

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

2016-02-10

Sox2 is an important transcriptional factor that has multiple functions in stem cell maintenance and tumorigenesis. To investigate the transcriptional regulation of the Sox2 gene, a luciferase knock-in reporter system was established in HEK293 cells by placing the luciferase gene in the genome under the control of the Sox2 gene promoter using a transcription activator-like effector nuclease (TALEN)-mediated genome editing technique. PCR and Southern blot results confirmed the site-specific integration of a single copy of the exogenous luciferase gene into the genome. To prove the reliability and sensitivity of this novel luciferase knock-in system, a CRISPR/Cas transcription activation system for the Sox2 gene was constructed and applied to the knock-in system. The results indicated that luciferase activity was directly correlated with the activity of the Sox2 endogenous promoter. This novel system will be a useful tool to study the transcriptional regulation of Sox2, and has great potential in medical and industrial applications. Copyright © 2015 Elsevier B.V. All rights reserved.

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

International Nuclear Information System (INIS)

Martinez-Cadenas, Conrado; Lozoya, Rafael; Boldó, Enrique; Bosch, Nuria; Peñas, Lucas; Flores-Couce, Esther; Ochoa, Enrique; Munárriz, Javier; Aracil, Juan P; Tajahuerce, Marcos; Royo, Ramón

2011-01-01

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. 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. 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

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

Directory of Open Access Journals (Sweden)

Tajahuerce Marcos

2011-08-01

Full Text Available Abstract 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.

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

Energy Technology Data Exchange (ETDEWEB)

Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

2014-04-20

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

A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes

DEFF Research Database (Denmark)

Seefried, Lothar; Mueller-Deubert, Sigrid; Wentzer, Thomas Schwarz

2010-01-01

of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching...

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

Science.gov (United States)

Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

2016-10-01

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

Science.gov (United States)

Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

2017-01-01

Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

A case report on ATP6V0A4 gene mutation: Forecast of familial ...

African Journals Online (AJOL)

The patient does not have hearing impairment. ATP6V0A4 gene ... This case report emphasizes the significance of early diagnosis and genetic screening of recessive forms of ... Buffer defect /low buffer type distal RTA also involves the loss of ... weight of 4 Kg with no emergency treatment at the time of birth. Afterwards, his ...

Aarskog-Scott syndrome: clinical update and report of nine novel mutations of the FGD1 gene.

Science.gov (United States)

Orrico, A; Galli, L; Faivre, L; Clayton-Smith, J; Azzarello-Burri, S M; Hertz, J M; Jacquemont, S; Taurisano, R; Arroyo Carrera, I; Tarantino, E; Devriendt, K; Melis, D; Thelle, T; Meinhardt, U; Sorrentino, V

2010-02-01

Mutations in the FGD1 gene have been shown to cause Aarskog-Scott syndrome (AAS), or facio-digito-genital dysplasia (OMIM#305400), an X-linked disorder characterized by distinctive genital and skeletal developmental abnormalities with a broad spectrum of clinical phenotypes. To date, 20 distinct mutations have been reported, but little phenotypic data are available on patients with molecularly confirmed AAS. In the present study, we report on our experience of screening for mutations in the FGD1 gene in a cohort of 60 European patients with a clinically suspected diagnosis of AAS. We identified nine novel mutations in 11 patients (detection rate of 18.33%), including three missense mutations (p.R402Q; p.S558W; p.K748E), four truncating mutations (p.Y530X; p.R656X; c.806delC; c.1620delC), one in-frame deletion (c.2020_2022delGAG) and the first reported splice site mutation (c.1935+3A>C). A recurrent mutation (p.R656X) was detected in three independent families. We did not find any evidence for phenotype-genotype correlations between type and position of mutations and clinical features. In addition to the well-established phenotypic features of AAS, other clinical features are also reported and discussed. Copyright 2010 Wiley-Liss, Inc.

A case report of novel mutation in PRF1 gene, which causes familial autosomal recessive hemophagocytic lymphohistiocytosis.

Science.gov (United States)

Bordbar, Mohammad Reza; Modarresi, Farzaneh; Farazi Fard, Mohammad Ali; Dastsooz, Hassan; Shakib Azad, Nader; Faghihi, Mohammad Ali

2017-05-03

Hemophagocytic Lymphohistiocytosis (HLH) is a life-threatening immunodeficiency and multi-organ disease that affects people of all ages and ethnic groups. Common symptoms and signs of this disease are high fever, hepatosplenomegaly, and cytopenias. Familial form of HLH disease, which is an autosomal recessive hematological disorder is due to disease-causing mutations in several genes essential for NK and T-cell granule-mediated cytotoxic function. For an effective cytotoxic response from cytotoxic T lymphocyte or NK cell encountering an infected cell or tumor cell, different processes are required, including trafficking, docking, priming, membrane fusion, and entry of cytotoxic granules into the target cell leading to apoptosis. Therefore, genes involved in these steps play important roles in the pathogenesis of HLH disease which include PRF1, UNC13D (MUNC13-4), STX11, and STXBP2 (MUNC18-2). Here, we report a novel missense mutation in an 8-year-old boy suffered from hepatosplenomegaly, hepatitis, epilepsy and pancytopenia. The patient was born to a first-cousin parents with no previous documented disease in his parents. To identify mutated gene in the proband, Whole Exome Sequencing (WES) utilizing next generation sequencing was used on an Illumina HiSeq 2000 platform on DNA sample from the patient. Results showed a novel deleterious homozygous missense mutation in PRF1 gene (NM_001083116: exon3: c. 1120 T > G, p.W374G) in the patient and then using Sanger sequencing it was confirmed in the proband and his parents. Since his parents were heterozygous for the identified mutation, autosomal recessive pattern of inheritance was confirmed in the family. Our study identified a rare new pathogenic missense mutation in PRF1 gene in patient with HLH disease and it is the first report of mutation in PRF1 in Iranian patients with this disease.

Development of a New Reporter Gene System-dsRed/Xanthine Phosphoribosyltransferase-Xanthine for Molecular Imaging of Processes Behind the Intact Blood-Brain Barrier

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Mikhail Doubrovin

2003-04-01

Full Text Available We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications. The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional. The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of [14C]-xanthine was in vitro (Ki = 0.124 ± 0.008 vs. 0.00031 ± 0.00005 mL/min/g in parental cell line, and [*]-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (logP = 0.69, meeting requirements for the blood-brain barrier (BBB penetrating tracer. In the in vivo experiment, the concentration of [* C]-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration. The accumulation in vivo in the transfected flank tumor was to 2.4 ± 0.3% dose/g, compared to 0.78 ± 0.02% dose/g and 0.64 ± 0.05% dose/g in the control flank tumors and intact muscle, respectively. [14C]-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT, which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections. The images of endogenous gene expression with the “sensory system” have to be normalized for the transfection efficiency based on the “beacon system” image data. Such an approach requires two different “reporter genes” and two different “reporter substrates.” Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB.

The construction and identification of hypoxia-regulated recombinant plasmid with reporter gene hNIS

International Nuclear Information System (INIS)

Hu Qunchao; Wu Jinchang; Zhou Jundong; Gu Ke

2011-01-01

Objective: To construct pShuttle-5 × HRE-CMV-NIS recombinant plasmid regulated by hypoxia-responsive element, which can possibly by used to detect the expression of hypoxia induced factor-α (HIF-1α) gene under hypoxia condition. Methods: Artificially synthesize the nucleotide sequences of five copies of hypoxia response elements (HREs) were cloned into pGL3-promoter vector to construct pGL3-promoter-5 × HRE vector. Human sodium/iodide symporter (hNIS) gene cDNA was amplified from human genome by RT-PCR, and subcloned into pGL3-promoter-5 × HRE vector then was sequenced. After treated with CoCl 2 as hypoxia mimic, HEK293 cells were transfected with recombinant plasmid with hNIS gene, while cells treated with DMSO as the control. Meanwhile, pcDNA3.1-HIF-1α and recombinant hNIS gene vectors were transfected into HEK293 cells at the ratio of 3 to 1, while co-transfection with pcDNA3.1 and pShuttle-NIS vectors cells were taken as the control. NIS mRNA expression was analyzed by qRT-PCR while function of NIS protein was tested by 99m TcO 4 - -uptake. Results: The sequence data of hNIS gene in recombinant plasmid were in accordance with those reported in the literatures. Compared with control groups, HEK293 cells co-transfected with both pShuttle-5 × HRE-CMV-NIS and HIF-1α gene vectors and CoCl 2 -treated after pShuttle-NIS transfecting presented higher mRNA expressions of NIS and 99m TcO 4 - uptake (P<0.01). Conclusion: HIF-1α can be bound to and activate pShuttle-5 × HRE-CMV-NIS in cells to accumulate radioactive nuclide 99m TcO 4 - and this technique is potential for detection of expression and activity of HIF-1α, the indicator of cell hypoxia. (authors)

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

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Md. Zahidus Sayeed

2014-01-01

Full Text Available 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.

Novel biallelic mutations in MSH6 and PMS2 genes: gene conversion as a likely cause of PMS2 gene inactivation.

Science.gov (United States)

Auclair, Jessie; Leroux, Dominique; Desseigne, Françoise; Lasset, Christine; Saurin, Jean Christophe; Joly, Marie Odile; Pinson, Stéphane; Xu, Xiao Li; Montmain, Gilles; Ruano, Eric; Navarro, Claudine; Puisieux, Alain; Wang, Qing

2007-11-01

Since the first report by our group in 1999, more than 20 unrelated biallelic mutations in DNA mismatch repair genes (MMR) have been identified. In the present report, we describe two novel cases: one carrying compound heterozygous mutations in the MSH6 gene; and the other, compound heterozygous mutations in the PMS2 gene. Interestingly, the inactivation of one PMS2 allele was likely caused by gene conversion. Although gene conversion has been suggested to be a mutation mechanism underlying PMS2 inactivation, this is the first report of its involvement in a pathogenic mutation. The clinical features of biallelic mutation carriers were similar to other previously described patients, with the presence of café-au-lait spots (CALS), early onset of brain tumors, and colorectal neoplasia. Our data provide further evidence of the existence, although rare, of a distinct recessively inherited syndrome on the basis of MMR constitutional inactivation. The identification of this syndrome should be useful for genetic counseling, especially in families with atypical hereditary nonpolyposis colon cancer (HNPCC) associated with childhood cancers, and for the clinical surveillance of these mutation carriers. 2007 Wiley-Liss, Inc.

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs

OpenAIRE

Chetverina, Darya; Savitskaya, Ekaterina; Maksimenko, Oksana; Melnikova, Larisa; Zaytseva, Olga; Parshikov, Alexander; Galkin, Alexander V.; Georgiev, Pavel

2007-01-01

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3′UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does ...

Structure and function of the human metallothionein gene family: Final technical report

International Nuclear Information System (INIS)

Karin, M.

1986-01-01

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-I gene whose amino acid sequence is in complete agreement with the published sequence of the human MT-I proteins. Therefore it is likely to be an active gene encoding a functional protein. However, since we have just completed the sequence analysis, we have not characterized this gene further yet. The hMT-I/sub B/ gene is closely linked to the hMT-I/sub A/ gene, and two pseudogenes, hMT-I/sub C/ and hMT-I/sub D/ separate the two. From its nucleotide sequence hMT-I/sub B/ seems to be an active gene, encoding a functional protein even though it differs in four positions from the published sequence of human MT-I proteins. This gene is expressed in a human hepatoma cell line, HepG2, and its expression is stimulated by Cd ++ . Using gene fusions to the viral thymidine-kinase gene we find that hMT-I/sub B/, like the hMT-I/sub A/ and hMT-II/sub A/ genes, contains a heavy metal responsive promoterregulatory element within its 5' flanking region. We analyzed the level of hMT-I/sub B/ mRNA in a variety of human cell lines by the S1 nuclease technique, and compared it to the expression of the hMT-II/sub A/ gene. While the hMT-II/sub A/ gene was expressed in all of the cell lines analyzed, the hMT-I/sub B/ gene was expressed in liver and kidney derived cell lines cells. This suggest that the expression of the hMT-I/sub B/ gene is controlled in a tissue specific manner. 13 refs

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

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Dunham Stephen

2009-11-01

Full Text Available Abstract Background Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions. Results A nitroreductase- encoding gene was identified in the N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of ~3 × 10-6 - 8 × 10-8 among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene. Conclusion We found that nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.

Antisense gene silencing

DEFF Research Database (Denmark)

Nielsen, Troels T; Nielsen, Jørgen E

2013-01-01

Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi) has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied...

Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

NARCIS (Netherlands)

Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

2001-01-01

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2

Combination therapy and evaluation of therapeutic effect in hepatocellular carcinoma cell using triple reporter genes; containing for NIS, HSV1-sr39tk and GFP

Energy Technology Data Exchange (ETDEWEB)

Lee, You La; Lee, Yong Jin; Ahn, Sohn Joo; Ahn, Byeong Cheol; Lee, Sang Woo; Yoo, Jeong Soo; Lee, Jae Tae [Kyungpook National University, Daegu (Korea, Republic of)

2007-07-01

To identify therapeutic effect after combine Sodium Iodine Symporter (NIS) and Mutant Herpes-simplex virus type 1 sr39tk (HSV1-sr39tk) expression in hepatocellular carcinoma cell, we transfected triple gene and investigated the properties of these gene ability in hepatocellular carcinoma cell line. After making vector with gene encoding a fusion protein comprised of HSV1-sr39tk and green florescence protein (GFP), to make triple reporter genes NIS gene was further fused to the vector using IRES vector. The vector expressing triple reporter gene was transfected to the Huh-7 cell line using liposome. Functions of hNIS and HSV1-sr39tk expression were confirmed by radio iodine uptake with and without perchlorate and [3H]-penciclovir (3-H PCV) uptake, respectively. To evaluate therapeutic effect in vitro, GCV and I-131 was treated in Huh-7/NTG cell and dual therapy performed. An animal imaging acquired using Optix and microPET in vivo. I-125 uptake was increased up to 100-fold compare to that of non-transfected cells. The transfected cell accumulated H-3 PCV up to 53 times higher at 2 hour than that of non-transfected cells. With fluorescence microscopy, green fluorescence was detected in the transfected cell. In cytotoxic studies, the cell viability of Huh-7/NTG cell was decreased to 41 % of control cell at 10ug/ml GCV concentrations. The survival rate of the Huh-7/NTG cell treated with I-131 decreased up to 16%. In I-131 and GCV dual therapy, Huh-7/NTG cell survival rate decreased up to 4%. In animal studies, Huh-7/NTG tumors showed higher uptake of 18F-FHBG and I-124 than Huh-7 tumors. GFP signal is also higher in Huh-7/NTG tumor than control. We successfully constructed a vector with delivery two therapeutic genes and one reporter gene and transfected the vector to a Huh-7 cell. The hepatocellular carcinoma cell transfected with the vector can be treated with GCV and I-131. The effect of dual gene therapy could be easily assessed by the optical reporter gene imaging.

β-Globin gene sequencing of hemoglobin Austin revises the historically reported electrophoretic migration pattern.

Science.gov (United States)

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

2014-06-01

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 β-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. 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 β-globin gene were performed. 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. 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.

Gene cluster statistics with gene families.

Science.gov (United States)

Raghupathy, Narayanan; Durand, Dannie

2009-05-01

analysis in genomes of various sizes and illustrate the utility of our methods by applying them to gene clusters recently reported in the literature. Mathematical code to compute cluster probabilities using our methods is available as supplementary material.

The use of molecular imaging of gene expression by radiotracers in gene therapy

International Nuclear Information System (INIS)

Richard-Fiardo, P.; Franken, P.R.; Harrington, K.J.; Vassaux, G.; Cambien, B.

2011-01-01

Introduction: Progress with gene-based therapies has been hampered by difficulties in monitoring the biodistribution and kinetics of vector-mediated gene expression. Recent developments in non-invasive imaging have allowed researchers and clinicians to assess the location, magnitude and persistence of gene expression in animals and humans. Such advances should eventually lead to improvement in the efficacy and safety of current clinical protocols for future treatments. Areas Covered: The molecular imaging techniques for monitoring gene therapy in the living subject, with a specific highlight on the key reporter gene approaches that have been developed and validated in preclinical models using the latest imaging modalities. The applications of molecular imaging to biotherapy, with a particular emphasis on monitoring of gene and vector biodistribution and on image-guided radiotherapy. Expert Opinion: Among the reporter gene/probe combinations that have been described so far, one stands out, in our view, as the most versatile and easy to implement: the Na/I symporter. This strategy, exploiting more than 50 years of experience in the treatment of differentiated thyroid carcinomas, has been validated in different types of experimental cancers and with different types of oncolytic viruses and is likely to become a key tool in the implementation of human gene therapy. (authors)

Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

Directory of Open Access Journals (Sweden)

Lee SH

2016-04-01

Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

Report of Chinese family with severe dermatitis, multiple allergies and metabolic wasting syndrome caused by novel homozygous desmoglein-1 gene mutation.

Science.gov (United States)

Cheng, Ruhong; Yan, Ming; Ni, Cheng; Zhang, Jia; Li, Ming; Yao, Zhirong

2016-10-01

Recently, homozygous mutations in the desmoglein-1 (DSG1) gene and heterozygous mutation in the desmoplakin (DSP) gene have been demonstrated to be associated with severe dermatitis, multiple allergies and metabolic wasting (SAM) syndrome (Mendelian Inheritance in Man no. 615508). We aim to identify the molecular basis for a Chinese pedigree of SAM syndrome. A Chinese pedigree of SAM syndrome was subjected to mutation detection in the DSG1 gene. Sequence analysis of the DSG1 gene and quantitative reverse transcriptase polymerase chain reaction analysis for gene expression of DSG1 using cDNA derived from the epidermis of patients and controls were both performed. Skin biopsies were also taken from patients for pathological study and transmission electron microscopy observation. Novel homozygous splicing mutation c.1892-1delG in the exon-intron border of the DSG1 gene has been demonstrated to be associated with SAM syndrome. We report a new family of SAM syndrome of Asian decent and expand the spectrum of mutations in the DSG1 gene. © 2016 Japanese Dermatological Association.

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

International Nuclear Information System (INIS)

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

2011-01-01

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

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

International Nuclear Information System (INIS)

VanEtten, H.

1997-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

VanEtten, H.

1997-06-01

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

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

Science.gov (United States)

Salmond, G P; Lutkenhaus, J F; Donachie, W D

1980-01-01

We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

Science.gov (United States)

Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

2009-01-01

The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

Fluorescence recovery allows theimplementation of a fluorescence reporter gene platform applicable for the detection and quantification of horizontal gene transfer in anoxic environments

DEFF Research Database (Denmark)

Pinilla-Redondo, Rafael; Riber, Leise; Sørensen, Søren Johannes

2018-01-01

H limitations, and provide experimental tools that will help broaden its horizon of application to other fields.IMPORTANCEMany anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT......). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated...

Assessment of a 44 gene classifier for the evaluation of chronic fatigue syndrome from peripheral blood mononuclear cell gene expression.

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Daniel Frampton

Full Text Available Chronic fatigue syndrome (CFS is a clinically defined illness estimated to affect millions of people worldwide causing significant morbidity and an annual cost of billions of dollars. Currently there are no laboratory-based diagnostic methods for CFS. However, differences in gene expression profiles between CFS patients and healthy persons have been reported in the literature. Using mRNA relative quantities for 44 previously identified reporter genes taken from a large dataset comprising both CFS patients and healthy volunteers, we derived a gene profile scoring metric to accurately classify CFS and healthy samples. This metric out-performed any of the reporter genes used individually as a classifier of CFS.To determine whether the reporter genes were robust across populations, we applied this metric to classify a separate blind dataset of mRNA relative quantities from a new population of CFS patients and healthy persons with limited success. Although the metric was able to successfully classify roughly two-thirds of both CFS and healthy samples correctly, the level of misclassification was high. We conclude many of the previously identified reporter genes are study-specific and thus cannot be used as a broad CFS diagnostic.

Role of PET in gene therapy

International Nuclear Information System (INIS)

Lee, Kyung Han

2002-01-01

In addition to the well-established use of positron emission tomography (PET) in clinical oncology, novel roles for PET are rapidly emerging in the field of gene therapy. Methods for controlled gene delivery to living bodies, made available through advances in molecular biology, are currently being employed in animals for reasearch purposes and in humans to treat diseases such as cancer. Although gene therapy is still in its early developmental stage, it is perceived that many serious illnesses could be treated successfully by the use of therapeutic gene delivery. A major challenge for the widespread use of human gene therapy is to achieve a controlled and effective delivery of foreign genes to target cells and subsequently, adequate levels of expression. As such, the availability of noninvasive imaging methods to accurately assess the location, duration, and level of transgene expression is critical for optimizing gene therapy strategies. Current endeavors to achieve this goal include methods that utilize magnetic resonance imaging, optical imaging, and nuclear imaging techniques. As for PET, reporter systems that utilize gene encoding enzymes that accumulate postion labeled substrates and those transcribing surface receptors that bind specific positron labeled ligands have been successfully developed. More recent advances in this area include improved reporter gene constructs and radiotracers, introduction of potential strategies to monitor endogenous gene expression, and human pilot studies evaluating the distribution and safety of reporter PET tracers. The remarkably rapid progress occuring in gene imaging technology indicates its importance and wide range of application. As such, gene imaging is likely to become a major and exciting new area for future application of PET technology

Role of PET in gene therapy

Energy Technology Data Exchange (ETDEWEB)

Lee, Kyung Han [School of Medicine, Sungkyunkwan Univ., Seoul (Korea, Republic of)

2002-02-01

In addition to the well-established use of positron emission tomography (PET) in clinical oncology, novel roles for PET are rapidly emerging in the field of gene therapy. Methods for controlled gene delivery to living bodies, made available through advances in molecular biology, are currently being employed in animals for reasearch purposes and in humans to treat diseases such as cancer. Although gene therapy is still in its early developmental stage, it is perceived that many serious illnesses could be treated successfully by the use of therapeutic gene delivery. A major challenge for the widespread use of human gene therapy is to achieve a controlled and effective delivery of foreign genes to target cells and subsequently, adequate levels of expression. As such, the availability of noninvasive imaging methods to accurately assess the location, duration, and level of transgene expression is critical for optimizing gene therapy strategies. Current endeavors to achieve this goal include methods that utilize magnetic resonance imaging, optical imaging, and nuclear imaging techniques. As for PET, reporter systems that utilize gene encoding enzymes that accumulate postion labeled substrates and those transcribing surface receptors that bind specific positron labeled ligands have been successfully developed. More recent advances in this area include improved reporter gene constructs and radiotracers, introduction of potential strategies to monitor endogenous gene expression, and human pilot studies evaluating the distribution and safety of reporter PET tracers. The remarkably rapid progress occuring in gene imaging technology indicates its importance and wide range of application. As such, gene imaging is likely to become a major and exciting new area for future application of PET technology.

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

Energy Technology Data Exchange (ETDEWEB)

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

2002-11-30

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

Hereditary neuropathy with liability to pressure palsy (HNPP): report of a family with a new point mutation in PMP22 gene.

Science.gov (United States)

Fusco, Carlo; Spagnoli, Carlotta; Salerno, Grazia Gabriella; Pavlidis, Elena; Frattini, Daniele; Pisani, Francesco

2017-10-27

Hereditary neuropathy with liability to pressure palsy (HNPP) is an autosomal dominant disorder most commonly presenting with acute-onset, non-painful focal sensory and motor mononeuropathy. Approximately 80% of patients carry a 1.5 Mb deletion of chromosome 17p11.2 involving the peripheral myelin protein 22 gene (PMP22), the same duplicated in Charcot-Marie-Tooth 1A patients. In a small proportion of patients the disease is caused by PMP22 point mutations. We report on a familial case harbouring a new point mutation in the PMP22 gene. The proband is a 4-years-old girl with acute onset of focal numbness and weakness in her right hand. Electroneurography demonstrated transient sensory and motor radial nerves involvement. In her father, reporting chronic symptoms (cramps and exercise-induced myalgia), we uncovered mild atrophy and areflexia on clinical examination and a mixed (predominantly demyelinating) polyneuropathy with sensory-motor involvement on electrophysiological study. Both carried a nucleotidic substitution c.178 + 2 T > C on intron 3 of the PMP22 gene, involving the splicing donor site, not reported on databases but predicted to be likely pathogenic. We described a previously unreported point mutation in PMP22 gene, which led to the development of a HNPP phenotype in a child and her father. In children evaluated for a sensory and motor transient episode, HNPP disorder due to PMP22 mutations should be suspected. Clinical and electrophysiological studies should be extended to all family members even in the absence of previous episodes suggestive for HNPP.

Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean.

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Aldrin Kay-Yuen Yim

Full Text Available Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq datasets (26 sequencing libraries in total to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable reference genes when more soybean RNA-Seq data from different growth stages and treatments are used.

Noninvasive imaging of transplanted living functional cells transfected with a reporter estrogen receptor gene

Energy Technology Data Exchange (ETDEWEB)

Takamatsu, Shinji [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan)]. E-mail: shinjit@fmsrsa.fukui-med.ac.jp; Furukawa, Takako [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Yonekura, Yoshiharu [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan)

2005-11-01

The transplantation of functional cells such as dopaminergic cells into damaged tissue is now clinically ongoing, but at present the population of surviving cells at the transplantation site mostly cannot be noninvasively examined. To visualize surviving transplanted functional cells using a noninvasive method, we chose the estrogen receptor ligand binding domain (ERL) as a reporter molecule and 16{alpha}-[{sup 18}F]-fluoro-17{beta}-estradiol (FES) for its ligand. We used a mouse embryonic stem (ES) cell line for recipient cells as a model. To obtain ES cells that constitutively or inducibly express ERL, we transfected two types of expression vectors into EB5 parental ES cell line using the lipofection method and obtained about 30 clones for each of the two types of transfectants. Then, to examine the expression level of ERL, we performed Western blotting analysis. Ligand uptake experiments were carried out using [{sup 3}H]-estradiol with or without excessive unlabeled estradiol for control cells and ERL transfectants. Each selected clone was also used for in vivo positron emission tomography (PET) imaging studies involving FES in nude mice transplanted with control cells and ERL transfectants. In some of the clones transfected with the inducible-type ERL gene, protein was expressed much higher than in the controls. However, constitutive-type ERL gene-transfected ES cells showed no protein production in spite of their gene expression activity being considerably high. All clones also expressed equal levels of the Oct-3/4 gene, a marker of pluripotency, in comparison with the parental cells. Also, the specific uptake of [{sup 3}H]-estradiol was over 30 times higher in inducer-treated ERL-expressing ES cells compared to untreated control cells. Finally, by performing dynamic PET imaging, we successfully visualized ERL-expressing teratomas using FES.

Noninvasive imaging of transplanted living functional cells transfected with a reporter estrogen receptor gene

International Nuclear Information System (INIS)

Takamatsu, Shinji; Furukawa, Takako; Mori, Tetsuya; Yonekura, Yoshiharu; Fujibayashi, Yasuhisa

2005-01-01

The transplantation of functional cells such as dopaminergic cells into damaged tissue is now clinically ongoing, but at present the population of surviving cells at the transplantation site mostly cannot be noninvasively examined. To visualize surviving transplanted functional cells using a noninvasive method, we chose the estrogen receptor ligand binding domain (ERL) as a reporter molecule and 16α-[ 18 F]-fluoro-17β-estradiol (FES) for its ligand. We used a mouse embryonic stem (ES) cell line for recipient cells as a model. To obtain ES cells that constitutively or inducibly express ERL, we transfected two types of expression vectors into EB5 parental ES cell line using the lipofection method and obtained about 30 clones for each of the two types of transfectants. Then, to examine the expression level of ERL, we performed Western blotting analysis. Ligand uptake experiments were carried out using [ 3 H]-estradiol with or without excessive unlabeled estradiol for control cells and ERL transfectants. Each selected clone was also used for in vivo positron emission tomography (PET) imaging studies involving FES in nude mice transplanted with control cells and ERL transfectants. In some of the clones transfected with the inducible-type ERL gene, protein was expressed much higher than in the controls. However, constitutive-type ERL gene-transfected ES cells showed no protein production in spite of their gene expression activity being considerably high. All clones also expressed equal levels of the Oct-3/4 gene, a marker of pluripotency, in comparison with the parental cells. Also, the specific uptake of [ 3 H]-estradiol was over 30 times higher in inducer-treated ERL-expressing ES cells compared to untreated control cells. Finally, by performing dynamic PET imaging, we successfully visualized ERL-expressing teratomas using FES

Analysis of 60 reported glioma risk SNPs replicates published GWAS findings but fails to replicate associations from published candidate-gene studies.

Science.gov (United States)

Walsh, Kyle M; Anderson, Erik; Hansen, Helen M; Decker, Paul A; Kosel, Matt L; Kollmeyer, Thomas; Rice, Terri; Zheng, Shichun; Xiao, Yuanyuan; Chang, Jeffrey S; McCoy, Lucie S; Bracci, Paige M; Wiemels, Joe L; Pico, Alexander R; Smirnov, Ivan; Lachance, Daniel H; Sicotte, Hugues; Eckel-Passow, Jeanette E; Wiencke, John K; Jenkins, Robert B; Wrensch, Margaret R

2013-02-01

Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes. © 2012 WILEY PERIODICALS, INC.

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

Energy Technology Data Exchange (ETDEWEB)

Fields, C.A.

1996-06-01

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

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

Science.gov (United States)

Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

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

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Heffer Alison

2011-03-01

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

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

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Haddley Kate

2011-05-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Plotan, Monika; Elliott, Christopher T. [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom); Scippo, Marie Louise [Department of Food Sciences, University of Liege, 4000 Liege (Belgium); Muller, Marc [Molecular Biology and Genetic Engineering GIGA-R, University of Liege, 4000 Liege (Belgium); Antignac, Jean-Philippe [LABERCA, ENVN, USC INRA 2013, BP 50707, 44 307, Nantes (France); Malone, Edward [The State Laboratory, Young' s Cross, Celbridge, Co. Kildare (Ireland); Bovee, Toine F.H. [RIKILT Institute of Food Safety, P.O. Box 230, AE Wageningen 6700 (Netherlands); Mitchell, Samuel [Agri-Food and Biosciences Institute, Belfast BT9 5PX (United Kingdom); Connolly, Lisa, E-mail: l.connolly@qub.ac.uk [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom)

2011-08-26

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

Comparative GO: a web application for comparative gene ontology and gene ontology-based gene selection in bacteria.

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Mario Fruzangohar

Full Text Available The primary means of classifying new functions for genes and proteins relies on Gene Ontology (GO, which defines genes/proteins using a controlled vocabulary in terms of their Molecular Function, Biological Process and Cellular Component. The challenge is to present this information to researchers to compare and discover patterns in multiple datasets using visually comprehensible and user-friendly statistical reports. Importantly, while there are many GO resources available for eukaryotes, there are none suitable for simultaneous, graphical and statistical comparison between multiple datasets. In addition, none of them supports comprehensive resources for bacteria. By using Streptococcus pneumoniae as a model, we identified and collected GO resources including genes, proteins, taxonomy and GO relationships from NCBI, UniProt and GO organisations. Then, we designed database tables in PostgreSQL database server and developed a Java application to extract data from source files and loaded into database automatically. We developed a PHP web application based on Model-View-Control architecture, used a specific data structure as well as current and novel algorithms to estimate GO graphs parameters. We designed different navigation and visualization methods on the graphs and integrated these into graphical reports. This tool is particularly significant when comparing GO groups between multiple samples (including those of pathogenic bacteria from different sources simultaneously. Comparing GO protein distribution among up- or down-regulated genes from different samples can improve understanding of biological pathways, and mechanism(s of infection. It can also aid in the discovery of genes associated with specific function(s for investigation as a novel vaccine or therapeutic targets.http://turing.ersa.edu.au/BacteriaGO.

Molecular Imaging with Activatable Reporter Systems

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Gang Niu, Xiaoyuan Chen

2012-01-01

Full Text Available Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes, the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.

The Impact of GFP Reporter Gene Transduction and Expression on Metabolomics of Placental Mesenchymal Stem Cells Determined by UHPLC-Q/TOF-MS

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Jinfeng Yang

2017-01-01

Full Text Available Introduction. Green fluorescent protein (GFP is widely used as a reporter gene in regenerative medicine research to label and track stem cells. Here, we examined whether expressing GFP gene may impact the metabolism of human placental mesenchymal stem cells (hPMSCs. Methods. The GFP gene was transduced into hPMSCs using lentiviral-based infection to establish GFP+hPMSCs. A sensitive 13C/12C-dansyl labeling LC-MS method targeting the amine/phenol submetabolome was used for in-depth cell metabolome profiling. Results. A total of 1151 peak pairs or metabolites were detected from 12 LC-MS runs. Principal component analysis and partial least squares discriminant analysis showed poor separation, and the volcano plots demonstrated that most of the metabolites were not significantly changed when hPMSCs were tagged with GFP. Overall, 739 metabolites were positively or putatively identified. Only 11 metabolites showed significant changes. Metabolic pathway analyses indicated that three of the identified metabolites were involved in nine pathways. However, these metabolites are unlikely to have a large impact on the metabolic pathways due to their nonessential roles and limited hits in pathway analysis. Conclusion. This study indicated that the expression of ectopic GFP reporter gene did not significantly alter the metabolomics pathways covered by the amine/phenol submetabolome.

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

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Paul D Thomas

Full Text Available 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

Case report: value of gene expression profiling in the diagnosis of atypical neuroblastoma.

Science.gov (United States)

Harttrampf, Anne C; Chen, Qingrong; Jüttner, Eva; Geiger, Julia; Vansant, Gordon; Khan, Javed; Kontny, Udo

2017-08-17

Nephroblastoma and neuroblastoma belong to the most common abdominal malignancies in childhood. Similarities in the initial presentation may provide difficulties in distinguishing between these two entities, especially if unusual variations to prevalent patterns of disease manifestation occur. Because of the risk of tumor rupture, European protocols do not require biopsy for diagnosis, which leads to misdiagnosis in some cases. We report on a 4½-year-old girl with a renal tumor displaying radiological and laboratory characteristics supporting the diagnosis of nephroblastoma. Imaging studies showed tumor extension into the inferior vena cava and bilateral lung metastases while urine catecholamines and MIBG-scintigraphy were negative. Preoperative chemotherapy with vincristine, actinomycine D and adriamycin according to the SIOP2001/GPOH protocol for the treatment of nephroblastoma was initiated and followed by surgical tumor resection. Histopathology revealed an undifferentiated tumor with expression of neuronal markers, suggestive of neuroblastoma. MYCN amplification could not be detected. DNA-microarray analysis was performed using Affymetrix genechip human genome U133 plus 2.0 and artificial neural network analysis. Results were confirmed by multiplex RT-PCR. Principal component analysis using 84 genes showed that the patient sample was clearly clustering with neuroblastoma tumors. This was confirmed by hierarchical clustering of the multiplex RT-PCR data. The patient underwent treatment for high-risk neuroblastoma comprising chemotherapy including cisplatin, etoposide, vindesine, dacarbacine, ifosfamide, vincristine, adriamycine and autologous stem cell transplantation followed by maintenance therapy with 13-cis retinoic acid (GPOH NB2004 High Risk Trial Protocol) and is in complete long-term remission. The use of gene expression profiling in an individual patient strongly contributed to clarification in a diagnostic dilemma which finally led to a change of

First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

Science.gov (United States)

Zouheir Habbal, Mohammad; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F

2014-01-01

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

Directory of Open Access Journals (Sweden)

Mohammad Zouheir Habbal

Full Text Available Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD. Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb, one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

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

International Nuclear Information System (INIS)

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

2004-01-01

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 (Δ45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Δ45HSV1-tk/GFP/luciferase (Δ45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Δ45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Δ45-TGL cells compared to nontransduced control cells. The Ki of 14 C-FIAU was 0.49±0.02, 0.51±0.03, and 0.003±0.001 ml/min/g in U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [ 131 I]FIAU (7.4 MBq/animal) or [ 124 I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity

PlantTribes: a gene and gene family resource for comparative genomics in plants

OpenAIRE

Wall, P. Kerr; Leebens-Mack, Jim; Müller, Kai F.; Field, Dawn; Altman, Naomi S.; dePamphilis, Claude W.

2007-01-01

The PlantTribes database (http://fgp.huck.psu.edu/tribe.html) is a plant gene family database based on the inferred proteomes of five sequenced plant species: Arabidopsis thaliana, Carica papaya, Medicago truncatula, Oryza sativa and Populus trichocarpa. We used the graph-based clustering algorithm MCL [Van Dongen (Technical Report INS-R0010 2000) and Enright et al. (Nucleic Acids Res. 2002; 30: 1575–1584)] to classify all of these species’ protein-coding genes into putative gene families, ca...

Final Technical Report

Energy Technology Data Exchange (ETDEWEB)

Simon Silver

2009-05-28

The work done with DOE support during this 15 year period was extensive and successful. It is best summarized by the list of 58 publications (below) which reported progress made with DOE support. These are from the grant period and a few more recent reporting on grant research. Mostly these are primary research reports in reviewed journals. There are also, however, many summary reviews in review journals and in scientific monographs, as they also are key places for reporting research progress. What we did during this grant period (and much longer) was to characterize genetic determinants for bacterial resistances to additional toxic heavy metals of DOE concern, through starting with phenotypic properties of the resistant bacteria to DNA sequence determination and characterization of the genes involved. Over the years (and as shown in the list of publications), the toxic metal-forming elements we have studied included Ag, As, Cd, Cr, and Hg. In each case, we started with basically nothing (or very little) known, progressed through quite detailed understanding, until other laboratory groups also became strongly involved in related studies. More recently, with DOE support, we were the first laboratory group in the world to identify genes for bacterial resistance to silver salts (sil genes) and the closely related silver-and-copper resistance genes cus. This was initially reported in detail in Gupta et al. (1999; see publications list below). We also identified the first toxic metal 'gene island' (multiple transcripts and perhaps 25 genes each in need of detailed study) which encodes the subunits of arsenite oxidase (which we called aso; Silver and Phung, 2005; but most other researchers have subsequently settled on aox for the gene mnemonic). Both of these systems were firsts. Now a few years later, a search on GenBank shows that each is now represented by gene families with more than a dozen examples that have been identified and sequenced. Most of the

A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2: A case report.

Science.gov (United States)

Shi, Yunfang; Li, Xiaozhou; Ju, Duan; Li, Yan; Zhang, Xiuling; Zhang, Ying

2016-04-01

Waardenburg syndrome (WS) is an autosomal dominant disorder with varying degrees of sensorineural hearing loss, and accumulation of pigmentation in hair, skin and iris. There are four types of WS (WS1-4) with differing characteristics. Mutations in six genes [paired box gene 3 ( PAX3 ), microphthalmia-associated transcription factor ( MITF ), endothelin 3 ( END3 ), endothelin receptor type B ( EDNRB ), SRY (sex determining region Y)-box 10 ( SOX10 ) and snail homolog 2 ( SNAI2 )] have been identified to be associated with the various types. This case report describes the investigation of genetic mutations in three patients with WS2 from a single family. Genomic DNA was extracted, and the six WS-related genes were sequenced using next-generation sequencing technology. In addition to mutations in PAX3, EDNRB and SOX10, a novel heterozygous MITF mutation, p.Δ315Arg (c.944_946delGAA) on exon 8 was identified. This is predicted to be a candidate disease-causing mutation that may affect the structure and function of the enzyme.

The first case report of a large deletion of the BRCA1 gene in Croatia: A case report.

Science.gov (United States)

Musani, Vesna; Sušac, Ilona; Ozretić, Petar; Eljuga, Domagoj; Levanat, Sonja

2017-12-01

Breast cancer is one of the most common cancers in women, and it is the leading cause of cancer related deaths in Croatia. BRCA1 and BRCA2 gene mutations are the most common cause of hereditary breast cancer. In this report we describe a Croatian patient with no apparent family history of cancer, who developed breast cancer first at 29, and again at 33. Due to the early development of first breast cancer and triple negative status of the second, the attending physician suspected a hereditary aspect. Patient was sent to BRCA1 genetic testing. Subsequently, her mother and sister were sent to check for the mutation found in the patient. BRCA1 exons 4-6 deletion was determined and sequencing confirmed the deletion as NG_005905.2:g.107648_117905del10257. Mother and sister were not affected, but since there were no available family members on the fathers' side, it was not possible to determine if this was a case of de novo mutation. Until now, only in three reports with the similar mutation the exact mutation borders were determined. The mutation in this case was not the same as previously reported and was more than twice in size. All large deletions should be described at the nucleotide level, so that in cases with missing family data it would be possible to deduce if the mutation is already known. If the mutation is already known, it is probably not a de novo event, since it is unlikely that the breakpoints would be exactly the same more than once.

Polymorphisms in dopaminergic system genes; association with criminal behavior and self-reported aggression in violent prison inmates from Pakistan.

Directory of Open Access Journals (Sweden)

Muhammad Imran Qadeer

Full Text Available Genetic factors contribute to antisocial and criminal behavior. Dopamine transporter DAT-1 (SLC6A3 and DRD2 gene for the dopamine-2 receptor are dopaminergic system genes that regulate dopamine reuptake and signaling, and may be part of the pathogenesis of psychiatric disorders including antisocial behaviors and traits. No previous studies have analyzed DAT-1 and DRD2 polymorphisms in convicted murderers, particularly from Indian subcontinent. In this study we investigated the association of 40 bp VNTR polymorphism of DAT-1 and Taq1 variant of DRD2 gene (rs1800479 with criminal behavior and self-reported aggression in 729 subjects, including 370 men in Pakistani prisons convicted of first degree murder(s and 359 control men without any history of violence or criminal tendency. The 9R allele of DAT-1 VNTR polymorphism was more prevalent in convicted murderers compared with control samples, for either one or two risk alleles (OR = 1.49 and 3.99 respectively, P = 0.003. This potential association of DAT-1 9R allele polymorphism with murderer phenotype was confirmed assuming different genetic models of inheritance. However, no genetic association was found for DRD2 Taq1 polymorphism. In addition, a combined haplotype (9R-A2 of DAT-1 and DRD2 genes was associated with this murderer phenotype. Further, 9R allele of DAT-1 was also associated with response to verbal abuse and parental marital complications, but not with other measures pertinent to self-reported aggression. These results suggest that 9R allele, which may influence levels of intra-synaptic dopamine in the brain, may contribute to criminal tendency in this sample of violent murderers of Pakistani origin. Future studies are needed to replicate this finding in other populations of murderers and see if this finding extends to other forms of violence and lesser degrees of aggression.

A New Nonsense Mutation in CDKL5 Gene in a Male Patient with Early Onset Refractory Epilepsy: a Case Report

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Soudeh Ghafouri-Fard

2016-02-01

Full Text Available Background The X-linked cyclin-dependent kinase like 5 (CDKL5/STK9 gene has been shown to be responsible for a severe encephalopathy condition characterized by early onset of epilepsy and severe developmental delay. CDKL5 mutations have been shown to be more frequent among female patients. Results Here we report a 6- month male patient, second child of a healthy non consanguineous in the Iranian population. He has been affected by early onset epileptic refractory seizures and developmental delay. Whole-exome sequencing (WES has revealed a base substitution c.173T>A in CDKL5 gene, resulting in the formation of stop codon p.L58X. This mutation resides in the catalytic domain of the corresponding protein and is expected to result in premature RNA break down with no CDKL5 resulting protein. Conclusion   The present report highlights the importance of CDKL5 mutation analysis in male patients affected with early onset refractory epilepsy.

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

Science.gov (United States)

Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin-ichi

2014-10-08

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.

Transient expression of β-glucuronidase reporter gene in ...

African Journals Online (AJOL)

STORAGESEVER

2009-05-18

May 18, 2009 ... Therefore, special attention must be given to the use of genetic manipulation for .... of gus gene was used as a visual marker in transformation. Bacteria ... selective culture (SC) medium containing 200 mg/l carbenicillin and. 50 mg/l .... clones had caused the difference of host-plant response to bacterial ...

Novel mutations detected in avirulence genes overcoming tomato Cf resistance genes in isolates of a Japanese population of Cladosporium fulvum

NARCIS (Netherlands)

Iida, Y.; Hof, van 't P.M.J.; Beenen, H.G.; Mesarich, C.H.; Kubota, M.; Stergiopoulos, I.; Mehrabi, A.; Notsu, A.; Fujiwara, K.; Bahkali, A.; Abd-Elsalam, K.; Collemare, J.; Wit, de P.J.G.M.

2015-01-01

Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction

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

Science.gov (United States)

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

2016-02-01

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

Gene-wide analysis detects two new susceptibility genes for Alzheimer's disease.

Science.gov (United States)

Escott-Price, Valentina; Bellenguez, Céline; Wang, Li-San; Choi, Seung-Hoan; Harold, Denise; Jones, Lesley; Holmans, Peter; Gerrish, Amy; Vedernikov, Alexey; Richards, Alexander; DeStefano, Anita L; Lambert, Jean-Charles; Ibrahim-Verbaas, Carla A; Naj, Adam C; Sims, Rebecca; Jun, Gyungah; Bis, Joshua C; Beecham, Gary W; Grenier-Boley, Benjamin; Russo, Giancarlo; Thornton-Wells, Tricia A; Denning, Nicola; Smith, Albert V; Chouraki, Vincent; Thomas, Charlene; Ikram, M Arfan; Zelenika, Diana; Vardarajan, Badri N; Kamatani, Yoichiro; Lin, Chiao-Feng; Schmidt, Helena; Kunkle, Brian; Dunstan, Melanie L; Vronskaya, Maria; Johnson, Andrew D; Ruiz, Agustin; Bihoreau, Marie-Thérèse; Reitz, Christiane; Pasquier, Florence; Hollingworth, Paul; Hanon, Olivier; Fitzpatrick, Annette L; Buxbaum, Joseph D; Campion, Dominique; Crane, Paul K; Baldwin, Clinton; Becker, Tim; Gudnason, Vilmundur; Cruchaga, Carlos; Craig, David; Amin, Najaf; Berr, Claudine; Lopez, Oscar L; De Jager, Philip L; Deramecourt, Vincent; Johnston, Janet A; Evans, Denis; Lovestone, Simon; Letenneur, Luc; Hernández, Isabel; Rubinsztein, David C; Eiriksdottir, Gudny; Sleegers, Kristel; Goate, Alison M; Fiévet, Nathalie; Huentelman, Matthew J; Gill, Michael; Brown, Kristelle; Kamboh, M Ilyas; Keller, Lina; Barberger-Gateau, Pascale; McGuinness, Bernadette; Larson, Eric B; Myers, Amanda J; Dufouil, Carole; Todd, Stephen; Wallon, David; Love, Seth; Rogaeva, Ekaterina; Gallacher, John; George-Hyslop, Peter St; Clarimon, Jordi; Lleo, Alberto; Bayer, Anthony; Tsuang, Debby W; Yu, Lei; Tsolaki, Magda; Bossù, Paola; Spalletta, Gianfranco; Proitsi, Petra; Collinge, John; Sorbi, Sandro; Garcia, Florentino Sanchez; Fox, Nick C; Hardy, John; Naranjo, Maria Candida Deniz; Bosco, Paolo; Clarke, Robert; Brayne, Carol; Galimberti, Daniela; Scarpini, Elio; Bonuccelli, Ubaldo; Mancuso, Michelangelo; Siciliano, Gabriele; Moebus, Susanne; Mecocci, Patrizia; Zompo, Maria Del; Maier, Wolfgang; Hampel, Harald; Pilotto, Alberto; Frank-García, Ana; Panza, Francesco; Solfrizzi, Vincenzo; Caffarra, Paolo; Nacmias, Benedetta; Perry, William; Mayhaus, Manuel; Lannfelt, Lars; Hakonarson, Hakon; Pichler, Sabrina; Carrasquillo, Minerva M; Ingelsson, Martin; Beekly, Duane; Alvarez, Victoria; Zou, Fanggeng; Valladares, Otto; Younkin, Steven G; Coto, Eliecer; Hamilton-Nelson, Kara L; Gu, Wei; Razquin, Cristina; Pastor, Pau; Mateo, Ignacio; Owen, Michael J; Faber, Kelley M; Jonsson, Palmi V; Combarros, Onofre; O'Donovan, Michael C; Cantwell, Laura B; Soininen, Hilkka; Blacker, Deborah; Mead, Simon; Mosley, Thomas H; Bennett, David A; Harris, Tamara B; Fratiglioni, Laura; Holmes, Clive; de Bruijn, Renee F A G; Passmore, Peter; Montine, Thomas J; Bettens, Karolien; Rotter, Jerome I; Brice, Alexis; Morgan, Kevin; Foroud, Tatiana M; Kukull, Walter A; Hannequin, Didier; Powell, John F; Nalls, Michael A; Ritchie, Karen; Lunetta, Kathryn L; Kauwe, John S K; Boerwinkle, Eric; Riemenschneider, Matthias; Boada, Mercè; Hiltunen, Mikko; Martin, Eden R; Schmidt, Reinhold; Rujescu, Dan; Dartigues, Jean-François; Mayeux, Richard; Tzourio, Christophe; Hofman, Albert; Nöthen, Markus M; Graff, Caroline; Psaty, Bruce M; Haines, Jonathan L; Lathrop, Mark; Pericak-Vance, Margaret A; Launer, Lenore J; Van Broeckhoven, Christine; Farrer, Lindsay A; van Duijn, Cornelia M; Ramirez, Alfredo; Seshadri, Sudha; Schellenberg, Gerard D; Amouyel, Philippe; Williams, Julie

2014-01-01

Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls. In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10-6) and 14 (IGHV1-67 p = 7.9×10-8) which indexed novel susceptibility loci. The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.

Gene-wide analysis detects two new susceptibility genes for Alzheimer's disease.

Directory of Open Access Journals (Sweden)

Valentina Escott-Price

Full Text Available Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls.In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10-6 and 14 (IGHV1-67 p = 7.9×10-8 which indexed novel susceptibility loci.The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.

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

International Nuclear Information System (INIS)

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

2001-01-01

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

Genetic components for radiosensitivity. Gene expression in radiosensitive monocygotic twins. Final report

International Nuclear Information System (INIS)

Dikomey, Ekkehard

2012-01-01

The underlying hypothesis of this project was that the variation of individual radiosensitivity is determined by the different expression of single gens. This concept was tested using 60 monozygotic twin pairs, followed by an evaluation with 80 prostate cancer patients. Radiosensitivity was assessed for both G0- as well as G2-phase using chromosomal assays. G0- radiosensitivity is determined by lethal chromosomal aberrations and reflects the individual amount of cell killing, while G2-sensitivity is determined by chromatid breaks and is taken as an indicator of individual cancer risk. For both populations, G0- and G2-radiosensitivity are characterized by substantial variation with a CV of 11 and 14% or 27 and 21%, respectively. While the mean G0-sensitivity is the same for both populations, there is a slight difference for G2. The slightly higher value of G2-sensitivity found for prostate cancer patients might result from the higher age of this group. For both populations gene expression profiles were determined using the Affymetrix chip HG-U133+2.0. Overall gene expression was characterized by a huge variation covering more than four decades. However, for single genes, expression showed little variation with CV generally ranging only between 2 and 8%. Analysis of data using several different methods revealed that variation of both G0- as well as G2-radiosensitivity cannot be ascribed to the different expression of single genes. For twins, random forests can be used to identify 8 to 10 genes than are relevant either for G0- or G2-radiosensitivity. However, these genes cannot be confirmed by an evaluation with 80 prostate cancer patients. This finding clearly demonstrates that the hypothesis, due to which variation of individual radiosensitivity is caused by different expression of single genes, has to be rejected. It appears more likely that this parameter is determined by complex interactions of several genes in functional networks. (orig.)

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U. S. Environmental Protection Agency Gene-Tox Program

Energy Technology Data Exchange (ETDEWEB)

Zimmermann, F.K.; von Borstel, R.C.; von Halle, E.S.; Parry, J.M.; Siebert, D.; Zetterberg, G.; Barale, R.; Loprieno, N.

1984-01-01

This review article with over 200 references summarizes the results of mutation screening tests with 492 chemicals using saccharomyces cerevisiae as the test organism. In addition, an extensive description of S. cerevisiae as a test organism is given. Yeast can be used to study genetic effects both in mitotic and in meiotic cells because it can be cultured as a stable haploid or a stable diploid. The most commonly used genetic endpoint has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the gene-tox program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.

Evolutionary signatures amongst disease genes permit novel methods for gene prioritization and construction of informative gene-based networks.

Directory of Open Access Journals (Sweden)

Nolan Priedigkeit

2015-02-01

Full Text Available Genes involved in the same function tend to have similar evolutionary histories, in that their rates of evolution covary over time. This coevolutionary signature, termed Evolutionary Rate Covariation (ERC, is calculated using only gene sequences from a set of closely related species and has demonstrated potential as a computational tool for inferring functional relationships between genes. To further define applications of ERC, we first established that roughly 55% of genetic diseases posses an ERC signature between their contributing genes. At a false discovery rate of 5% we report 40 such diseases including cancers, developmental disorders and mitochondrial diseases. Given these coevolutionary signatures between disease genes, we then assessed ERC's ability to prioritize known disease genes out of a list of unrelated candidates. We found that in the presence of an ERC signature, the true disease gene is effectively prioritized to the top 6% of candidates on average. We then apply this strategy to a melanoma-associated region on chromosome 1 and identify MCL1 as a potential causative gene. Furthermore, to gain global insight into disease mechanisms, we used ERC to predict molecular connections between 310 nominally distinct diseases. The resulting "disease map" network associates several diseases with related pathogenic mechanisms and unveils many novel relationships between clinically distinct diseases, such as between Hirschsprung's disease and melanoma. Taken together, these results demonstrate the utility of molecular evolution as a gene discovery platform and show that evolutionary signatures can be used to build informative gene-based networks.

A fruit quality gene map of Prunus

Directory of Open Access Journals (Sweden)

Bliss Fredrick A

2009-12-01

Full Text Available Abstract Background Prunus fruit development, growth, ripening, and senescence includes major biochemical and sensory changes in texture, color, and flavor. The genetic dissection of these complex processes has important applications in crop improvement, to facilitate maximizing and maintaining stone fruit quality from production and processing through to marketing and consumption. Here we present an integrated fruit quality gene map of Prunus containing 133 genes putatively involved in the determination of fruit texture, pigmentation, flavor, and chilling injury resistance. Results A genetic linkage map of 211 markers was constructed for an intraspecific peach (Prunus persica progeny population, Pop-DG, derived from a canning peach cultivar 'Dr. Davis' and a fresh market cultivar 'Georgia Belle'. The Pop-DG map covered 818 cM of the peach genome and included three morphological markers, 11 ripening candidate genes, 13 cold-responsive genes, 21 novel EST-SSRs from the ChillPeach database, 58 previously reported SSRs, 40 RAFs, 23 SRAPs, 14 IMAs, and 28 accessory markers from candidate gene amplification. The Pop-DG map was co-linear with the Prunus reference T × E map, with 39 SSR markers in common to align the maps. A further 158 markers were bin-mapped to the reference map: 59 ripening candidate genes, 50 cold-responsive genes, and 50 novel EST-SSRs from ChillPeach, with deduced locations in Pop-DG via comparative mapping. Several candidate genes and EST-SSRs co-located with previously reported major trait loci and quantitative trait loci for chilling injury symptoms in Pop-DG. Conclusion The candidate gene approach combined with bin-mapping and availability of a community-recognized reference genetic map provides an efficient means of locating genes of interest in a target genome. We highlight the co-localization of fruit quality candidate genes with previously reported fruit quality QTLs. The fruit quality gene map developed here is a

Novel gene sets improve set-level classification of prokaryotic gene expression data.

Science.gov (United States)

Holec, Matěj; Kuželka, Ondřej; Železný, Filip

2015-10-28

Set-level classification of gene expression data has received significant attention recently. In this setting, high-dimensional vectors of features corresponding to genes are converted into lower-dimensional vectors of features corresponding to biologically interpretable gene sets. The dimensionality reduction brings the promise of a decreased risk of overfitting, potentially resulting in improved accuracy of the learned classifiers. However, recent empirical research has not confirmed this expectation. Here we hypothesize that the reported unfavorable classification results in the set-level framework were due to the adoption of unsuitable gene sets defined typically on the basis of the Gene ontology and the KEGG database of metabolic networks. We explore an alternative approach to defining gene sets, based on regulatory interactions, which we expect to collect genes with more correlated expression. We hypothesize that such more correlated gene sets will enable to learn more accurate classifiers. We define two families of gene sets using information on regulatory interactions, and evaluate them on phenotype-classification tasks using public prokaryotic gene expression data sets. From each of the two gene-set families, we first select the best-performing subtype. The two selected subtypes are then evaluated on independent (testing) data sets against state-of-the-art gene sets and against the conventional gene-level approach. The novel gene sets are indeed more correlated than the conventional ones, and lead to significantly more accurate classifiers. The novel gene sets are indeed more correlated than the conventional ones, and lead to significantly more accurate classifiers. Novel gene sets defined on the basis of regulatory interactions improve set-level classification of gene expression data. The experimental scripts and other material needed to reproduce the experiments are available at http://ida.felk.cvut.cz/novelgenesets.tar.gz.

Nance-Horan syndrome: a contiguous gene syndrome involving deletion of the amelogenin gene? A case report and molecular analysis.

Science.gov (United States)

Franco, E; Hodgson, S; Lench, N; Roberts, G J

1995-03-01

A case of Nance-Horan syndrome in a male is presented, with some features of the condition in his carrier mother and her mother. It is proposed that Nance-Horan syndrome might be a contiguous gene syndrome mapping to chromosome Xp21.2-p22.3. The proband had congenital cataract microphthalmia and dental abnormalities including screwdriver shaped incisors and evidence of enamel pitting hypoplasia. The region Xp21.2-p22.3 also contains the tooth enamel protein gene, amelogenin (AMGX). Using molecular genetic techniques, we have shown that there is no evidence that the AMGX gene is deleted in this case of the Nance-Horan syndrome.

In vitro reporter gene assays for assessment of PPAR- and Nrf2-mediated health effects of tomato and its bioactive constituents

NARCIS (Netherlands)

Gijsbers, L.

2013-01-01

The consumption of food products with health-promoting properties, such as for example margarines with plant sterols, fruit juice enriched with calcium and cereals with (soluble) fibre, has increased rapidly during the last years. The present thesis provides proof-of-principle that reporter gene

In Vivo Imaging of Retinoic Acid Receptor Activity using a Sodium/Iodide Symporter and Luciferase Dual Imaging Reporter Gene

Directory of Open Access Journals (Sweden)

Min Kyung So

2004-07-01

Full Text Available Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/NL. After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SK-RARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention.

Transient expression of β-glucuronidase reporter gene in ...

African Journals Online (AJOL)

Agrobacterium tumefaciens strain LBA4404 carrying the pBI.121 binary plasmid was used in transformation to introduce the gus (ß-glucuronidase/GUS) gene into teak shoot-tissues. In vitro regenerated shoots from various teak clones, i.e. the ITB, GT, P97, P96, P75, P20, and P108 clones were vacuum-infiltrated for 5 min in ...

Radiosensitivity and genes

Energy Technology Data Exchange (ETDEWEB)

Qiyue, Hu; Mingyue, Lun [Suzhou Medical Coll., JS (China)

1995-07-01

Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G{sub 1} phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM{sub 9} cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation.

Radiosensitivity and genes

International Nuclear Information System (INIS)

Hu Qiyue; Lun Mingyue

1995-07-01

Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G 1 phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM 9 cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation

Sea urchin neural alpha2 tubulin gene: isolation and promoter analysis.

Science.gov (United States)

Costa, S; Ragusa, M A; Drago, G; Casano, C; Alaimo, G; Guida, N; Gianguzza, F

2004-04-02

Expression of Talpha2 gene, during sea urchin Paracentrotus lividus development, is spatially and temporally regulated. In order to characterize this gene, we isolated the relevant genomic sequences and scanned the isolated 5'-flanking region in searching for cis-regulatory elements required for proper expression. Gel mobility shift and footprinting assays, as well as reporter gene (CAT and beta-gal) expression assays, were used to address cis-regulatory elements involved in regulation. Here we report that an upstream 5'-flanking fragment of PlTalpha2 gene drives temporal expression of reporter genes congruent with that of endogenous Talpha2 gene. The fragment contains cis-elements able to bind nuclear proteins from the gastrula stage (at which the Talpha2 gene is expressed) whose sequences could be consistent with the consensus sequences for transcription factors present in data bank.

A Gene Implicated in Activation of Retinoic Acid Receptor Targets Is a Novel Renal Agenesis Gene in Humans

DEFF Research Database (Denmark)

Brophy, Patrick D.; Rasmussen, Maria; Parida, Mrutyunjaya

2017-01-01

investigations have identified several gene variants that cause RA, including EYA1, LHX1, and WT1 However, whereas compound null mutations of genes encoding α and γ retinoic acid receptors (RARs) cause RA in mice, to date there have been no reports of variants in RAR genes causing RA in humans. In this study, we...... in humans....

Positron emission tomography and gene therapy: basic concepts and experimental approaches for in vivo gene expression imaging.

Science.gov (United States)

Peñuelas, Iván; Boán, JoséF; Martí-Climent, Josep M; Sangro, Bruno; Mazzolini, Guillermo; Prieto, Jesús; Richter, José A

2004-01-01

More than two decades of intense research have allowed gene therapy to move from the laboratory to the clinical setting, where its use for the treatment of human pathologies has been considerably increased in the last years. However, many crucial questions remain to be solved in this challenging field. In vivo imaging with positron emission tomography (PET) by combination of the appropriate PET reporter gene and PET reporter probe could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy. PET imaging could be used to define parameters not available by other techniques that are of substantial interest not only for the proper understanding of the gene therapy process, but also for its future development and clinical application in humans. This review focuses on the molecular biology basis of gene therapy and molecular imaging, describing the fundamentals of in vivo gene expression imaging by PET, and the application of PET to gene therapy, as a technology that can be used in many different ways. It could be applied to avoid invasive procedures for gene therapy monitoring; accurately diagnose the pathology for better planning of the most adequate therapeutic approach; as treatment evaluation to image the functional effects of gene therapy at the biochemical level; as a quantitative noninvasive way to monitor the location, magnitude and persistence of gene expression over time; and would also help to a better understanding of vector biology and pharmacology devoted to the development of safer and more efficient vectors.

Homozygous germ-line mutation of the PMS2 mismatch repair gene: a unique case report of constitutional mismatch repair deficiency (CMMRD)

OpenAIRE

Ramchander, N. C.; Ryan, N. A. J.; Crosbie, E. J.; Evans, D. G.

2017-01-01

BackgroundConstitutional mismatch repair deficiency syndrome results from bi-allelic inheritance of mutations affecting the key DNA mismatch repair genes: MLH1, MSH2, MSH6 or PMS2. Individuals with bi-allelic mutations have a dysfunctional mismatch repair system from birth; as a result, constitutional mismatch repair deficiency syndrome is characterised by early onset malignancies. Fewer than 150 cases have been reported in the literature over the past 20 years. This is the first report of th...

Report of space experiment project, 'Rad Gene', performed in the International Space Station Kibo

International Nuclear Information System (INIS)

Ohnishi, Takeo; Takahashi, Akihisa; Nagamatsu, Aiko

2010-01-01

This report summarizes results of the project in the title adopted by Japan Aerospace Exploration Agency (JAXA) (in 2000) aiming to elucidate the biological effect of space environment, and contains 3 major parts of the process of the experiment, and of findings by analysis after flight and in radioadaptive response. The process for the experiment includes training of the experimenter crew (Dr. S. Magnus) in JAXA, preparation of samples (frozen cells with normal and mutated p53 genes derived from human lymphoblast TK6) and their transfer to the Space Shuttle Endeavour STS-126 launched on Nov. 15, 2008 (Japanese time) for cell culturing in Feb., 2009. Analyses after flight back to the Kennedy Space Center on Mar. 29, 2009, done on the ground in Japan thereafter include the physical evaluation, confirmation of DNA damage, and phenotypic expression with DNA- and protein-arrays (genes induced for expression of p53-related phenotypes in those cells which were stored frozen in the space, thawed on the ground and then cultured, genes induced for expressing the phenotypes and p53-related proteins expressed in cells cultured in space). Physically, total absorbed dose and dose equivalent are found to be respectively 43.5 mGy and 71.2 mSv (0.5 mSv/day). Interestingly, the biologically estimated dose by DNA-double strand breaks detected by γH2AX staining, 94.5 mSv (0.7 mSv/day), in living, frozen cells in space, is close to the above physical dose. Expression experiments of p53-related phenotypes have revealed that expression of 750 or more genes in 41,000 genes in the array is changed: enhanced or suppressed by space radiation, micro-gravity and/or their mixed effects in space environment. In 642 protein antibodies in the array, 2 proteins are found enhanced and 8, suppressed whereas heat-shock protein is unchanged. Radioadaptive response is the acquisition of radio-resistance to acute exposure by previous irradiation of small dose (window width 20-100 mSv) in normal p53

Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry

NARCIS (Netherlands)

Jonker, W.; Lamoree, M.H.; Houtman, C.J.; Hamers, T.; Somsen, G.W.; Kool, J.

2015-01-01

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was

Discovering genes underlying QTL

Energy Technology Data Exchange (ETDEWEB)

Vanavichit, Apichart [Kasetsart University, Kamphaengsaen, Nakorn Pathom (Thailand)

2002-02-01

A map-based approach has allowed scientists to discover few genes at a time. In addition, the reproductive barrier between cultivated rice and wild relatives has prevented us from utilizing the germ plasm by a map-based approach. Most genetic traits important to agriculture or human diseases are manifested as observable, quantitative phenotypes called Quantitative Trait Loci (QTL). In many instances, the complexity of the phenotype/genotype interaction and the general lack of clearly identifiable gene products render the direct molecular cloning approach ineffective, thus additional strategies like genome mapping are required to identify the QTL in question. Genome mapping requires no prior knowledge of the gene function, but utilizes statistical methods to identify the most likely gene location. To completely characterize genes of interest, the initially mapped region of a gene location will have to be narrowed down to a size that is suitable for cloning and sequencing. Strategies for gene identification within the critical region have to be applied after the sequencing of a potentially large clone or set of clones that contains this gene(s). Tremendous success of positional cloning has been shown for cloning many genes responsible for human diseases, including cystic fibrosis and muscular dystrophy as well as plant disease resistance genes. Genome and QTL mapping, positional cloning: the pre-genomics era, comparative approaches to gene identification, and positional cloning: the genomics era are discussed in the report. (M. Suetake)

A case report: Becker muscular dystrophy presenting with epilepsy and dysgnosia induced by duplication mutation of Dystrophin gene.

Science.gov (United States)

Miao, Jing; Feng, Jia-Chun; Zhu, Dan; Yu, Xue-Fan

2016-12-12

Becker muscular dystrophy (BMD), a genetic disorder of X-linked recessive inheritance, typically presents with gradually progressive muscle weakness. The condition is caused by mutations of Dystrophin gene located at Xp21.2. Epilepsy is an infrequent manifestation of BMD, while cases of BMD with dysgnosia are extremely rare. We describe a 9-year-old boy with BMD, who presented with epilepsy and dysgnosia. Serum creatine kinase level was markedly elevated (3665 U/L). Wechsler intelligence tests showed a low intelligence quotient (IQ = 65). Electromyogram showed slight myogenic changes and skeletal muscle biopsy revealed muscular dystrophy. Immunohistochemical staining showed partial positivity of sarcolemma for dystrophin-N. Multiplex ligation-dependent probe amplification revealed a duplication mutation in exons 37-44 in the Dystrophin gene. The present case report helps to better understand the clinical and genetic features of BMD.

Development of a codon optimization strategy using the efor RED reporter gene as a test case

Science.gov (United States)

Yip, Chee-Hoo; Yarkoni, Orr; Ajioka, James; Wan, Kiew-Lian; Nathan, Sheila

2018-04-01

Synthetic biology is a platform that enables high-level synthesis of useful products such as pharmaceutically related drugs, bioplastics and green fuels from synthetic DNA constructs. Large-scale expression of these products can be achieved in an industrial compliant host such as Escherichia coli. To maximise the production of recombinant proteins in a heterologous host, the genes of interest are usually codon optimized based on the codon usage of the host. However, the bioinformatics freeware available for standard codon optimization might not be ideal in determining the best sequence for the synthesis of synthetic DNA. Synthesis of incorrect sequences can prove to be a costly error and to avoid this, a codon optimization strategy was developed based on the E. coli codon usage using the efor RED reporter gene as a test case. This strategy replaces codons encoding for serine, leucine, proline and threonine with the most frequently used codons in E. coli. Furthermore, codons encoding for valine and glycine are substituted with the second highly used codons in E. coli. Both the optimized and original efor RED genes were ligated to the pJS209 plasmid backbone using Gibson Assembly and the recombinant DNAs were transformed into E. coli E. cloni 10G strain. The fluorescence intensity per cell density of the optimized sequence was improved by 20% compared to the original sequence. Hence, the developed codon optimization strategy is proposed when designing an optimal sequence for heterologous protein production in E. coli.

Molecular Study of Three Lebanese and Syrian Patients with Waardenburg Syndrome and Report of Novel Mutations in the EDNRB and MITF Genes

Science.gov (United States)

Haddad, N.M.; Ente, D.; Chouery, E.; Jalkh, N.; Mehawej, C.; Khoueir, Z.; Pingault, V.; Mégarbané, A.

2011-01-01

Waardenburg syndrome (WS) is a genetic disorder characterized primarily by depigmentation of the skin and hair, heterochromia of the irides, sensorineural deafness, and sometimes by dystopia canthorum, and Hirschsprung disease. WS presents a large clinical and genetic heterogeneity. Four different types have been individualized and linked to 5 different genes. We report 2 cases of WS type II and 1 case of WS type IV from Lebanon and Syria. The genetic studies revealed 2 novel mutations in the MITF gene of the WS type II cases and 1 novel homozygous mutation in the EDNRB gene of the WS type IV case. This is the first molecular study of patients from the Arab world. Additional cases will enable a more detailed description of the clinical spectrum of Waardenburg syndrome in this region. PMID:21373256

Molecular Study of Three Lebanese and Syrian Patients with Waardenburg Syndrome and Report of Novel Mutations in the EDNRB and MITF Genes.

Science.gov (United States)

Haddad, N M; Ente, D; Chouery, E; Jalkh, N; Mehawej, C; Khoueir, Z; Pingault, V; Mégarbané, A

2011-01-01

Waardenburg syndrome (WS) is a genetic disorder characterized primarily by depigmentation of the skin and hair, heterochromia of the irides, sensorineural deafness, and sometimes by dystopia canthorum, and Hirschsprung disease. WS presents a large clinical and genetic heterogeneity. Four different types have been individualized and linked to 5 different genes. We report 2 cases of WS type II and 1 case of WS type IV from Lebanon and Syria. The genetic studies revealed 2 novel mutations in the MITF gene of the WS type II cases and 1 novel homozygous mutation in the EDNRB gene of the WS type IV case. This is the first molecular study of patients from the Arab world. Additional cases will enable a more detailed description of the clinical spectrum of Waardenburg syndrome in this region.

Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

DEFF Research Database (Denmark)

Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

1999-01-01

Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrog......Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential...... antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were...... calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands...

The Medicago truncatula lysine motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes

NARCIS (Netherlands)

Arrighi, J.F.; Barre, A.; Amor, Ben B.; Bersoult, A.; Campos Soriano, L.; Mirabella, R.; Carvalho-Niebel, de F.; Journet, E.P.; Ghérardi, M.; Huguet, T.; Geurts, R.; Dénarié, J.; Rougé, P.; Gough, C.

2006-01-01

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide

Behavioral science and the study of gene-nutrition and gene-physical activity interactions in obesity research.

Science.gov (United States)

Faith, Myles S

2008-12-01

This report summarizes emerging opportunities for behavioral science to help advance the field of gene-environment and gene-behavior interactions, based on presentations at The National Cancer Institute (NCI) Workshop, "Gene-Nutrition and Gene-Physical Activity Interactions in the Etiology of Obesity." Three opportunities are highlighted: (i) designing potent behavioral "challenges" in experiments, (ii) determining viable behavioral phenotypes for genetics studies, and (iii) identifying specific measures of the environment or environmental exposures. Additional points are underscored, including the need to incorporate novel findings from neuroimaging studies regarding motivation and drive for eating and physical activity. Advances in behavioral science theory and methods can play an important role in advancing understanding of gene-brain-behavior relationships in obesity onset.

Cloning and Expression of Three New Azotobacter vinelandii Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

OpenAIRE

Svanem, Britt Iren Glærum; Skjåk-Bræk, Gudmund; Ertesvåg, Helga; Valla, Svein

1999-01-01

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of β-d-mannuronic acid to α-l-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced...

Using riboswitches to regulate gene expression and define gene function in mycobacteria.

Science.gov (United States)

Van Vlack, Erik R; Seeliger, Jessica C

2015-01-01

Mycobacteria include both environmental species and many pathogenic species such as Mycobacterium tuberculosis, an intracellular pathogen that is the causative agent of tuberculosis in humans. Inducible gene expression is a powerful tool for examining gene function and essentiality, both in in vitro culture and in host cell infections. The theophylline-inducible artificial riboswitch has recently emerged as an alternative to protein repressor-based systems. The riboswitch is translationally regulated and is combined with a mycobacterial promoter that provides transcriptional control. We here provide methods used by our laboratory to characterize the riboswitch response to theophylline in reporter strains, recombinant organisms containing riboswitch-regulated endogenous genes, and in host cell infections. These protocols should facilitate the application of both existing and novel artificial riboswitches to the exploration of gene function in mycobacteria. © 2015 Elsevier Inc. All rights reserved.

THE USE OF gusA REPORTER GENE TO MONITOR THE SURVIVAL OF INTRODUCED BACTERIA IN THE SOIL

Directory of Open Access Journals (Sweden)

Edi Husen

2013-07-01

Full Text Available An effective marker to monitor the survival of introduced bacteria in the soil is required for further evaluation of their beneficial effects on plant growth. This study tested the use of gusA gene as a marker to trace the fate of three Gram negative bacteria in the root, rhizosphere, and soil. The study was conducted at the laboratory and greenhouse of the National Institute of Molecular Biology and Biotechnology, Philippines from January to December 2001. Isolates TCaR 61 and TCeRe 60, and Azotobacter vinelandii Mac 259 were selected as test bacteria based on their ability to produce indole-3acetic acid and solubilize precipitated phosphate, which may promote plant growth in the field. These bacteria were marked with gusA reporter gene from Escherichia coli strain S17-1(λ-pir containing mTn5SSgusA21. The gusA (β-glucuronidase gene from the donor (E. coli was transferred to each bacterium (recipient through bacterial conjugation in mating procedures using tryptone-yeast agar followed by the selection of the transconjugants (bacteria receiving gusA in tryptone-yeast agar supplemented with double antibiotics and X-GlcA (5bromo-4chloro- 3indoxyl-β-D-glucuronic acid. The antibiotics used were rifampicin and either streptomycin or spectinomycin based on antibiotic profiles of the donor and recipients. The results showed that the insertion of gusA gene into bacterial genomes of the recipient did not impair its phenotypic traits; the growth rates of the transconjugants as well as their ability to produce indole-3acetic acid and solubilize precipitated phosphate in pure culture were similar to their wild types. All transconjugants colonized the roots of hot pepper (Capsicum annuum L. and survived in the rhizosphere and soil until the late of vegetative growth stage. The distinct blue staining of transconjugants as the expression of gusA gene in media containing X-GlcA coupled with their resistance to rifampicin and streptomycin or spectinomycin

Radiopharmaceuticals to monitor gene transfer

International Nuclear Information System (INIS)

Wiebe, L. I.; Morin, K. W.; Knaus, E. E.

1997-01-01

Advances in genetic engineering and molecular biology have opened the door to disease treatment by transferring genes to cells that are responsible for the pathological condition being addressed. These genes can serve to supplement or introduce the function of indigenous genes that are either inadequately expressed or that are congenitally absent in the patient. They can introduce new functions such as drug sensitization to provide a unique therapeutic target. Gene transfer is readily monitored in vitro using a range of histochemical and biochemical tests that are ''built in'' to the therapeutic gene cassette. In vivo, in situ monitoring of the gene transfer and gene expression processes can be achieved with these tests only if biopsy is possible. Scintigraphic imaging can offer unique information on both the extent and location of gene expression, provided that an appropriate reporter gene is included in the therapeutic cassette. This overview includes a brief orientation to gene transfer therapy and is followed by a review of current approaches to gene therapy imaging. The concluding section deals with imaging based on radiolabelled nucleoside substrates for herpes simplex type-1 thymidine kinase, with emphasis on IVFRU, a stable potent and selective HSV-1 TK substrate developed in their laboratories

Relaxation rates of gene expression kinetics reveal the feedback signs of autoregulatory gene networks

Science.gov (United States)

Jia, Chen; Qian, Hong; Chen, Min; Zhang, Michael Q.

2018-03-01

The transient response to a stimulus and subsequent recovery to a steady state are the fundamental characteristics of a living organism. Here we study the relaxation kinetics of autoregulatory gene networks based on the chemical master equation model of single-cell stochastic gene expression with nonlinear feedback regulation. We report a novel relation between the rate of relaxation, characterized by the spectral gap of the Markov model, and the feedback sign of the underlying gene circuit. When a network has no feedback, the relaxation rate is exactly the decaying rate of the protein. We further show that positive feedback always slows down the relaxation kinetics while negative feedback always speeds it up. Numerical simulations demonstrate that this relation provides a possible method to infer the feedback topology of autoregulatory gene networks by using time-series data of gene expression.

Function analysis of unknown genes

DEFF Research Database (Denmark)

Rogowska-Wrzesinska, A.

2002-01-01

  This thesis entitled "Function analysis of unknown genes" presents the use of proteome analysis for the characterisation of yeast (Saccharomyces cerevisiae) genes and their products (proteins especially those of unknown function). This study illustrates that proteome analysis can be used...... to describe different aspects of molecular biology of the cell, to study changes that occur in the cell due to overexpression or deletion of a gene and to identify various protein modifications. The biological questions and the results of the described studies show the diversity of the information that can...... genes and proteins. It reports the first global proteome database collecting 36 yeast single gene deletion mutants and selecting over 650 differences between analysed mutants and the wild type strain. The obtained results show that two-dimensional gel electrophoresis and mass spectrometry based proteome...

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

Science.gov (United States)

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

2010-02-01

Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer ((123)I(-), (124)I(-), (99m)TcO4(-)), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells.

Gene-Environment Interaction in Parkinson's Disease

DEFF Research Database (Denmark)

Chuang, Yu-Hsuan; Lill, Christina M; Lee, Pei-Chen

2016-01-01

BACKGROUND AND PURPOSE: Drinking caffeinated coffee has been reported to provide protection against Parkinson's disease (PD). Caffeine is an adenosine A2A receptor (encoded by the gene ADORA2A) antagonist that increases dopaminergic neurotransmission and Cytochrome P450 1A2 (gene: CYP1A2...

A Transgenic Tri-Modality Reporter Mouse

OpenAIRE

Yan, Xinrui; Ray, Pritha; Paulmurugan, Ramasamy; Tong, Ricky; Gong, Yongquan; Sathirachinda, Ataya; Wu, Joseph C.; Gambhir, Sanjiv S.

2013-01-01

Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent...

Correction of gene expression data

DEFF Research Database (Denmark)

Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin

2014-01-01

This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

Application of HSVtk suicide gene to X-SCID gene therapy: Ganciclovir treatment offsets gene corrected X-SCID B cells

International Nuclear Information System (INIS)

Uchiyama, Toru; Kumaki, Satoru; Ishikawa, Yoshinori; Onodera, Masafumi; Sato, Miki; Du, Wei; Sasahara, Yoji; Tanaka, Nobuyuki; Sugamura, Kazuo; Tsuchiya, Shigeru

2006-01-01

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human γc chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the γc chain, the cells were treated with ganciclovir (GCV). The γc chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the γc chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial

Delimiting Coalescence Genes (C-Genes) in Phylogenomic Data Sets.

Science.gov (United States)

Springer, Mark S; Gatesy, John

2018-02-26

coalescence methods have emerged as a popular alternative for inferring species trees with large genomic datasets, because these methods explicitly account for incomplete lineage sorting. However, statistical consistency of summary coalescence methods is not guaranteed unless several model assumptions are true, including the critical assumption that recombination occurs freely among but not within coalescence genes (c-genes), which are the fundamental units of analysis for these methods. Each c-gene has a single branching history, and large sets of these independent gene histories should be the input for genome-scale coalescence estimates of phylogeny. By contrast, numerous studies have reported the results of coalescence analyses in which complete protein-coding sequences are treated as c-genes even though exons for these loci can span more than a megabase of DNA. Empirical estimates of recombination breakpoints suggest that c-genes may be much shorter, especially when large clades with many species are the focus of analysis. Although this idea has been challenged recently in the literature, the inverse relationship between c-gene size and increased taxon sampling in a dataset-the 'recombination ratchet'-is a fundamental property of c-genes. For taxonomic groups characterized by genes with long intron sequences, complete protein-coding sequences are likely not valid c-genes and are inappropriate units of analysis for summary coalescence methods unless they occur in recombination deserts that are devoid of incomplete lineage sorting (ILS). Finally, it has been argued that coalescence methods are robust when the no-recombination within loci assumption is violated, but recombination must matter at some scale because ILS, a by-product of recombination, is the raison d'etre for coalescence methods. That is, extensive recombination is required to yield the large number of independently segregating c-genes used to infer a species tree. If coalescent methods are powerful

Isolation and identification of differentially expressed genes between ...

African Journals Online (AJOL)

Plants have evolved sophisticated molecular defense mechanisms in order to survive disease conditions. So far, a number of pathogen resistance (R) genes have been reported in plants. These R genes are thought to be involved in activating the signals that lead to disease resistance. The structural specificity of R genes ...

Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish.

Directory of Open Access Journals (Sweden)

Wanlada Klangnurak

Full Text Available We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm, were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

Case reports of juvenile GM1 gangliosidosisis type II caused by mutation in GLB1 gene.

Science.gov (United States)

Karimzadeh, Parvaneh; Naderi, Samaneh; Modarresi, Farzaneh; Dastsooz, Hassan; Nemati, Hamid; Farokhashtiani, Tayebeh; Shamsian, Bibi Shahin; Inaloo, Soroor; Faghihi, Mohammad Ali

2017-07-17

Type II or juvenile GM1-gangliosidosis is an autosomal recessive lysosomal storage disorder, which is clinically distinct from infantile form of the disease by the lack of characteristic cherry-red spot and hepatosplenomegaly. The disease is characterized by slowly progressive neurodegeneration and mild skeletal changes. Due to the later age of onset and uncharacteristic presentation, diagnosis is frequently puzzled with other ataxic and purely neurological disorders. Up to now, 3-4 types of GM1-gangliosidosis have been reported and among them type I is the most common phenotype with the age of onset around 6 months. Various forms of GM1-gangliosidosis are caused by GLB1 gene mutations but severity of the disease and age of onset are directly related to the position and the nature of deleterious mutations. However, due to its unique genetic cause and overlapping clinical features, some researchers believe that GM1 gangliosidosis represents an overlapped disease spectrum instead of four distinct types. Here, we report a less frequent type of autosomal recessive GM1 gangliosidosis with perplexing clinical presentation in three families in the southwest part of Iran, who are unrelated but all from "Lurs" ethnic background. To identify disease-causing mutations, Whole Exome Sequencing (WES) utilizing next generation sequencing was performed. Four patients from three families were investigated with the age of onset around 3 years old. Clinical presentations were ataxia, gate disturbances and dystonia leading to wheelchair-dependent disability, regression of intellectual abilities, and general developmental regression. They all were born in consanguineous families with no previous documented similar disease in their parents. A homozygote missense mutation in GLB1 gene (c. 601 G > A, p.R201C) was found in all patients. Using Sanger sequencing this identified mutation was confirmed in the proband, their parents, grandparents, and extended family members, confirming

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

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Orozco Javier I

2011-10-01

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

Why commercialization of gene therapy stalled; examining the life cycles of gene therapy technologies.

Science.gov (United States)

Ledley, F D; McNamee, L M; Uzdil, V; Morgan, I W

2014-02-01

This report examines the commercialization of gene therapy in the context of innovation theories that posit a relationship between the maturation of a technology through its life cycle and prospects for successful product development. We show that the field of gene therapy has matured steadily since the 1980s, with the congruent accumulation of >35 000 papers, >16 000 US patents, >1800 clinical trials and >$4.3 billion in capital investment in gene therapy companies. Gene therapy technologies comprise a series of dissimilar approaches for gene delivery, each of which has introduced a distinct product architecture. Using bibliometric methods, we quantify the maturation of each technology through a characteristic life cycle S-curve, from a Nascent stage, through a Growing stage of exponential advance, toward an Established stage and projected limit. Capital investment in gene therapy is shown to have occurred predominantly in Nascent stage technologies and to be negatively correlated with maturity. Gene therapy technologies are now achieving the level of maturity that innovation research and biotechnology experience suggest may be requisite for efficient product development. Asynchrony between the maturation of gene therapy technologies and capital investment in development-focused business models may have stalled the commercialization of gene therapy.

Congenital Hypothyroidism Caused by a PAX8 Gene Mutation Manifested as Sodium/Iodide Symporter Gene Defect

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Wakako Jo

2010-01-01

Full Text Available Loss-of-function mutations of the PAX8 gene are considered to mainly cause congenital hypothyroidism (CH due to thyroid hypoplasia. However, some patients with PAX8 mutation have demonstrated a normal-sized thyroid gland. Here we report a CH patient caused by a PAX8 mutation, which manifested as iodide transport defect (ITD. Hypothyroidism was detected by neonatal screening and L-thyroxine replacement was started immediately. Although 123I scintigraphy at 5 years of age showed that the thyroid gland was in the normal position and of small size, his iodide trapping was low. The ratio of the saliva/plasma radioactive iodide was low. He did not have goiter; however laboratory findings suggested that he had partial ITD. Gene analyses showed that the sodium/iodide symporter (NIS gene was normal; instead, a mutation in the PAX8 gene causing R31H substitution was identified. The present report demonstrates that individuals with defective PAX8 can have partial ITD, and thus genetic analysis is useful for differential diagnosis.

Gene therapy for ocular diseases.

Science.gov (United States)

Liu, Melissa M; Tuo, Jingsheng; Chan, Chi-Chao

2011-05-01

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.

Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes

Science.gov (United States)

2010-01-01

Background Horizontal gene transfer (HGT) is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants. Results In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR) survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR) were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT)-PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes. Conclusions This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native mitochondrial copies suggests

Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes

Directory of Open Access Journals (Sweden)

Hao Weilong

2010-12-01

Full Text Available Abstract Background Horizontal gene transfer (HGT is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants. Results In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT-PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes. Conclusions This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

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Zhimin Dai

Full Text Available Biological nitrogen fixation is an essential function of acid mine drainage (AMD microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

Science.gov (United States)

Dai, Zhimin; Guo, Xue; Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

2014-01-01

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

Science.gov (United States)

Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

2014-01-01

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community. PMID:24498417

Gene mining a marama bean expressed sequence tags (ESTs ...

African Journals Online (AJOL)

The authors reported the identification of genes associated with embryonic development and microsatellite sequences. The future direction will entail characterization of these genes using gene over-expression and mutant assays. Key words: Namibia, simple sequence repeats (SSR), data mining, homology searches, ...

The barley Jip23b gene

DEFF Research Database (Denmark)

Müller-Uri, Frieder; Cameron-Mills, Verena; Mundy, John

2002-01-01

The barley gene (Jip23) encoding a 23,000-Da protein of unknown function was isolated and shown to be induced by jasmonate methyl ester (MeJA) in leaves. 5'upstream Jip23 sequence was isolated and fused to the beta-glucuronidase gene (GUS), and this reporter was introduced by particle bombardment...

A new type of gene-disruption cassette with a rescue gene for Pichia pastoris.

Science.gov (United States)

Shibui, Tatsuro; Hara, Hiroyoshi

2017-09-01

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017. © 2017 American Institute of Chemical Engineers.

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

NARCIS (Netherlands)

Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.

2006-01-01

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

NARCIS (Netherlands)

Boerboom, A.M.J.F.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.

2006-01-01

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

DEFF Research Database (Denmark)

Choi, Kyeong Rok; Cho, Jae Sung; Cho, In Jin

2018-01-01

Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable...... plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest....

Stochastic fluctuations and distributed control of gene expression impact cellular memory.

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Guillaume Corre

Full Text Available Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.

beta. -Amyloid gene dosage in Alzheimer's disease

Energy Technology Data Exchange (ETDEWEB)

Murdoch, G H; Manuelidis, L; Kim, J H; Manuelidis, E E

1988-01-11

The 4-5 kd amyloid ..beta..-peptide is a major constituent of the characteristic amyloid plaque of Alzheimer's disease. It has been reported that some cases of sporatic Alzheimer's disease are associated with at least a partial duplication of chromosome 21 containing the gene corresponding to the 695 residue precursor of this peptide. To contribute to an understanding of the frequency to such a duplication event in the overall Alzheimer's population, the authors have determined the gene dosage of the ..beta..-amyloid gene in this collection of cases. All cases had a clinical diagnosis of Alzheimer's confirmed neuropathologically. Each Alzheimer's case had an apparent normal diploid ..beta..-amyloid gene dosage, while control Down's cases had the expected triploid dosage. Thus partial duplication of chromosome 21 may be a rare finding in Alzheimer's disease. Similar conclusions were just reported in several studies of the Harvard Alzheimer collection.

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

Science.gov (United States)

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

2009-01-01

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

A Novel Missense Mutation of the NSD1 Gene Associated with Overgrowth in Three Generations of an Italian Family: Case Report, Differential Diagnosis, and Review of Mutations of NSD1 Gene in Familial Sotos Syndrome

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Gianluigi Laccetta

2017-11-01

Full Text Available Sotos syndrome (SoS is characterized by overgrowth of prenatal onset, learning disability, and characteristic facial appearance; it is usually due to haploinsufficiency of NSD1 gene at chromosome 5q35. An Italian child was born at 37 weeks of gestation (weight 2,910 g, 25th–50th centiles; length 50 cm, 75th centile; head circumference 36 cm, 97th centile showing cryptorchidism on the right side, hypertelorism, dolichocephaly, broad and prominent forehead, and narrow jaw; the pregnancy was worsened by maternal preeclampsia and gestational diabetes, and his mother had a previous history of four early miscarriages. The patient showed neonatal jaundice, hypotonia, feeding difficulties, frequent vomiting, and gastroesophageal reflux. After the age of 6 months, his weight, length, and head circumference were above the 97th centile; psychomotor development was delayed. At the age of 9 years, the patient showed also joint laxity and scoliosis. DNA sequence analysis of NSD1 gene detected a novel heterozygous mutation (c.521T>A, p.Val174Asp in exon 2. The same mutant allele was also found in the mother and in the maternal grandfather of the proband; both the mother and the maternal grandfather of the proband showed isolated overgrowth with height above the 97th centile in absence of other features of SoS. At present 23 familial cases of SoS have been described (two cases with mutation in exon 2 of NSD1 gene; no familial cases of SoS with mutation of NSD1 gene and isolated overgrowth have been reported. Probably, point mutations of NSD1 gene, and particularly mutations between exon 20 and exon 23, are not likely to affect reproductive fitness. Epigenetic mechanisms and intrauterine environment may influence phenotypes, therefore genetic tests are not useful to predict the phenotype but they are indispensable for the diagnosis of SoS. This is the first Italian familial case of SoS with genetic confirmation and the third report in which a

Mining tissue specificity, gene connectivity and disease association to reveal a set of genes that modify the action of disease causing genes

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Reverter Antonio

2008-09-01

Full Text Available Abstract Background The tissue specificity of gene expression has been linked to a number of significant outcomes including level of expression, and differential rates of polymorphism, evolution and disease association. Recent studies have also shown the importance of exploring differential gene connectivity and sequence conservation in the identification of disease-associated genes. However, no study relates gene interactions with tissue specificity and disease association. Methods We adopted an a priori approach making as few assumptions as possible to analyse the interplay among gene-gene interactions with tissue specificity and its subsequent likelihood of association with disease. We mined three large datasets comprising expression data drawn from massively parallel signature sequencing across 32 tissues, describing a set of 55,606 true positive interactions for 7,197 genes, and microarray expression results generated during the profiling of systemic inflammation, from which 126,543 interactions among 7,090 genes were reported. Results Amongst the myriad of complex relationships identified between expression, disease, connectivity and tissue specificity, some interesting patterns emerged. These include elevated rates of expression and network connectivity in housekeeping and disease-associated tissue-specific genes. We found that disease-associated genes are more likely to show tissue specific expression and most frequently interact with other disease genes. Using the thresholds defined in these observations, we develop a guilt-by-association algorithm and discover a group of 112 non-disease annotated genes that predominantly interact with disease-associated genes, impacting on disease outcomes. Conclusion We conclude that parameters such as tissue specificity and network connectivity can be used in combination to identify a group of genes, not previously confirmed as disease causing, that are involved in interactions with disease causing

Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.

Science.gov (United States)

Homma, Kohei; Usui, Sumiko; Kaneda, Makoto

2017-03-01

Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Methylated genes as new cancer biomarkers

DEFF Research Database (Denmark)

Brunner, Nils; Duffy, M.J; Napieralski, R.

2009-01-01

Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that meas......Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested...... that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2...... for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene...

Organization and evolution of the rat tyrosine hydroxylase gene

International Nuclear Information System (INIS)

Brown, E.R.; Coker, G.T. III; O'Malley, K.L.

1987-01-01

This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylases genes are members of a gene family which has a common evolutionary origin. The authors predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family

A Strategy for Identifying Quantitative Trait Genes Using Gene Expression Analysis and Causal Analysis

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Akira Ishikawa

2017-11-01

Full Text Available Large numbers of quantitative trait loci (QTL affecting complex diseases and other quantitative traits have been reported in humans and model animals. However, the genetic architecture of these traits remains elusive due to the difficulty in identifying causal quantitative trait genes (QTGs for common QTL with relatively small phenotypic effects. A traditional strategy based on techniques such as positional cloning does not always enable identification of a single candidate gene for a QTL of interest because it is difficult to narrow down a target genomic interval of the QTL to a very small interval harboring only one gene. A combination of gene expression analysis and statistical causal analysis can greatly reduce the number of candidate genes. This integrated approach provides causal evidence that one of the candidate genes is a putative QTG for the QTL. Using this approach, I have recently succeeded in identifying a single putative QTG for resistance to obesity in mice. Here, I outline the integration approach and discuss its usefulness using my studies as an example.

A Strategy for Identifying Quantitative Trait Genes Using Gene Expression Analysis and Causal Analysis.

Science.gov (United States)

Ishikawa, Akira

2017-11-27

Large numbers of quantitative trait loci (QTL) affecting complex diseases and other quantitative traits have been reported in humans and model animals. However, the genetic architecture of these traits remains elusive due to the difficulty in identifying causal quantitative trait genes (QTGs) for common QTL with relatively small phenotypic effects. A traditional strategy based on techniques such as positional cloning does not always enable identification of a single candidate gene for a QTL of interest because it is difficult to narrow down a target genomic interval of the QTL to a very small interval harboring only one gene. A combination of gene expression analysis and statistical causal analysis can greatly reduce the number of candidate genes. This integrated approach provides causal evidence that one of the candidate genes is a putative QTG for the QTL. Using this approach, I have recently succeeded in identifying a single putative QTG for resistance to obesity in mice. Here, I outline the integration approach and discuss its usefulness using my studies as an example.

Spectrum of CFTR gene mutations in Ecuadorian cystic fibrosis patients: the second report of the p.H609R mutation.

Science.gov (United States)

Ortiz, Sofía C; Aguirre, Santiago J; Flores, Sofía; Maldonado, Claudio; Mejía, Juan; Salinas, Lilian

2017-11-01

High heterogeneity in the CFTR gene mutations disturbs the molecular diagnosis of cystic fibrosis (CF). In order to improve the diagnosis of CF in our country, the present study aims to define a panel of common CFTR gene mutations by sequencing 27 exons of the gene in Ecuadorian Cystic Fibrosis patients. Forty-eight Ecuadorian individuals with suspected/confirmed CF diagnosis were included. Twenty-seven exons of CFTR gene were sequenced to find sequence variations. Prevalence of pathogenic variations were determined and compared with other countries' data. We found 70 sequence variations. Eight of these are CF-causing mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. Also this study is the second report of p.H609R in Ecuadorian population. Mutation prevalence differences between Ecuadorian population and other Latin America countries were found. The panel of mutations suggested as an initial screening for the Ecuadorian population with cystic fibrosis should contain the mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. © 2017 NETLAB Laboratorios Especializados. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Stochastic gene expression in Arabidopsis thaliana.

Science.gov (United States)

Araújo, Ilka Schultheiß; Pietsch, Jessica Magdalena; Keizer, Emma Mathilde; Greese, Bettina; Balkunde, Rachappa; Fleck, Christian; Hülskamp, Martin

2017-12-14

Although plant development is highly reproducible, some stochasticity exists. This developmental stochasticity may be caused by noisy gene expression. Here we analyze the fluctuation of protein expression in Arabidopsis thaliana. Using the photoconvertible KikGR marker, we show that the protein expressions of individual cells fluctuate over time. A dual reporter system was used to study extrinsic and intrinsic noise of marker gene expression. We report that extrinsic noise is higher than intrinsic noise and that extrinsic noise in stomata is clearly lower in comparison to several other tissues/cell types. Finally, we show that cells are coupled with respect to stochastic protein expression in young leaves, hypocotyls and roots but not in mature leaves. Our data indicate that stochasticity of gene expression can vary between tissues/cell types and that it can be coupled in a non-cell-autonomous manner.

Applications of the Preclinical Molecular Imaging in Biomedicine: Gene Therapy

International Nuclear Information System (INIS)

Collantes, M.; Peñuelas, I.

2014-01-01

Gene therapy constitutes a promising option for efficient and targeted treatment of several inherited disorders. Imaging techniques using ionizing radiation as PET or SPECT are used for non-invasive monitoring of the distribution and kinetics of vector-mediated gene expression. In this review the main reporter gene/reporter probe strategies are summarized, as well as the contribution of preclinical models to the development of this new imaging modality previously to its application in clinical arena. [es

Transgenic Arabidopsis Gene Expression System

Science.gov (United States)

Ferl, Robert; Paul, Anna-Lisa

2009-01-01

The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

Extensive lineage-specific gene duplication and evolution of the spiggin multi-gene family in stickleback

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Nishida Mutsumi

2007-11-01

Full Text Available Abstract Background The threespine stickleback (Gasterosteus aculeatus has a characteristic reproductive mode; mature males build nests using a secreted glue-like protein called spiggin. Although recent studies reported multiple occurrences of genes that encode this glue-like protein spiggin in threespine and ninespine sticklebacks, it is still unclear how many genes compose the spiggin multi-gene family. Results Genome sequence analysis of threespine stickleback showed that there are at least five spiggin genes and two pseudogenes, whereas a single spiggin homolog occurs in the genomes of other fishes. Comparative genome sequence analysis demonstrated that Muc19, a single-copy mucous gene in human and mouse, is an ortholog of spiggin. Phylogenetic and molecular evolutionary analyses of these sequences suggested that an ancestral spiggin gene originated from a member of the mucin gene family as a single gene in the common ancestor of teleosts, and gene duplications of spiggin have occurred in the stickleback lineage. There was inter-population variation in the copy number of spiggin genes and positive selection on some codons, indicating that additional gene duplication/deletion events and adaptive evolution at some amino acid sites may have occurred in each stickleback population. Conclusion A number of spiggin genes exist in the threespine stickleback genome. Our results provide insight into the origin and dynamic evolutionary process of the spiggin multi-gene family in the threespine stickleback lineage. The dramatic evolution of genes for mucous substrates may have contributed to the generation of distinct characteristics such as "bio-glue" in vertebrates.

Gene study within the 5' flanking regions of growth hormone gene of ...

African Journals Online (AJOL)

user

2011-01-17

Jan 17, 2011 ... Expression of more than one gene for GH has been reported, indicating ..... hormone levels of palsmáticos IGF-1 and carcass traits in beef cattle. Dissertation ... Structure-function relation of somatotropin with reference to ...

Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

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Fu Juanjuan

2011-07-01

Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

Genes2Networks: connecting lists of gene symbols using mammalian protein interactions databases

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Ma'ayan Avi

2007-10-01

Full Text Available Abstract Background In recent years, mammalian protein-protein interaction network databases have been developed. The interactions in these databases are either extracted manually from low-throughput experimental biomedical research literature, extracted automatically from literature using techniques such as natural language processing (NLP, generated experimentally using high-throughput methods such as yeast-2-hybrid screens, or interactions are predicted using an assortment of computational approaches. Genes or proteins identified as significantly changing in proteomic experiments, or identified as susceptibility disease genes in genomic studies, can be placed in the context of protein interaction networks in order to assign these genes and proteins to pathways and protein complexes. Results Genes2Networks is a software system that integrates the content of ten mammalian interaction network datasets. Filtering techniques to prune low-confidence interactions were implemented. Genes2Networks is delivered as a web-based service using AJAX. The system can be used to extract relevant subnetworks created from "seed" lists of human Entrez gene symbols. The output includes a dynamic linkable three color web-based network map, with a statistical analysis report that identifies significant intermediate nodes used to connect the seed list. Conclusion Genes2Networks is powerful web-based software that can help experimental biologists to interpret lists of genes and proteins such as those commonly produced through genomic and proteomic experiments, as well as lists of genes and proteins associated with disease processes. This system can be used to find relationships between genes and proteins from seed lists, and predict additional genes or proteins that may play key roles in common pathways or protein complexes.

Targeting Herpetic Keratitis by Gene Therapy

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Hossein Mostafa Elbadawy

2012-01-01

Full Text Available Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1 can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.

Oestrogenic activity of a textile industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN gene-reporter luciferase assay

Energy Technology Data Exchange (ETDEWEB)

Schiliro, Tiziana, E-mail: tiziana.schiliro@unito.it [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Porfido, Arianna [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy); Spina, Federica; Varese, Giovanna Cristina [Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino (Italy); Gilli, Giorgio [Department of Public Health and Microbiology, University of Torino, Via Santena 5bis, 10126 Torino (Italy)

2012-08-15

This study quantified the biological oestrogenic activity in the effluent of a textile industrial wastewater treatment plant (IWWTP) in northwestern Italy. Samples of the IWWTP effluent were collected monthly, both before and after tertiary treatment (ozonation). After solid phase extraction, all samples were subjected to two in vitro tests of total estrogenic activity, the human breast cancer cell line (MCF-7 BUS) proliferation assay, or E-screen test, and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay, to measure the 17{beta}-oestradiol equivalent quantity (EEQ). In the E-screen test, the mean EEQ values were 2.35 {+-} 1.68 ng/L pre-ozonation and 0.72 {+-} 0.58 ng/L post-ozonation; in the MELN gene-reporter luciferase assay, the mean EEQ values were 4.18 {+-} 3.54 ng/L pre-ozonation and 2.53 {+-} 2.48 ng/L post-ozonation. These results suggest that the post-ozonation IWWTP effluent had a lower oestrogenic activity (simple paired t-tests, p < 0.05). The average reduction of estrogenic activity of IWWTP effluent after ozonation was 67 {+-} 26% and 52 {+-} 27% as measured by E-screen test and MELN gene-reporter luciferase assay, respectively. There was a positive and significant correlation between the two tests (Rho S = 0.650, p = 0.022). This study indicates that the environmental risk is low because oestrogenic substances are deposited into the river via IWWTP at concentrations lower than those at which chronic exposure has been reported to affect the endocrine system of living organisms. -- Highlights: Black-Right-Pointing-Pointer The two in vitro tests are suited for oestrogenic activity assessment in textile WWTP. Black-Right-Pointing-Pointer There is a significant correlation between the results of the two in vitro tests. Black-Right-Pointing-Pointer The oestrogenic activity of the effluent is reduced by ozonation. Black-Right-Pointing-Pointer The input of estrogenic substances into the river via textile WWTP is low.

Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins.

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Stefano Varrella

Full Text Available Marine organisms possess a series of cellular strategies to counteract the negative effects of toxic compounds, including the massive reorganization of gene expression networks. Here we report the modulated dose-dependent response of activated genes by diatom polyunsaturated aldehydes (PUAs in the sea urchin Paracentrotus lividus. PUAs are secondary metabolites deriving from the oxidation of fatty acids, inducing deleterious effects on the reproduction and development of planktonic and benthic organisms that feed on these unicellular algae and with anti-cancer activity. Our previous results showed that PUAs target several genes, implicated in different functional processes in this sea urchin. Using interactomic Ingenuity Pathway Analysis we now show that the genes targeted by PUAs are correlated with four HUB genes, NF-κB, p53, δ-2-catenin and HIF1A, which have not been previously reported for P. lividus. We propose a working model describing hypothetical pathways potentially involved in toxic aldehyde stress response in sea urchins. This represents the first report on gene networks affected by PUAs, opening new perspectives in understanding the cellular mechanisms underlying the response of benthic organisms to diatom exposure.

Two fundamentally different classes of microbial genes.

Science.gov (United States)

Wolf, Yuri I; Makarova, Kira S; Lobkovsky, Alexander E; Koonin, Eugene V

2016-11-07

The evolution of bacterial and archaeal genomes is highly dynamic and involves extensive horizontal gene transfer and gene loss 1-4 . Furthermore, many microbial species appear to have open pangenomes, where each newly sequenced genome contains more than 10% ORFans, that is, genes without detectable homologues in other species 5,6 . Here, we report a quantitative analysis of microbial genome evolution by fitting the parameters of a simple, steady-state evolutionary model to the comparative genomic data on the gene content and gene order similarity between archaeal genomes. The results reveal two sharply distinct classes of microbial genes, one of which is characterized by effectively instantaneous gene replacement, and the other consists of genes with finite, distributed replacement rates. These findings imply a conservative estimate of the size of the prokaryotic genomic universe, which appears to consist of at least a billion distinct genes. Furthermore, the same distribution of constraints is shown to govern the evolution of gene complement and gene order, without the need to invoke long-range conservation or the selfish operon concept 7 .

Development of a cell-based reporter assay for screening of inhibitors of hypoxia-inducible factor 2-induced gene expression.

Science.gov (United States)

Woldemichael, Girma M; Vasselli, James R; Gardella, Roberta S; McKee, Tawnya C; Linehan, W Marston; McMahon, James B

2006-09-01

Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

Differentially expressed genes in the midgut of Silkworm infected ...

African Journals Online (AJOL)

In this report, we employed suppression subtractive hybridization to compare differentially expressed genes in the midguts of CPV-infected and normal silkworm larvae. 36 genes and 20 novel ESTs were obtained from 2 reciprocal subtractive libraries. Three up-regulated genes (ferritin, rpL11 and alkaline nuclease) and 3 ...

A family with hereditary hemochromatosis carrying HFE gene splice site mutation: a case report

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NING Huibin

2017-01-01

Full Text Available ObjectiveTo investigate a new type of HFE gene mutation in a family with hereditary hemochromatosis (HH. MethodsThe analysis of HFE gene was performed for one patient with a confirmed diagnosis of HH and five relatives. Blood genomic DNA was extracted and PCR multiplication was performed for the exon and intron splice sequences of related HFE, HJV, HAMP, transferrin receptor 2 (TfR2, and SLC40A1 genes. After agarose gel electrophoresis and purification, bi-directional direct sequencing was performed to detect mutation sites. ResultsThe proband had abnormal liver function and increases in serum iron, total iron binding capacity, serum ferritin, and transferrin saturation, as well as T→C homozygous mutation in the fourth base of intron 2 in the intervening sequence of the exon EXON2 of HFE gene (IVs 2+4T→C, C/C homozygous, splicing, abnormal. There were no abnormalities in HJV, HAMP, TfR2, and SLC40A1 genes. The proband′s son had the same homozygous mutation, three relatives had heterozygous mutations, and one relative had no abnormal mutations. ConclusionGene detection plays an important role in the diagnosis of hemochromatosis, and IVs 2+4T→C mutation may be a new pathogenic mutation for HH in China.

Mutation update for the PORCN gene

DEFF Research Database (Denmark)

Lombardi, Maria Paola; Bulk, Saskia; Celli, Jacopo

2011-01-01

Mutations in the PORCN gene were first identified in Goltz-Gorlin syndrome patients in 2007. Since then, several reports have been published describing a large variety of genetic defects resulting in the Goltz-Gorlin syndrome, and mutations or deletions were also reported in angioma serpiginosum......, the pentalogy of Cantrell and Limb-Body Wall Complex. Here we present a review of the published mutations in the PORCN gene to date and report on seven new mutations together with the corresponding clinical data. Based on the review we have created a Web-based locus-specific database that lists all identified...... variants and allows the inclusion of future reports. The database is based on the Leiden Open (source) Variation Database (LOVD) software, and is accessible online at http://www.lovd.nl/porcn. At present, the database contains 106 variants, representing 68 different mutations, scattered along the whole...

Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

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Daniell Henry

2011-06-01

Full Text Available Abstract Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun" delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI methods. Results Plasmid DNA carrying the firefly luciferase (LUC reporter gene under the control of the human Cytomegalovirus (CMV promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter using different DNA Loading Ratios (DLRs, and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50 at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results

Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

International Nuclear Information System (INIS)

Golubovskaya, Vita M.; Ho, Baotran; Conroy, Jeffrey; Liu, Song; Wang, Dan; Cance, William G.

2014-01-01

Focal Adhesion Kinase (FAK) is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane) compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53 +/+ and p53 −/− cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05) in HCT116 p53 +/+ cells but not in p53 −/− cells. Among up-regulated genes in HCT p53 +/+ cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53 +/+ colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach

A simple protocol for preparation of a liposomal vesicle with encapsulated plasmid DNA that mediate high accumulation and reporter gene activity in tumor tissue

DEFF Research Database (Denmark)

Gjetting, Torben; Andresen, Thomas Lars; Christensen, Camilla Laulund

2011-01-01

The systemic delivery of gene therapeutics by non-viral methods has proven difficult. Transfection systems that are performing well in vitro have been reported to have disadvantageous properties such as rapid clearance and short circulation time often resulting in poor transfection efficiency whe...

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress.

Science.gov (United States)

Chen, Jingchao; Huang, Zhaofeng; Huang, Hongjuan; Wei, Shouhui; Liu, Yan; Jiang, Cuilan; Zhang, Jie; Zhang, Chaoxian

2017-04-21

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species.

Isolation and characterization of LHY homolog gene expressed in ...

African Journals Online (AJOL)

STORAGESEVER

2008-05-02

May 2, 2008 ... responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene. Key words: LHY gene, circadian ...

Visualization of the Dynamics of Gene Expression in the Living Mouse

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Amy Ryan

2004-01-01

Full Text Available Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals. We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac Operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse. Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus. The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal.

Mutation update for the PORCN gene

NARCIS (Netherlands)

Lombardi, Maria Paola; Bulk, Saskia; Celli, Jacopo; Lampe, Anne; Gabbett, Michael T.; Ousager, Lillian Bomme; van der Smagt, Jasper J.; Soller, Maria; Stattin, Eva-Lena; Mannens, Marcel A. M. M.; Smigiel, Robert; Hennekam, Raoul C.

2011-01-01

Mutations in the PORCN gene were first identified in Goltz-Gorlin syndrome patients in 2007. Since then, several reports have been published describing a large variety of genetic defects resulting in the Goltz-Gorlin syndrome, and mutations or deletions were also reported in angioma serpiginosum,

Gene Dosage Analysis in a Clinical Environment: Gene-Targeted Microarrays as the Platform-of-Choice

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Donald R. Love

2013-03-01

Full Text Available The role of gene deletion and duplication in the aetiology of disease has become increasingly evident over the last decade. In addition to the classical deletion/duplication disorders diagnosed using molecular techniques, such as Duchenne Muscular Dystrophy and Charcot-Marie-Tooth Neuropathy Type 1A, the significance of partial or whole gene deletions in the pathogenesis of a large number single-gene disorders is becoming more apparent. A variety of dosage analysis methods are available to the diagnostic laboratory but the widespread application of many of these techniques is limited by the expense of the kits/reagents and restrictive targeting to a particular gene or portion of a gene. These limitations are particularly important in the context of a small diagnostic laboratory with modest sample throughput. We have developed a gene-targeted, custom-designed comparative genomic hybridisation (CGH array that allows twelve clinical samples to be interrogated simultaneously for exonic deletions/duplications within any gene (or panel of genes on the array. We report here on the use of the array in the analysis of a series of clinical samples processed by our laboratory over a twelve-month period. The array has proven itself to be robust, flexible and highly suited to the diagnostic environment.

Biolistic transformation of Schistosoma mansoni: Studies with modified reporter-gene constructs containing regulatory regions of protease genes

Czech Academy of Sciences Publication Activity Database

Dvořák, Jan; Beckmann, S.; Lim, K.-C.; Engel, J. C.; Grevelding, C. G.; McKerrow, J. H.; Caffrey, C. R.

2010-01-01

Roč. 170, č. 1 (2010), s. 37-40 ISSN 0166-6851 Institutional research plan: CEZ:AV0Z60220518 Keywords : Schistosoma * Protease * Transgene * Gene promoter * Biolistics * Electroporation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.875, year: 2010

Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors

DEFF Research Database (Denmark)

Dahlgaard, Katja; Troelsen, Jesper

2018-01-01

Several tumor suppressors possess gene regulatory activity. Here, we describe how promoter and promoter/enhancer reporter assays can be used to characterize a colorectal tumor suppressor proteins’ gene regulatory activity of possible target genes. In the first part, a bioinformatic approach...... of the quick and efficient In-Fusion cloning method, and how to carry out transient transfections of Caco-2 colon cancer cells with the produced luciferase reporter plasmids using polyethyleneimine (PEI). A plan describing how to set up and carry out the luciferase expression assay is presented. The luciferase...... to identify relevant gene regulatory regions of potential target genes is presented. In the second part, it is demonstrated how to prepare and carry out the functional assay. We explain how to clone the bioinformatically identified gene regulatory regions into luciferase reporter plasmids by the use...

pTRA - A reporter system for monitoring the intracellular dynamics of gene expression.

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Wagner, Sabine G; Ziegler, Martin; Löwe, Hannes; Kremling, Andreas; Pflüger-Grau, Katharina

2018-01-01

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

Gene-Gene Interactions in the Folate Metabolic Pathway and the Risk of Conotruncal Heart Defects

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Philip J. Lupo

2010-01-01

Full Text Available Conotruncal and related heart defects (CTRD are common, complex malformations. Although there are few established risk factors, there is evidence that genetic variation in the folate metabolic pathway influences CTRD risk. This study was undertaken to assess the association between inherited (i.e., case and maternal gene-gene interactions in this pathway and the risk of CTRD. Case-parent triads (n=727, ascertained from the Children's Hospital of Philadelphia, were genotyped for ten functional variants of nine folate metabolic genes. Analyses of inherited genotypes were consistent with the previously reported association between MTHFR A1298C and CTRD (adjusted P=.02, but provided no evidence that CTRD was associated with inherited gene-gene interactions. Analyses of the maternal genotypes provided evidence of a MTHFR C677T/CBS 844ins68 interaction and CTRD risk (unadjusted P=.02. This association is consistent with the effects of this genotype combination on folate-homocysteine biochemistry but remains to be confirmed in independent study populations.

[Metatropic dysplasia in a girl with c.1811_1812delinsAT mutation in exon 11 of the TRPV4 gene not previously reported].

Science.gov (United States)

Cammarata-Scalisi, Francisco; Matysiak-Scholze, Uta; Heinze, Jessica; Barrera, Albaro; Lacruz-Rengel, María Angelina; Bracho, Ana; Guerrero, Yudith

2015-01-01

Metatropic dysplasia is a skeletal disorder with clinical heterogeneity, characterized by craniofacial dysmorphy including frontal bossing and midface hypoplasia, short trunk,progressive kyphoscoliosis and shortened limbs. The TRPV4 gene is located on 12q24.11, coding a cation channel with nonselective permeability to calcium; it is expressed and involved in many physiological processes through responses to different stimuli. Over 50 mutations in TRPV4 have been described. We present a seven months old girl with heterozygous mutation c.1811_1812delinsAT; p.I604N in intron 11 not previously reported in the TRPV4 gene and with clinical findings compatible with metatropic dysplasia.

Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

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Øvstebø Reidun

2010-05-01

Full Text Available Abstract Background Gene expression in lipopolysaccharide (LPS-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase or ACTB (beta-actin as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. Results Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B, B2M (beta-2-microglobulin and PPIA (cyclophilin A as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha and IL10 (interleukin 10 expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. Conclusions Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

The use of hygromycin phosphotransferase gene (hpt) with an ...

African Journals Online (AJOL)

Previously, we have developed a marker-off system to truncate a selec marker in transgenic plants by locating one end of the transposon in the intron of the marker gene (glyphosate-tolerant epsps gene). In order to expand this technique to those marker genes without intron in this report, we created an artificial intron ...

Genes and Gene Therapy

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

Gene therapy for CNS diseases – Krabbe disease

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Mohammad A. Rafi

2016-06-01

Full Text Available This is a brief report of the 19th Annual Meeting of the American Society of Gene and Cell Therapy that took place from May 4th through May 7th, 2016 in Washington, DC, USA. While the meeting provided many symposiums, lectures, and scientific sessions this report mainly focuses on one of the sessions on the "Gene Therapy for central nervous system (CNS Diseases" and specifically on the "Gene Therapy for the globoid cell leukodystrophy or Krabbe disease. Two presentations focused on this subject utilizing two animal models of this disease: mice and dog models. Different serotypes of adeno-associate viral vectors (AAV alone or in combination with bone marrow transplantations were used in these research projects. The Meeting of the ASGCT reflected continuous growth in the fields of gene and cell therapy and brighter forecast for efficient treatment options for variety of human diseases.

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species

International Nuclear Information System (INIS)

Manning, Gillian E.; Farmahin, Reza; Crump, Doug; Jones, Stephanie P.; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

2012-01-01

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R 2 ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect their relative

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

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Jacob, Tiago R; Peres, Nalu T A; Persinoti, Gabriela F; Silva, Larissa G; Mazucato, Mendelson; Rossi, Antonio; Martinez-Rossi, Nilce M

2012-05-01

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Impaired expression of mitochondrial and adipogenic genes in adipose tissue from a patient with acquired partial lipodystrophy (Barraquer-Simons syndrome: a case report

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Guallar Jordi P

2008-08-01

Full Text Available Abstract Introduction Acquired partial lipodystrophy or Barraquer-Simons syndrome is a rare form of progressive lipodystrophy. The etiopathogenesis of adipose tissue atrophy in these patients is unknown. Case presentation This is a case report of a 44-year-old woman with acquired partial lipodystrophy. To obtain insight into the molecular basis of lipoatrophy in acquired partial lipodystrophy, we examined gene expression in adipose tissue from this patient newly diagnosed with acquired partial lipodystrophy. A biopsy of subcutaneous adipose tissue was obtained from the patient, and DNA and RNA were extracted in order to evaluate mitochondrial DNA abundance and mRNA expression levels. Conclusion The expression of marker genes of adipogenesis and adipocyte metabolism, including the master regulator PPARγ, was down-regulated in subcutaneous adipose tissue from this patient. Adiponectin mRNA expression was also reduced but leptin mRNA levels were unaltered. Markers of local inflammatory status were unaltered. Expression of genes related to mitochondrial function was reduced despite unaltered levels of mitochondrial DNA. It is concluded that adipogenic and mitochondrial gene expression is impaired in adipose tissue in this patient with acquired partial lipodystrophy.

Aneuploids of wheat and chromosomal localization of genes

African Journals Online (AJOL)

Administrator

2011-06-22

Jun 22, 2011 ... chromosome location of such genes is critical for effective utilization and subsequent manipulation. Further, chromosomal localization will lead to the identification of ... Various cytogenetic stocks and techniques have been previously reported useful in localizing genes on wheat chromosomes. The objective ...

A simple protocol for preparation of a liposomal vesicle with encapsulated plasmid DNA that mediate high accumulation and reporter gene activity in tumor tissue

DEFF Research Database (Denmark)

Gjetting, Torben; Andresen, Thomas Lars; Christensen, Camilla Laulund

2011-01-01

The systemic delivery of gene therapeutics by non-viral methods has proven dif¿cult. Transfection systems that are performing well in vitro have been reported to have disadvantageous properties such as rapid clearance and short circulation time often resulting in poor transfection ef¿ciency when ...

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

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Walchli John

2009-04-01

Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

Circular DNA Intermediate in the Duplication of Nile Tilapia vasa Genes

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Fujimura, Koji; Conte, Matthew A.; Kocher, Thomas D.

2011-01-01

vasa is a highly conserved RNA helicase involved in animal germ cell development. Among vertebrate species, it is typically present as a single copy per genome. Here we report the isolation and sequencing of BAC clones for Nile tilapia vasa genes. Contrary to a previous report that Nile tilapia have a single copy of the vasa gene, we find evidence for at least three vasa gene loci. The vasa gene locus was duplicated from the original site and integrated into two distant novel sites. For one of these insertions we find evidence that the duplication was mediated by a circular DNA intermediate. This mechanism of gene duplication may explain the origin of isolated gene duplicates during the evolution of fish genomes. These data provide a foundation for studying the role of multiple vasa genes in the development of tilapia gonads, and will contribute to investigations of the molecular mechanisms of sex determination and evolution in cichlid fishes. PMID:22216289

Identification of the first large deletion in the CLDN16 gene in a patient with FHHNC and late-onset of chronic kidney disease: case report.

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Yamaguti, Paulo Marcio; dos Santos, Pollyanna Almeida Costa; Leal, Bruno Sakamoto; Santana, Viviane Brandão Bandeira de Mello; Mazzeu, Juliana Forte; Acevedo, Ana Carolina; Neves, Francisco de Assis Rocha

2015-07-02

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is a rare autosomal recessive renal disease characterized by tubular disorders at the thick ascending limb of Henle's loop. It is caused by mutations in the tight junction structural proteins claudin-16 or claudin-19, which are encoded by the CLDN16 and CLDN19 genes, respectively. Patients exhibit excessive wasting of calcium and magnesium, nephrocalcinosis, chronic kidney disease, and early progression to end-stage renal failure during infancy. We here report the phenotype and molecular analysis of a female Brazilian patient with a novel large homozygous deletion in the CLDN16 gene. The proband, born from consanguineous parents, presented the first symptoms at age 20. Clinical examination revealed hypocalcemia, hypomagnesemia, nephrocalcinosis, mild myopia, high serum levels of uric acid and intact parathyroid hormone, and moderate chronic kidney disease (stage 3). She and her mother were subjected to CLDN16 and CLDN19 mutational analysis. In addition, the multiplex ligation-dependent probe amplification method was used to confirm a CLDN16 multi-exon deletion. Direct sequencing revealed a normal CLDN19 sequence and suggested a large deletion in the CLDN16 gene. Multiplex ligation-dependent probe amplification showed a homozygous CLDN16 multi-exon deletion (E2_E5del). The patient initiated conventional treatment for familial hypomagnesemia with hypercalciuria and nephrocalcinosis and progressed to end-stage kidney disease after five years. This study provides the first report of a large homozygous deletion in the CLDN16 gene causing familial hypomagnesemia with hypercalciuria and nephrocalcinosis with late onset of the first symptoms. This description expands the phenotypic and genotypic characterization of the disease. The late-onset chronic kidney disease in the presence of a homozygous deletion in the CLDN16 gene reinforces the great variability of genotype-phenotype manifestation in patients with

Phosphatase and tensin homolog (PTEN) gene mutations and autism: literature review and a case report of a patient with Cowden syndrome, autistic disorder, and epilepsy.

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Conti, Sara; Condò, Maria; Posar, Annio; Mari, Francesca; Resta, Nicoletta; Renieri, Alessandra; Neri, Iria; Patrizi, Annalisa; Parmeggiani, Antonia

2012-03-01

Phosphatase and tensin homolog (PTEN) gene mutations are associated with a spectrum of clinical disorders characterized by skin lesions, macrocephaly, hamartomatous overgrowth of tissues, and an increased risk of cancers. Autism has rarely been described in association with these variable clinical features. At present, 24 patients with phosphatase and tensin homolog gene mutation, autism, macrocephaly, and some clinical findings described in phosphatase and tensin homolog syndromes have been reported in the literature. We describe a 14-year-old boy with autistic disorder, focal epilepsy, severe and progressive macrocephaly, and multiple papular skin lesions and palmoplantar punctate keratoses, characteristic of Cowden syndrome. The boy has a de novo phosphatase and tensin homolog gene mutation. Our patient is the first case described to present a typical Cowden syndrome and autism associated with epilepsy.

Identification of SNP c.-22G>A in the melanophilin gene from a dog with color dilution alopecia: case report

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L.M. Santos

Full Text Available ABSTRACT Mutant color alopecia is an ectodermical defection of color dilution, characterized by partial alopecia, dry, shine-less hair, and peeling and papule. Melanization damages also occur on the cortical structure of the affected hair. The animals affected have big melanin grains with irregular shape on the basal keratinocytes, also on the hair matrix cells and rod. Therefore, there is not a specific treatment that makes any difference on the syndrome evolution. Although in some animals, it is possible to use weekly showers with benzyl peroxide to reduce seborrhea formation and secondary infections. There is evidence that the condition in dogs is caused by a single nucleotide polymorphism in the gene encoding the melanophilin protein. In the present study the identification of the SNP c.-22G>A in the melanophilin gene of a Dachshund breed dog with clinical and histopathologic evidence of color dilution alopecia is reported.

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours

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Merron, Andrew; McNeish, Iain A. [Queen Mary' s School of Medicine and Dentistry, Centre for Molecular Oncology, Institute of Cancer, London (United Kingdom); Baril, Patrick; Tran, Lucile; Vassaux, Georges [CHU Hotel Dieu, INSERM, Nantes (France); CHU de Nantes, Institut des Maladies de l' Appareil Digestif, Nantes (France); Martin-Duque, Pilar [Instituto Aragones de Ciencias de la Salud, Zaragoza (Spain); Vieja, Antonio de la [Instituto de Investigaciones Biomedicas, Madrid (Spain); Briat, Arnaud [INSERM U877, Grenoble (France); Harrington, Kevin J. [Chester Beatty Laboratories, Institute of Cancer Research, London (United Kingdom)

2010-07-15

In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects. (orig.)

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours

International Nuclear Information System (INIS)

Merron, Andrew; McNeish, Iain A.; Baril, Patrick; Tran, Lucile; Vassaux, Georges; Martin-Duque, Pilar; Vieja, Antonio de la; Briat, Arnaud; Harrington, Kevin J.

2010-01-01

In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects. (orig.)

A de novo whole gene deletion of XIAP detected by exome sequencing analysis in very early onset inflammatory bowel disease: a case report.

Science.gov (United States)

Kelsen, Judith R; Dawany, Noor; Martinez, Alejandro; Martinez, Alejuandro; Grochowski, Christopher M; Maurer, Kelly; Rappaport, Eric; Piccoli, David A; Baldassano, Robert N; Mamula, Petar; Sullivan, Kathleen E; Devoto, Marcella

2015-11-18

Children with very early-onset inflammatory bowel disease (VEO-IBD), those diagnosed at less than 5 years of age, are a unique population. A subset of these patients present with a distinct phenotype and more severe disease than older children and adults. Host genetics is thought to play a more prominent role in this young population, and monogenic defects in genes related to primary immunodeficiencies are responsible for the disease in a small subset of patients with VEO-IBD. We report a child who presented at 3 weeks of life with very early-onset inflammatory bowel disease (VEO-IBD). He had a complicated disease course and remained unresponsive to medical and surgical therapy. The refractory nature of his disease, together with his young age of presentation, prompted utilization of whole exome sequencing (WES) to detect an underlying monogenic primary immunodeficiency and potentially target therapy to the identified defect. Copy number variation analysis (CNV) was performed using the eXome-Hidden Markov Model. Whole exome sequencing revealed 1,380 nonsense and missense variants in the patient. Plausible candidate variants were not detected following analysis of filtered variants, therefore, we performed CNV analysis of the WES data, which led us to identify a de novo whole gene deletion in XIAP. This is the first reported whole gene deletion in XIAP, the causal gene responsible for XLP2 (X-linked lymphoproliferative Disease 2). XLP2 is a syndrome resulting in VEO-IBD and can increase susceptibility to hemophagocytic lymphohistocytosis (HLH). This identification allowed the patient to be referred for bone marrow transplantation, potentially curative for his disease and critical to prevent the catastrophic sequela of HLH. This illustrates the unique etiology of VEO-IBD, and the subsequent effects on therapeutic options. This cohort requires careful and thorough evaluation for monogenic defects and primary immunodeficiencies.

Efficient transformation and expression of gfp gene in Valsa mali var. mali.

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Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

2015-01-01

Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

Cis-regulatory timers for developmental gene expression.

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Lionel Christiaen

2013-10-01

Full Text Available How does a fertilized egg decode its own genome to eventually develop into a mature animal? Each developing cell must activate a battery of genes in a timely manner and according to the function it will ultimately perform, but how? During development of the notochord--a structure akin to the vertebrate spine--in a simple marine invertebrate, an essential protein called Brachyury binds to specific sites in its target genes. A study just published in PLOS Biology reports that if the target gene contains multiple Brachyury-binding sites it will be activated early in development but if it contains only one site it will be activated later. Genes that contain no binding site can still be activated by Brachyury, but only indirectly by an earlier Brachyury-dependent gene product, so later than the directly activated genes. Thus, this study shows how several genes can interpret the presence of a single factor differently to become active at distinct times in development.

Gene structure, phylogeny and expression profile of the sucrose synthase gene family in cacao (Theobroma cacao L.).

Science.gov (United States)

Li, Fupeng; Hao, Chaoyun; Yan, Lin; Wu, Baoduo; Qin, Xiaowei; Lai, Jianxiong; Song, Yinghui

2015-09-01

In higher plants, sucrose synthase (Sus, EC 2.4.1.13) is widely considered as a key enzyme involved in sucrose metabolism. Although, several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, to date detailed information about the Sus genes is lacking for cacao. This study reports the identification of six novel Sus genes from economically important cacao tree. Analyses of the gene structure and phylogeny of the Sus genes demonstrated evolutionary conservation in the Sus family across cacao and other plant species. The expression of cacao Sus genes was investigated via real-time PCR in various tissues, different developmental phases of leaf, flower bud and pod. The Sus genes exhibited distinct but partially redundant expression profiles in cacao, with TcSus1, TcSus5 and TcSus6, being the predominant genes in the bark with phloem, TcSus2 predominantly expressing in the seed during the stereotype stage. TcSus3 and TcSus4 were significantly detected more in the pod husk and seed coat along the pod development, and showed development dependent expression profiles in the cacao pod. These results provide new insights into the evolution, and basic information that will assist in elucidating the functions of cacao Sus gene family.

State-related alterations of gene expression in bipolar disorder

DEFF Research Database (Denmark)

Munkholm, Klaus; Vinberg, Maj; Berk, Michael

2012-01-01

Munkholm K, Vinberg M, Berk M, Kessing LV. State-related alterations of gene expression in bipolar disorder: a systematic review. Bipolar Disord 2012: 14: 684-696. © 2012 The Authors. Journal compilation © 2012 John Wiley & Sons A/S. Objective:  Alterations in gene expression in bipolar disorder...... have been found in numerous studies. It is unclear whether such alterations are related to specific mood states. As a biphasic disorder, mood state-related alterations in gene expression have the potential to point to markers of disease activity, and trait-related alterations might indicate...... vulnerability pathways. This review therefore evaluated the evidence for whether gene expression in bipolar disorder is state or trait related. Methods:  A systematic review, using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guideline for reporting systematic reviews, based...

Improvement Method of Gene Transfer in Kappaphycus Alvarezii

OpenAIRE

Triana, St. Hidayah; Alimuddin,; Widyastuti, Utut; Suharsono,; Suryati, Emma; Parenrengi, Andi

2016-01-01

Method of foreign gene transfer in red seaweed Kappaphycus alvarezii has been reported, however, li-mited number of transgenic F0 (broodstock) was obtained. This study was conducted to improve the method of gene transfer mediated by Agrobacterium tumefaciens in order to obtain high percentage of K. alvarezii transgenic. Superoxide dismutase gene from Melastoma malabatrichum (MmCu/Zn-SOD) was used as model towards increasing adaptability of K. alvarezii to environmental stress. The treat-ment...

Mosaic origins of a complex chimeric mitochondrial gene in Silene vulgaris.

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Helena Storchova

Full Text Available Chimeric genes are significant sources of evolutionary innovation that are normally created when portions of two or more protein coding regions fuse to form a new open reading frame. In plant mitochondria astonishingly high numbers of different novel chimeric genes have been reported, where they are generated through processes of rearrangement and recombination. Nonetheless, because most studies do not find or report nucleotide variation within the same chimeric gene, evolution after the origination of these chimeric genes remains unstudied. Here we identify two alleles of a complex chimera in Silene vulgaris that are divergent in nucleotide sequence, genomic position relative to other mitochondrial genes, and expression patterns. Structural patterns suggest a history partially influenced by gene conversion between the chimeric gene and functional copies of subunit 1 of the mitochondrial ATP synthase gene (atp1. We identified small repeat structures within the chimeras that are likely recombination sites allowing generation of the chimera. These results establish the potential for chimeric gene divergence in different plant mitochondrial lineages within the same species. This result contrasts with the absence of diversity within mitochondrial chimeras found in crop species.

A network of genes, genetic disorders, and brain areas.

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Satoru Hayasaka

Full Text Available The network-based approach has been used to describe the relationship among genes and various phenotypes, producing a network describing complex biological relationships. Such networks can be constructed by aggregating previously reported associations in the literature from various databases. In this work, we applied the network-based approach to investigate how different brain areas are associated to genetic disorders and genes. In particular, a tripartite network with genes, genetic diseases, and brain areas was constructed based on the associations among them reported in the literature through text mining. In the resulting network, a disproportionately large number of gene-disease and disease-brain associations were attributed to a small subset of genes, diseases, and brain areas. Furthermore, a small number of brain areas were found to be associated with a large number of the same genes and diseases. These core brain regions encompassed the areas identified by the previous genome-wide association studies, and suggest potential areas of focus in the future imaging genetics research. The approach outlined in this work demonstrates the utility of the network-based approach in studying genetic effects on the brain.

In Vitro and In Vivo Characterization of a Dual-Function Green Fluorescent Protein–HSV1-Thymidine Kinase Reporter Gene Driven by the Human Elongation Factor 1α Promoter

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Gary D. Luker

2002-04-01

Full Text Available Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK as a reporter gene driven by the promoter for human elongation factor 1α (EF-1α-EGFP-TK. Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV. As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutylguanine ([18F]FHBG ≈ 8-[3H]penciclovir (8-[3H]PCV < 2′-fluoro-2′-deoxy-5-iodouracil-beta-d-arabinofuranoside (2-[14C]FIAU. Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

GeneAnalytics: An Integrative Gene Set Analysis Tool for Next Generation Sequencing, RNAseq and Microarray Data.

Science.gov (United States)

Ben-Ari Fuchs, Shani; Lieder, Iris; Stelzer, Gil; Mazor, Yaron; Buzhor, Ella; Kaplan, Sergey; Bogoch, Yoel; Plaschkes, Inbar; Shitrit, Alina; Rappaport, Noa; Kohn, Asher; Edgar, Ron; Shenhav, Liraz; Safran, Marilyn; Lancet, Doron; Guan-Golan, Yaron; Warshawsky, David; Shtrichman, Ronit

2016-03-01

Postgenomics data are produced in large volumes by life sciences and clinical applications of novel omics diagnostics and therapeutics for precision medicine. To move from "data-to-knowledge-to-innovation," a crucial missing step in the current era is, however, our limited understanding of biological and clinical contexts associated with data. Prominent among the emerging remedies to this challenge are the gene set enrichment tools. This study reports on GeneAnalytics™ ( geneanalytics.genecards.org ), a comprehensive and easy-to-apply gene set analysis tool for rapid contextualization of expression patterns and functional signatures embedded in the postgenomics Big Data domains, such as Next Generation Sequencing (NGS), RNAseq, and microarray experiments. GeneAnalytics' differentiating features include in-depth evidence-based scoring algorithms, an intuitive user interface and proprietary unified data. GeneAnalytics employs the LifeMap Science's GeneCards suite, including the GeneCards®--the human gene database; the MalaCards-the human diseases database; and the PathCards--the biological pathways database. Expression-based analysis in GeneAnalytics relies on the LifeMap Discovery®--the embryonic development and stem cells database, which includes manually curated expression data for normal and diseased tissues, enabling advanced matching algorithm for gene-tissue association. This assists in evaluating differentiation protocols and discovering biomarkers for tissues and cells. Results are directly linked to gene, disease, or cell "cards" in the GeneCards suite. Future developments aim to enhance the GeneAnalytics algorithm as well as visualizations, employing varied graphical display items. Such attributes make GeneAnalytics a broadly applicable postgenomics data analyses and interpretation tool for translation of data to knowledge-based innovation in various Big Data fields such as precision medicine, ecogenomics, nutrigenomics, pharmacogenomics, vaccinomics

Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance quantitative trait loci

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Sehgal Deepmala

2012-01-01

Full Text Available Abstract Background Identification of genes underlying drought tolerance (DT quantitative trait loci (QTLs will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL. Results Seventy five single nucleotide polymorphism (SNP and conserved intron spanning primer (CISP markers were developed from available expressed sequence tags (ESTs using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions. Conclusions We have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene

Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

International Nuclear Information System (INIS)

Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

1988-01-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses

Potential hot spot for de novo mutations in PTCH1 gene in Gorlin syndrome patients: a case report of twins from Croatia.

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Musani, Vesna; Ozretić, Petar; Trnski, Diana; Sabol, Maja; Poduje, Sanja; Tošić, Mateja; Šitum, Mirna; Levanat, Sonja

2018-02-28

We describe a case of twins with sporadic Gorlin syndrome. Both twins had common Gorlin syndrome features including calcification of the falx cerebri, multiple jaw keratocysts, and multiple basal cell carcinomas, but with different expressivity. One brother also had benign testicular mesothelioma. We propose this tumor type as a possible new feature of Gorlin syndrome. Gorlin syndrome is a rare autosomal dominant disorder characterized by both developmental abnormalities and cancer predisposition, with variable expression of various developmental abnormalities and different types of tumors. The syndrome is primarily caused by mutations in the Patched 1 (PTCH1) gene, although rare mutations of Patched 2 (PTCH2) or Suppressor of Fused (SUFU) genes have also been found. Neither founder mutations nor hot spot locations have been described for PTCH1 in Gorlin syndrome patients. Although de novo mutations of the PTCH1 gene occur in almost 50% of Gorlin syndrome cases, there are a few recurrent mutations. Our twin patients were carriers of a de novo mutation in the PTCH1 gene, c.3364_3365delAT (p.Met1122ValfsX22). This is, to our knowledge, the first Gorlin syndrome-causing mutation that has been reported four independent times in distant geographical locations. Therefore, we propose the location of the described mutation as a potential hot spot for mutations in PTCH1.

A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD{sub 50} of polychlorinated biphenyls in avian species

Energy Technology Data Exchange (ETDEWEB)

Manning, Gillian E., E-mail: gmann017@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Farmahin, Reza, E-mail: mfarm070@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Jones, Stephanie P., E-mail: stephanie.jones@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Klein, Jeff, E-mail: jeffery@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Konstantinov, Alex, E-mail: alex@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Potter, Dave, E-mail: dpotter@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3M5 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)

2012-09-15

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R{sup 2} ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect

Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum.

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Yuichiro Iida

Full Text Available Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i frameshift mutations and (ii transposon insertions in Avr2, (iii point mutations in Avr4 and Avr4E, and (iv deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed.

Image of HSV1-TK gene expression with {sup 123}IVDU

Energy Technology Data Exchange (ETDEWEB)

Kim, S. Y.; Woo, K. S.; Chung, W. S. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2005-07-01

The liver is an important target organ for gene transfer due to its capacity for synthesizing serum protein and its involvement in numerous genetic diseases. So livertargeted gene transfer is significant tool for expanding the treatment options and gene function studies. Gene transfer methods commonly use recombinant viral vector. However, viral vectors also have various disadvantages for example immune recognition after adenoviral vector delivery and potential viralassociated toxicity including helper virus replication and insertional mutagenesis. In contrast, nonviral vectors such as naked plasmid DNA(pDNA) and cationic liposomal systems exhibit low immunogenicity and repeated administration is possible(Ledley et al.,1992; Nabel et al.,1993). These are attractive vectors for in vivo gene transfer because of their suitable characteristics such as biodegradability, minimal toxicity, nonimmunogenicity, and simplicity of use. But non-viral gene delivery, has problems associated with limited efficiency at gene expression. hydrodynamic-based produce has very high level efficiency of gene extraction in liver or soild tumor. In mice, hydrodynamic-based produce was reported that a high level of transgene expression could be obtained in the liver by intravenous injection of large volume( 8{approx}10% of body weight) and high-speed ( Kobayashi N et al., 2004 ). HSV1-TK is one of the most widely use effect gene systems sued for imaging gene expression, in association with its use as a suicide gene, or as a reporter gene In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have developed as prodrug for tumor proliferation imaging or as anti-viral drugs. Several 5-substituted uracil nucleoside derivatives have been identified to have high sensitivity and selective accumulation in HSV1-TK expression cell. This producer has been used hydrodynamic-based produce, we investigated to image of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene with (E

Genes, stress, and depression.

Science.gov (United States)

Wurtman, Richard J

2005-05-01

A relationship between genetic makeup and susceptibility to major depressive disorder (MDD) has long been suspected on the basis of family and twin studies. A metaanalysis of reports on the basis of twin studies has estimated MDD's degree of heritability to be 0.33 (confidence interval, 0.26-0.39). Among families exhibiting an increased prevalence of MDD, risk of developing the illness was enhanced in members exposed to a highly stressful environment. Aberrant genes can predispose to depression in a number of ways, for example, by diminishing production of growth factors that act during brain development. An aberrant gene could also increase or decrease a neurotransmitter's release into synapses, its actions, or its duration of activity. The gene products of greatest interest at present are those involved in the synthesis and actions of serotonin; among them, the serotonin-uptake protein localized within the terminals and dendrites of serotonin-releasing neurons. It has been found that the Vmax of platelet serotonin uptake is low in some patients with MDD; also, Vmax is highly correlated in twins. Antidepressant drugs such as the selective serotonin reuptake inhibitors act on this uptake protein. The specific genetic locus causing serotonin uptake to be lower in some patients with major depression involves a polymorphic region (5-HTTLPR) in the promoter region of the gene for the uptake protein. The gene itself exists as several alleles, the short "S" allele and the long "L" allele. The S variant is associated with less, and the L variant with more, of the uptake protein. The effect of stressful life events on depressive symptoms in young adults was found to be significantly stronger among SS or SL subjects than among LL subjects. Neuroimaging studies showed that people with the SS or SL alleles exhibited a greater activation of the amygdala in response to fearful stimuli than those with LL. It has been reported recently that mutations in the gene that controls

Gene therapy for human glioblastoma using neurotropic JC virus-like particles as a gene delivery vector.

Science.gov (United States)

Chao, Chun-Nun; Yang, Yu-Hsuan; Wu, Mu-Sheng; Chou, Ming-Chieh; Fang, Chiung-Yao; Lin, Mien-Chun; Tai, Chien-Kuo; Shen, Cheng-Huang; Chen, Pei-Lain; Chang, Deching; Wang, Meilin

2018-02-02

Glioblastoma multiforme (GBM), the most common malignant brain tumor, has a short period of survival even with recent multimodality treatment. The neurotropic JC polyomavirus (JCPyV) infects glial cells and oligodendrocytes and causes fatal progressive multifocal leukoencephalopathy in patients with AIDS. In this study, a possible gene therapy strategy for GBM using JCPyV virus-like particles (VLPs) as a gene delivery vector was investigated. We found that JCPyV VLPs were able to deliver the GFP reporter gene into tumor cells (U87-MG) for expression. In an orthotopic xenograft model, nude mice implanted with U87 cells expressing the near-infrared fluorescent protein and then treated by intratumoral injection of JCPyV VLPs carrying the thymidine kinase suicide gene, combined with ganciclovir administration, exhibited significantly prolonged survival and less tumor fluorescence during the experiment compared with controls. Furthermore, JCPyV VLPs were able to protect and deliver a suicide gene to distal subcutaneously implanted U87 cells in nude mice via blood circulation and inhibit tumor growth. These findings show that metastatic brain tumors can be targeted by JCPyV VLPs carrying a therapeutic gene, thus demonstrating the potential of JCPyV VLPs to serve as a gene therapy vector for the far highly treatment-refractory GBM.

Identification of multiple sites suitable for insertion of foreign genes in herpes simplex virus genomes.

Science.gov (United States)

Morimoto, Tomomi; Arii, Jun; Akashi, Hiroomi; Kawaguchi, Yasushi

2009-03-01

Information on sites in HSV genomes at which foreign gene(s) can be inserted without disrupting viral genes or affecting properties of the parental virus are important for basic research on HSV and development of HSV-based vectors for human therapy. The intergenic region between HSV-1 UL3 and UL4 genes has been reported to satisfy the requirements for such an insertion site. The UL3 and UL4 genes are oriented toward the intergenic region and, therefore, insertion of a foreign gene(s) into the region between the UL3 and UL4 polyadenylation signals should not disrupt any viral genes or transcriptional units. HSV-1 and HSV-2 each have more than 10 additional regions structurally similar to the intergenic region between UL3 and UL4. In the studies reported here, it has been demonstrated that insertion of a reporter gene expression cassette into several of the HSV-1 and HSV-2 intergenic regions has no effect on viral growth in cell culture or virulence in mice, suggesting that these multiple intergenic regions may be suitable HSV sites for insertion of foreign genes.

Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET

International Nuclear Information System (INIS)

Deroose, Christophe M.; Chitneni, Satish K.; Gijsbers, Rik; Vermaelen, Peter; Ibrahimi, Abdelilah; Balzarini, Jan; Baekelandt, Veerle; Verbruggen, Alfons; Nuyts, Johan; Debyser, Zeger; Bormans, Guy M.

2012-01-01

Introduction: Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. Methods: The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. Results: High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ∼ 1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. Conclusions: We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

Republished review: Gene therapy for ocular diseases.

Science.gov (United States)

Liu, Melissa M; Tuo, Jingsheng; Chan, Chi-Chao

2011-07-01

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.

Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae).

Science.gov (United States)

Park, Jeong-Mi; Manen, Jean-François; Schneeweiss, Gerald M

2007-06-01

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.

A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes.

Science.gov (United States)

Becker, A; Kaufmann, K; Freialdenhoven, A; Vincent, C; Li, M-A; Saedler, H; Theissen, G

2002-02-01

Class B floral homeotic genes specify the identity of petals and stamens during the development of angiosperm flowers. Recently, putative orthologs of these genes have been identified in different gymnosperms. Together, these genes constitute a clade, termed B genes. Here we report that diverse seed plants also contain members of a hitherto unknown sister clade of the B genes, termed B(sister) (B(s)) genes. We have isolated members of the B(s) clade from the gymnosperm Gnetum gnemon, the monocotyledonous angiosperm Zea mays and the eudicots Arabidopsis thaliana and Antirrhinum majus. In addition, MADS-box genes from the basal angiosperm Asarum europaeum and the eudicot Petunia hybrida were identified as B(s) genes. Comprehensive expression studies revealed that B(s) genes are mainly transcribed in female reproductive organs (ovules and carpel walls). This is in clear contrast to the B genes, which are predominantly expressed in male reproductive organs (and in angiosperm petals). Our data suggest that the B(s) genes played an important role during the evolution of the reproductive structures in seed plants. The establishment of distinct B and B(s) gene lineages after duplication of an ancestral gene may have accompanied the evolution of male microsporophylls and female megasporophylls 400-300 million years ago. During flower evolution, expression of B(s) genes diversified, but the focus of expression remained in female reproductive organs. Our findings imply that a clade of highly conserved close relatives of class B floral homeotic genes has been completely overlooked until recently and awaits further evaluation of its developmental and evolutionary importance. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00438-001-0615-8.

Meta-analysis and candidate gene mining of low-phosphorus tolerance in maize.

Science.gov (United States)

Zhang, Hongwei; Uddin, Mohammed Shalim; Zou, Cheng; Xie, Chuanxiao; Xu, Yunbi; Li, Wen-Xue

2014-03-01

Plants with tolerance to low-phosphorus (P) can grow better under low-P conditions, and understanding of genetic mechanisms of low-P tolerance can not only facilitate identifying relevant genes but also help to develop low-P tolerant cultivars. QTL meta-analysis was conducted after a comprehensive review of the reports on QTL mapping for low-P tolerance-related traits in maize. Meta-analysis produced 23 consensus QTL (cQTL), 17 of which located in similar chromosome regions to those previously reported to influence root traits. Meanwhile, candidate gene mining yielded 215 genes, 22 of which located in the cQTL regions. These 22 genes are homologous to 14 functionally characterized genes that were found to participate in plant low-P tolerance, including genes encoding miR399s, Pi transporters and purple acid phosphatases. Four cQTL loci (cQTL2-1, cQTL5-3, cQTL6-2, and cQTL10-2) may play important roles for low-P tolerance because each contains more original QTL and has better consistency across previous reports. © 2014 Institute of Botany, Chinese Academy of Sciences.

Androgen Receptor Gene Polymorphism, Aggression, and Reproduction in Tanzanian Foragers and Pastoralists

Science.gov (United States)

Butovskaya, Marina L.; Lazebny, Oleg E.; Vasilyev, Vasiliy A.; Dronova, Daria A.; Karelin, Dmitri V.; Mabulla, Audax Z. P.; Shibalev, Dmitri V.; Shackelford, Todd K.; Fink, Bernhard; Ryskov, Alexey P.

2015-01-01

The androgen receptor (AR) gene polymorphism in humans is linked to aggression and may also be linked to reproduction. Here we report associations between AR gene polymorphism and aggression and reproduction in two small-scale societies in northern Tanzania (Africa)—the Hadza (monogamous foragers) and the Datoga (polygynous pastoralists). We secured self-reports of aggression and assessed genetic polymorphism of the number of CAG repeats for the AR gene for 210 Hadza men and 229 Datoga men (aged 17–70 years). We conducted structural equation modeling to identify links between AR gene polymorphism, aggression, and number of children born, and included age and ethnicity as covariates. Fewer AR CAG repeats predicted greater aggression, and Datoga men reported more aggression than did Hadza men. In addition, aggression mediated the identified negative relationship between CAG repeats and number of children born. PMID:26291982

Polymorphism in TNP-1 gene of Murrah buffalo bulls | Panigrahi ...

African Journals Online (AJOL)

... spermatids, have been reported to be important for histone displacement and chromatin ... (TNP-1) gene was analyzed using polymerase chain reaction-single strand ... Analysis of TNP-1 gene sequence of Murrah buffalo revealed 3 single ...

Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

International Nuclear Information System (INIS)

Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

2013-01-01

Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

Energy Technology Data Exchange (ETDEWEB)

Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

2013-01-20

Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

Heritable and lineage-specific gene knockdown in zebrafish embryo.

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Mei Dong

Full Text Available BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA. The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.

Marfan syndrome with a complex chromosomal rearrangement including deletion of the FBN1 gene

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Colovati Mileny ES

2012-01-01

Full Text Available Abstract Background The majority of Marfan syndrome (MFS cases is caused by mutations in the fibrillin-1 gene (FBN1, mapped to chromosome 15q21.1. Only few reports on deletions including the whole FBN1 gene, detected by molecular cytogenetic techniques, were found in literature. Results We report here on a female patient with clinical symptoms of the MFS spectrum plus craniostenosis, hypothyroidism and intellectual deficiency who presents a 1.9 Mb deletion, including the FBN1 gene and a complex rearrangement with eight breakpoints involving chromosomes 6, 12 and 15. Discussion This is the first report of MFS with a complex chromosome rearrangement involving a deletion of FBN1 and contiguous genes. In addition to the typical clinical findings of the Marfan syndrome due to FBN1 gene haploinsufficiency, the patient presents features which may be due to the other gene deletions and possibly to the complex chromosome rearrangement.

Early evolution of the LIM homeobox gene family

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Srivastava, Mansi; Larroux, Claire; Lu, Daniel R; Mohanty, Kareshma; Chapman, Jarrod; Degnan, Bernard M; Rokhsar, Daniel S

2010-01-01

LIM homeobox (Lhx) transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known. We identified Lhx genes in two cnidarians and a placozoan and report the expression of Lhx genes during embryonic development in Nematostella and the demosponge Amphimedon. Members of the six major LIM homeobox subfamilies are represented in the genomes of the starlet sea anemone, Nematostella vectensis, and the placozoan Trichoplax adhaerens. The hydrozoan cnidarian, Hydra magnipapillata, has retained four of the six Lhx subfamilies, but apparently lost two others. Only three subfamilies are represented in the haplosclerid demosponge Amphimedon queenslandica. A tandem cluster of three Lhx genes of different subfamilies and a gene containing two LIM domains in the genome of T. adhaerens (an animal without any neurons) indicates that Lhx subfamilies were generated by tandem duplication. This tandem cluster in Trichoplax is likely a remnant of the original chromosomal context in which Lhx subfamilies first appeared. Three of the six Trichoplax Lhx genes are expressed in animals in laboratory culture, as are all Lhx genes in Hydra. Expression patterns of Nematostella Lhx genes correlate with neural territories in larval and juvenile polyp stages. In the aneural demosponge, A. queenslandica, the three Lhx genes are expressed widely during development, including in cells that are associated with the larval photosensory ring. The Lhx family expanded and diversified early in animal evolution, with all six subfamilies already diverged prior to the cnidarian-placozoan-bilaterian last common ancestor. In Nematostella, Lhx gene expression is correlated with neural

Early evolution of the LIM homeobox gene family

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Degnan Bernard M

2010-01-01

Full Text Available Abstract Background LIM homeobox (Lhx transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known. Results We identified Lhx genes in two cnidarians and a placozoan and report the expression of Lhx genes during embryonic development in Nematostella and the demosponge Amphimedon. Members of the six major LIM homeobox subfamilies are represented in the genomes of the starlet sea anemone, Nematostella vectensis, and the placozoan Trichoplax adhaerens. The hydrozoan cnidarian, Hydra magnipapillata, has retained four of the six Lhx subfamilies, but apparently lost two others. Only three subfamilies are represented in the haplosclerid demosponge Amphimedon queenslandica. A tandem cluster of three Lhx genes of different subfamilies and a gene containing two LIM domains in the genome of T. adhaerens (an animal without any neurons indicates that Lhx subfamilies were generated by tandem duplication. This tandem cluster in Trichoplax is likely a remnant of the original chromosomal context in which Lhx subfamilies first appeared. Three of the six Trichoplax Lhx genes are expressed in animals in laboratory culture, as are all Lhx genes in Hydra. Expression patterns of Nematostella Lhx genes correlate with neural territories in larval and juvenile polyp stages. In the aneural demosponge, A. queenslandica, the three Lhx genes are expressed widely during development, including in cells that are associated with the larval photosensory ring. Conclusions The Lhx family expanded and diversified early in animal evolution, with all six subfamilies already diverged prior to the cnidarian-placozoan-bilaterian last common ancestor. In

DDEC: Dragon database of genes implicated in esophageal cancer

International Nuclear Information System (INIS)

Essack, Magbubah; Radovanovic, Aleksandar; Schaefer, Ulf; Schmeier, Sebastian; Seshadri, Sundararajan V; Christoffels, Alan; Kaur, Mandeep; Bajic, Vladimir B

2009-01-01

Esophageal cancer ranks eighth in order of cancer occurrence. Its lethality primarily stems from inability to detect the disease during the early organ-confined stage and the lack of effective therapies for advanced-stage disease. Moreover, the understanding of molecular processes involved in esophageal cancer is not complete, hampering the development of efficient diagnostics and therapy. Efforts made by the scientific community to improve the survival rate of esophageal cancer have resulted in a wealth of scattered information that is difficult to find and not easily amendable to data-mining. To reduce this gap and to complement available cancer related bioinformatic resources, we have developed a comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer) with esophageal cancer related information, as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. Manually curated 529 genes differentially expressed in EC are contained in the database. We extracted and analyzed the promoter regions of these genes and complemented gene-related information with transcription factors that potentially control them. We further, precompiled text-mined and data-mined reports about each of these genes to allow for easy exploration of information about associations of EC-implicated genes with other human genes and proteins, metabolites and enzymes, toxins, chemicals with pharmacological effects, disease concepts and human anatomy. The resulting database, DDEC, has a useful feature to display potential associations that are rarely reported and thus difficult to identify. Moreover, DDEC enables inspection of potentially new 'association hypotheses' generated based on the precompiled reports. We hope that this resource will serve as a useful complement to the existing public resources and as a good starting point for researchers and physicians interested in EC genetics. DDEC is freely accessible to academic

Progress in Gene Therapy for Prostate Cancer

International Nuclear Information System (INIS)

Ahmed, Kamran A.; Davis, Brian J.; Wilson, Torrence M.; Wiseman, Gregory A.; Federspiel, Mark J.; Morris, John C.

2012-01-01

Gene therapy has held promise to correct various disease processes. Prostate cancer represents the second leading cause of cancer death in American men. A number of clinical trials involving gene therapy for the treatment of prostate cancer have been reported. The ability to efficiently transduce tumors with effective levels of therapeutic genes has been identified as a fundamental barrier to effective cancer gene therapy. The approach utilizing gene therapy in prostate cancer patients at our institution attempts to address this deficiency. The sodium-iodide symporter (NIS) is responsible for the ability of the thyroid gland to transport and concentrate iodide. The characteristics of the NIS gene suggest that it could represent an ideal therapeutic gene for cancer therapy. Published results from Mayo Clinic researchers have indicated several important successes with the use of the NIS gene and prostate gene therapy. Studies have demonstrated that transfer of the human NIS gene into prostate cancer using adenovirus vectors in vitro and in vivo results in efficient uptake of radioactive iodine and significant tumor growth delay with prolongation of survival. Preclinical successes have culminated in the opening of a phase I trial for patients with advanced prostate disease which is currently accruing patients. Further study will reveal the clinical promise of NIS gene therapy in the treatment of prostate as well as other malignancies.

Progress in Gene Therapy for Prostate Cancer

Energy Technology Data Exchange (ETDEWEB)

Ahmed, Kamran A.; Davis, Brian J. [Department of Radiation Oncology, Mayo Clinic, Rochester, MN (United States); Wilson, Torrence M. [Department of Urology, Mayo Clinic, Rochester, MN (United States); Wiseman, Gregory A. [Division of Nuclear Medicine, Mayo Clinic, Rochester, MN (United States); Federspiel, Mark J. [Department of Molecular Medicine, Mayo Clinic, Rochester, MN (United States); Morris, John C., E-mail: davis.brian@mayo.edu [Division of Endocrinology, Mayo Clinic, Rochester, MN (United States)

2012-11-19

Gene therapy has held promise to correct various disease processes. Prostate cancer represents the second leading cause of cancer death in American men. A number of clinical trials involving gene therapy for the treatment of prostate cancer have been reported. The ability to efficiently transduce tumors with effective levels of therapeutic genes has been identified as a fundamental barrier to effective cancer gene therapy. The approach utilizing gene therapy in prostate cancer patients at our institution attempts to address this deficiency. The sodium-iodide symporter (NIS) is responsible for the ability of the thyroid gland to transport and concentrate iodide. The characteristics of the NIS gene suggest that it could represent an ideal therapeutic gene for cancer therapy. Published results from Mayo Clinic researchers have indicated several important successes with the use of the NIS gene and prostate gene therapy. Studies have demonstrated that transfer of the human NIS gene into prostate cancer using adenovirus vectors in vitro and in vivo results in efficient uptake of radioactive iodine and significant tumor growth delay with prolongation of survival. Preclinical successes have culminated in the opening of a phase I trial for patients with advanced prostate disease which is currently accruing patients. Further study will reveal the clinical promise of NIS gene therapy in the treatment of prostate as well as other malignancies.

Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

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Sara E. Howden

2015-12-01

Full Text Available The derivation of genetically modified induced pluripotent stem (iPS cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

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Turner Renee J

2009-08-01

Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Novel exonic mutation inducing aberrant splicing in the IL10RA gene and resulting in infantile-onset inflammatory bowel disease: a case report.

Science.gov (United States)

Yanagi, Tadahiro; Mizuochi, Tatsuki; Takaki, Yugo; Eda, Keisuke; Mitsuyama, Keiichi; Ishimura, Masataka; Takada, Hidetoshi; Shouval, Dror S; Griffith, Alexandra E; Snapper, Scott B; Yamashita, Yushiro; Yamamoto, Ken

2016-01-28

Although deleterious mutations in interleukin-10 and its receptor molecules cause severe infantile-onset inflammatory bowel disease, there are no reports of mutations affecting this signaling pathway in Japanese patients. Here we report a novel exonic mutation in the IL10RA gene that caused unique splicing aberrations in a Japanese patient with infantile-onset of inflammatory bowel disease in association with immune thrombocytopenic purpura and a transient clinical syndrome mimicking juvenile myelomonocytic leukemia. A Japanese boy, who was the first child of non-consanguineous healthy parents, developed bloody diarrhea, perianal fistula, and folliculitis in early infancy and was diagnosed with inflammatory bowel disease. He also developed immune thrombocytopenic purpura and transient features mimicking juvenile myelomonocytic leukemia. The patient failed to respond to various treatments, including elemental diet, salazosulfapyridine, metronidazole, corticosteroid, infliximab, and adalimumab. We identified a novel mutation (c.537G > A, p.T179T) in exon 4 of the IL10RA gene causing unique splicing aberrations and resulting in lack of signaling through the interleukin-10 receptor. At 21 months of age, the patient underwent allogeneic hematopoietic stem cell transplantation and achieved clinical remission. We describe a novel exonic mutation in the IL10RA gene resulting in infantile-onset inflammatory bowel disease. This mutation might also be involved in his early-onset hematologic disorders. Physicians should be familiar with the clinical phenotype of IL-10 signaling defects in order to enable prompt diagnosis at an early age and referral for allogeneic hematopoietic stem cell transplantation.

Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

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Alnawaz Rehemtulla

2002-01-01

Full Text Available Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD along with an optical reporter gene (luciferase. Following intratumoral injection of the vector into orthotopic 9L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging

Large-scale inference of gene function through phylogenetic annotation of Gene Ontology terms: case study of the apoptosis and autophagy cellular processes.

Science.gov (United States)

Feuermann, Marc; Gaudet, Pascale; Mi, Huaiyu; Lewis, Suzanna E; Thomas, Paul D

2016-01-01

We previously reported a paradigm for large-scale phylogenomic analysis of gene families that takes advantage of the large corpus of experimentally supported Gene Ontology (GO) annotations. This 'GO Phylogenetic Annotation' approach integrates GO annotations from evolutionarily related genes across ∼100 different organisms in the context of a gene family tree, in which curators build an explicit model of the evolution of gene functions. GO Phylogenetic Annotation models the gain and loss of functions in a gene family tree, which is used to infer the functions of uncharacterized (or incompletely characterized) gene products, even for human proteins that are relatively well studied. Here, we report our results from applying this paradigm to two well-characterized cellular processes, apoptosis and autophagy. This revealed several important observations with respect to GO annotations and how they can be used for function inference. Notably, we applied only a small fraction of the experimentally supported GO annotations to infer function in other family members. The majority of other annotations describe indirect effects, phenotypes or results from high throughput experiments. In addition, we show here how feedback from phylogenetic annotation leads to significant improvements in the PANTHER trees, the GO annotations and GO itself. Thus GO phylogenetic annotation both increases the quantity and improves the accuracy of the GO annotations provided to the research community. We expect these phylogenetically based annotations to be of broad use in gene enrichment analysis as well as other applications of GO annotations.Database URL: http://amigo.geneontology.org/amigo. © The Author(s) 2016. Published by Oxford University Press.

Guidelines for the naming of genes, gene products, and mutants in the opportunistic protists.

Science.gov (United States)

Limper, Andrew H; Weiss, Louis M

2011-01-01

The opportunistic protists encompass a wide diversity of organisms including Pneumocystis, Toxoplasma, cryptosporidia, microsporidia, and related genera. Recent advances in the molecular biology and cellular biochemistry of these organisms have led to the identification of an ever growing numbers of key genes and their cognate proteins. Until now, these molecules have not been designated using any consistent nomenclature system, leading to considerable confusion. The participants of the 11th International Workshop on Opportunistic Protists met on August 3, 2010 to reach consensus of a nomenclature system for genes, gene products, and mutants in the opportunistic protists. The following summary reports the consensus agreement to move toward a unified nomenclature system for these organisms. The system is adapted from that used for Saccharomyces cerevisiae. © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

Science.gov (United States)

Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

2015-09-01

Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling.

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Stéphanie Cornen

Full Text Available Breast cancers (BCs of the luminal B subtype are estrogen receptor-positive (ER+, highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs, DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15 and UTRN (6q24, were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

Characterization of the global profile of genes expressed in cervical epithelium by Serial Analysis of Gene Expression (SAGE

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Piña-Sanchez Patricia

2005-09-01

Full Text Available Abstract Background Serial Analysis of Gene Expression (SAGE is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE, useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV, where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC. Results We report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation. Conclusion This first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs.

Science.gov (United States)

Chetverina, Darya; Savitskaya, Ekaterina; Maksimenko, Oksana; Melnikova, Larisa; Zaytseva, Olga; Parshikov, Alexander; Galkin, Alexander V; Georgiev, Pavel

2008-02-01

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3'UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari-Wari, Wari-gypsy or 1A2-Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.

A gene co-expression network in whole blood of schizophrenia patients is independent of antipsychotic-use and enriched for brain-expressed genes

DEFF Research Database (Denmark)

de Jong, Simone; Boks, Marco P M; Fuller, Tova F

2012-01-01

Despite large-scale genome-wide association studies (GWAS), the underlying genes for schizophrenia are largely unknown. Additional approaches are therefore required to identify the genetic background of this disorder. Here we report findings from a large gene expression study in peripheral blood...... of schizophrenia patients and controls. We applied a systems biology approach to genome-wide expression data from whole blood of 92 medicated and 29 antipsychotic-free schizophrenia patients and 118 healthy controls. We show that gene expression profiling in whole blood can identify twelve large gene co......, and regulated by the major histocompatibility (MHC) complex, which is intriguing in light of the fact that common allelic variants from the MHC region have been implicated in schizophrenia. This suggests that the MHC increases schizophrenia susceptibility via altered gene expression of regulatory genes...

Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion

NARCIS (Netherlands)

Jonkman, MF; Scheffer, H; Stulp, R; Pas, HH; Nijenhuis, Albertine; Heeres, K; Owaribe, K; Pulkkinen, L; Uitto, J

1997-01-01

Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction.

Science.gov (United States)

Tetteh, Antonia Y; Sun, Katherine H; Hung, Chiu-Yueh; Kittur, Farooqahmed S; Ibeanu, Gordon C; Williams, Daniel; Xie, Jiahua

2014-01-01

Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se(0)), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼ 50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼ 30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

Structured association analysis leads to insight into Saccharomyces cerevisiae gene regulation by finding multiple contributing eQTL hotspots associated with functional gene modules.

Science.gov (United States)

Curtis, Ross E; Kim, Seyoung; Woolford, John L; Xu, Wenjie; Xing, Eric P

2013-03-21

Association analysis using genome-wide expression quantitative trait locus (eQTL) data investigates the effect that genetic variation has on cellular pathways and leads to the discovery of candidate regulators. Traditional analysis of eQTL data via pairwise statistical significance tests or linear regression does not leverage the availability of the structural information of the transcriptome, such as presence of gene networks that reveal correlation and potentially regulatory relationships among the study genes. We employ a new eQTL mapping algorithm, GFlasso, which we have previously developed for sparse structured regression, to reanalyze a genome-wide yeast dataset. GFlasso fully takes into account the dependencies among expression traits to suppress false positives and to enhance the signal/noise ratio. Thus, GFlasso leverages the gene-interaction network to discover the pleiotropic effects of genetic loci that perturb the expression level of multiple (rather than individual) genes, which enables us to gain more power in detecting previously neglected signals that are marginally weak but pleiotropically significant. While eQTL hotspots in yeast have been reported previously as genomic regions controlling multiple genes, our analysis reveals additional novel eQTL hotspots and, more interestingly, uncovers groups of multiple contributing eQTL hotspots that affect the expression level of functional gene modules. To our knowledge, our study is the first to report this type of gene regulation stemming from multiple eQTL hotspots. Additionally, we report the results from in-depth bioinformatics analysis for three groups of these eQTL hotspots: ribosome biogenesis, telomere silencing, and retrotransposon biology. We suggest candidate regulators for the functional gene modules that map to each group of hotspots. Not only do we find that many of these candidate regulators contain mutations in the promoter and coding regions of the genes, in the case of the Ribi group

A gene expression analysis of cell wall biosynthetic genes in Malus × domestica infected by ‘Candidatus Phytoplasma mali’

Science.gov (United States)

Guerriero, Gea; Giorno, Filomena; Ciccotti, Anna Maria; Schmidt, Silvia; Baric, Sanja

2016-01-01

Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus × domestica Borkh., we investigated the expression of the cellulose (CesA) and callose synthase (CalS) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesAs showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several ‘knot’-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion. PMID:23086810

Cloning and characterization of human DNA repair genes

International Nuclear Information System (INIS)

Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Stewart, S.A.; Carrano, A.V.

1987-01-01

The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

Gametocidal genes of Aegilops: segregation distorters in wheat-Aegilops wide hybridization.

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Niranjana, M

2017-08-01

Aegilops is a genus belonging to the family Poaceace, which have played an indispensible role in the evolution of bread wheat and continues to do so by transferring genes by wide hybridization. Being the secondary gene pool of wheat, gene transfer from Aegilops poses difficulties and segregation distortion is common. Gametocidal genes are the most well characterized class of segregation distorters reported in interspecific crosses of wheat with Aegilops. These "selfish" genetic elements ensure their preferential transmission to progeny at the cost of gametes lacking them without providing any phenotypic benefits to the plant, thereby causing a proportional reduction in fertility. Gametocidal genes (Gc) have been reported in different species of Aegilops belonging to the sections Aegilops (Ae. geniculata and Ae. triuncialis), Cylindropyrum (Ae. caudata and Ae. cylindrica), and Sitopsis (Ae. longissima, Ae. sharonensis, and Ae. speltoides). Gametocidal activity is mostly confined to 2, 3, and 4 homeologous groups of C, S, S 1 , S sh , and M g genomes. Removal of such genes is necessary for successful alien gene introgression and can be achieved by mutagenesis or allosyndetic pairing. However, there are some instances where Gc genes are constructively utilized for development of deletion stocks in wheat, improving genetic variability and chromosome engineering.

Gene repressive mechanisms in the mouse brain involved in memory formation.

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Yu, Nam-Kyung; Kaang, Bong-Kiun

2016-04-01

Gene regulation in the brain is essential for long-term plasticity and memory formation. Despite this established notion, the quantitative translational map in the brain during memory formation has not been reported. To systematically probe the changes in protein synthesis during memory formation, our recent study exploited ribosome profiling using the mouse hippocampal tissues at multiple time points after a learning event. Analysis of the resulting database revealed novel types of gene regulation after learning. First, the translation of a group of genes was rapidly suppressed without change in mRNA levels. At later time points, the expression of another group of genes was downregulated through reduction in mRNA levels. This reduction was predicted to be downstream of inhibition of ESR1 (Estrogen Receptor 1) signaling. Overexpressing Nrsn1, one of the genes whose translation was suppressed, or activating ESR1 by injecting an agonist interfered with memory formation, suggesting the functional importance of these findings. Moreover, the translation of genes encoding the translational machineries was found to be suppressed, among other genes in the mouse hippocampus. Together, this unbiased approach has revealed previously unidentified characteristics of gene regulation in the brain and highlighted the importance of repressive controls. [BMB Reports 2016; 49(4): 199-200].

A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

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Mi Zhao

Full Text Available We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

Science.gov (United States)

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

2014-01-01

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

Science.gov (United States)

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

2014-01-01

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

Science.gov (United States)

Lai, Cheng-Wei; Chen, Hsiao-Ling; Yen, Chih-Ching; Wang, Jiun-Long; Yang, Shang-Hsun; Chen, Chuan-Mu

2016-12-01

Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

Science.gov (United States)

Gao, J; Naglich, J G; Laidlaw, J; Whaley, J M; Seizinger, B R; Kley, N

1995-02-15

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

A novel mutation of the fibrillin gene causing Ectopia lentis

Energy Technology Data Exchange (ETDEWEB)

Loennqvist, L.; Kainulainen, K.; Puhakka, L.; Peltonen, L. (National Public Health Institute, Helsinki (Finland)); Child, A. (St. George' s Hospital Medical School, London (United Kingdom)); Peltonen, L. (Duncan Guthrie Institute, Glasgow, Scotland (United Kingdom))

1994-02-01

Ectopia lentis (EL), a dominantly inherited connective tissue disorder, has been genetically linked to the fibrillin gene on chromosome 15 (FBN1) in earlier studies. Here, the authors report the first EL mutation in the FBN1 gene confirming that EL is caused by mutations of this gene. So far, several mutations in the FBN1 gene have been reported in patients with Marfan syndrome (MFS). EL and MFS are clinically related but distinct conditions with typical manifestations in the ocular and skeletal systems, the fundamental difference between them being the absence of cardiovascular involvement in EL. They report a point mutation, cosegregating with the disease in the described family, that displays EL over four generations. The mutation changes a conserved glutamic acid residue in an EGF-like motif, which is the major structural component of the fibrillin and is repeated throughout the polypeptide. In vitro mutagenetic studies have demonstrated the necessity of an analogous glutamic acid residue for calcium binding in an EGF-like repeat of human factor IX. This provides a possible explanation for the role of this mutation in the disease pathogenesis. 32 refs., 2 figs., 1 tab.

Real-time PCR detection of Fe-type nitrile hydratase genes from environmental isolates suggests horizontal gene transfer between multiple genera.

Science.gov (United States)

Coffey, Lee; Owens, Erica; Tambling, Karen; O'Neill, David; O'Connor, Laura; O'Reilly, Catherine

2010-11-01

Nitriles are widespread in the environment as a result of biological and industrial activity. Nitrile hydratases catalyse the hydration of nitriles to the corresponding amide and are often associated with amidases, which catalyze the conversion of amides to the corresponding acids. Nitrile hydratases have potential as biocatalysts in bioremediation and biotransformation applications, and several successful examples demonstrate the advantages. In this work a real-time PCR assay was designed for the detection of Fe-type nitrile hydratase genes from environmental isolates purified from nitrile-enriched soils and seaweeds. Specific PCR primers were also designed for amplification and sequencing of the genes. Identical or highly homologous nitrile hydratase genes were detected from isolates of numerous genera from geographically diverse sites, as were numerous novel genes. The genes were also detected from isolates of genera not previously reported to harbour nitrile hydratases. The results provide further evidence that many bacteria have acquired the genes via horizontal gene transfer. The real-time PCR assay should prove useful in searching for nitrile hydratases that could have novel substrate specificities and therefore potential in industrial applications.

New Mutation Identified in the SRY Gene High Mobility Group (HMG

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Feride İffet Şahin

2013-06-01

Full Text Available Mutations in the SRY gene prevent the differentiation of the fetal gonads to testes and cause developing female phenotype, and as a result sex reversal and pure gonadal dysgenesis (Swyer syndrome can be developed. Different types of mutations identified in the SRY gene are responsible for 15% of the gonadal dysgenesis. In this study, we report a new mutation (R132P in the High Mobility Group (HMG region of SRY gene was detected in a patient with primary amenorrhea who has 46,XY karyotype. This mutation leads to replacement of the polar and basic arginine with a nonpolar hydrophobic proline residue at aminoacid 132 in the nuclear localization signal region of the protein. With this case report we want to emphasize the genetic approach to the patients with gonadal dysgenesis. If Y chromosome is detected during cytogenetic analysis, revealing the presence of the SRY gene and identification of mutations in this gene by sequencing analysis is become important in.

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput.

Science.gov (United States)

Uwase, Grace; Enrico, Taylor P; Chelimo, David S; Keyser, Benjamin R; Johnson, Russell R

2018-03-30

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

The remnant of the European rabbit (Oryctolagus cuniculus IgD gene.

Directory of Open Access Journals (Sweden)

Dennis K Lanning

Full Text Available Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (Cδ gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit Cδ gene approximately 21 kb downstream of the IgM gene. The remnant Cδ locus lacks the δCH1 and hinge exons, but contains truncated δCH2 and δCH3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the Cδ gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

Science.gov (United States)

Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

2014-01-24

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

International Nuclear Information System (INIS)

Robinson, Claire; Kolb, Andreas F.

2009-01-01

The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A β-galactosidase reporter gene was inserted in place of the β-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the β-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal β-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the β-casein gene

Identification of an expressed gene in Dipylidium caninum.

Science.gov (United States)

Miranda, Rodrigo R C; Costa-Júnior, Livio M; Campos, Artur K; Santos, Hudson A; Rabelo, Elida M L

2004-10-01

Recombinant DNA studies have been focused on developing vaccines to different cestodes. But few studies involving Dipylidium caninum molecular biology and genes have been done. Only partial sequences of mitochondrial DNA and ribosomal RNA gene are available in databases. Any molecular work with this parasite, including epidemiology, study of drug-resistant strains, and vaccine development, is hampered by the lack of knowledge of its genome. Thus, the knowledge of specific genes of different developmental stages of D. caninum is crucial to locate potential targets to be used as candidates to develop a vaccine and/or new drugs against this parasite. Here we report, for the first time, the sequencing of a fragment of a D. caninum expressed gene.

A TAD further: exogenous control of gene activation.

Science.gov (United States)

Mapp, Anna K; Ansari, Aseem Z

2007-01-23

Designer molecules that can be used to impose exogenous control on gene transcription, artificial transcription factors (ATFs), are highly desirable as mechanistic probes of gene regulation, as potential therapeutic agents, and as components of cell-based devices. Recently, several advances have been made in the design of ATFs that activate gene transcription (activator ATFs), including reports of small-molecule-based systems and ATFs that exhibit potent activity. However, the many open mechanistic questions about transcriptional activators, in particular, the structure and function of the transcriptional activation domain (TAD), have hindered rapid development of synthetic ATFs. A compelling need thus exists for chemical tools and insights toward a more detailed portrait of the dynamic process of gene activation.

[Double mutant alleles in the EXT1 gene not previously reported in a teenager with hereditary multiple exostoses].

Science.gov (United States)

Cammarata-Scalisi, Francisco; Cozar, Mónica; Grinberg, Daniel; Balcells, Susana; Asteggiano, Carla G; Martínez-Domenech, Gustavo; Bracho, Ana; Sánchez, Yanira; Stock, Frances; Delgado-Luengo, Wilmer; Zara-Chirinos, Carmen; Chacín, José Antonio

2015-04-01

Hereditary forms of multiple exostoses, now called EXT1/EXT2-CDG within Congenital Disorders of Glycosylation, are the most common benign bone tumors in humans and clinical description consists of the formation of several cartilage-capped bone tumors, usually benign and localized in the juxta-epiphyseal region of long bones, although wide body dissemination in severe cases is not uncommon. Onset of the disease is variable ranging from 2-3 years up to 13-15 years with an estimated incidence ranging from 1/18,000 to 1/50,000 cases in European countries. We present a double mutant alleles in the EXT1 gene not previously reported in a teenager and her family with hereditary multiple exostoses.

Using SNP genetic markers to elucidate the linkage of the Co-34/Phg-3 anthracnose and angular leaf spot resistance gene cluster with the Ur-14 resistance gene

Science.gov (United States)

The Ouro Negro common bean cultivar contains the Co-34/Phg-3 gene cluster that confers resistance to the anthracnose (ANT) and angular leaf spot (ALS) pathogens. These genes are tightly linked on chromosome 4. Ouro Negro also has the Ur-14 rust resistance gene, reportedly in the vicinity of Co- 34; ...

A Comprehensive Classification and Evolutionary Analysis of Plant Homeobox Genes

OpenAIRE

Mukherjee, Krishanu; Brocchieri, Luciano; B?rglin, Thomas R.

2009-01-01

The full complement of homeobox transcription factor sequences, including genes and pseudogenes, was determined from the analysis of 10 complete genomes from flowering plants, moss, Selaginella, unicellular green algae, and red algae. Our exhaustive genome-wide searches resulted in the discovery in each class of a greater number of homeobox genes than previously reported. All homeobox genes can be unambiguously classified by sequence evolutionary analysis into 14 distinct classes also charact...

Large clusters of co-expressed genes in the Drosophila genome.

Science.gov (United States)

Boutanaev, Alexander M; Kalmykova, Alla I; Shevelyov, Yuri Y; Nurminsky, Dmitry I

2002-12-12

Clustering of co-expressed, non-homologous genes on chromosomes implies their co-regulation. In lower eukaryotes, co-expressed genes are often found in pairs. Clustering of genes that share aspects of transcriptional regulation has also been reported in higher eukaryotes. To advance our understanding of the mode of coordinated gene regulation in multicellular organisms, we performed a genome-wide analysis of the chromosomal distribution of co-expressed genes in Drosophila. We identified a total of 1,661 testes-specific genes, one-third of which are clustered on chromosomes. The number of clusters of three or more genes is much higher than expected by chance. We observed a similar trend for genes upregulated in the embryo and in the adult head, although the expression pattern of individual genes cannot be predicted on the basis of chromosomal position alone. Our data suggest that the prevalent mechanism of transcriptional co-regulation in higher eukaryotes operates with extensive chromatin domains that comprise multiple genes.

Characterization of the canine desmin (DES) gene and evaluation as a candidate gene for dilated cardiomyopathy in the Dobermann.

Science.gov (United States)

Stabej, Polona; Imholz, Sandra; Versteeg, Serge A; Zijlstra, Carla; Stokhof, Arnold A; Domanjko-Petric, Aleksandra; Leegwater, Peter A J; van Oost, Bernard A

2004-10-13

Canine-dilated cardiomyopathy (DCM) in dogs is a disease of the myocardium associated with dilatation and impaired contraction of the ventricles and is suspected to have a genetic cause. A missense mutation in the desmin gene (DES) causes DCM in a human family. Human DCM closely resembles the canine disease. In the present study, we evaluated whether DES gene mutations are responsible for DCM in Dobermann dogs. We have isolated bacterial artificial chromosome clones (BACs) containing the canine DES gene and determined the chromosomal location by fluorescence in situ hybridization (FISH). Using data deposited in the NCBI trace archive and GenBank, the canine DES gene DNA sequence was assembled and seven single nucleotide polymorphisms (SNPs) were identified. From the canine DES gene BAC clones, a polymorphic microsatellite marker was isolated. The microsatellite marker and four informative desmin SNPs were typed in a Dobermann family with frequent DCM occurrence, but the disease phenotype did not associate with a desmin haplotype. We concluded that mutations in the DES gene do not play a role in Dobermann DCM. Availability of the microsatellite marker, SNPs and DNA sequence reported in this study enable fast evaluation of the DES gene as a DCM candidate gene in other dog breeds with DCM occurrence.

Updated clusters of orthologous genes for Archaea: a complex ancestor of the Archaea and the byways of horizontal gene transfer

Directory of Open Access Journals (Sweden)

Wolf Yuri I

2012-12-01

Full Text Available Abstract Background Collections of Clusters of Orthologous Genes (COGs provide indispensable tools for comparative genomic analysis, evolutionary reconstruction and functional annotation of new genomes. Initially, COGs were made for all complete genomes of cellular life forms that were available at the time. However, with the accumulation of thousands of complete genomes, construction of a comprehensive COG set has become extremely computationally demanding and prone to error propagation, necessitating the switch to taxon-specific COG collections. Previously, we reported the collection of COGs for 41 genomes of Archaea (arCOGs. Here we present a major update of the arCOGs and describe evolutionary reconstructions to reveal general trends in the evolution of Archaea. Results The updated version of the arCOG database incorporates 91% of the pangenome of 120 archaea (251,032 protein-coding genes altogether into 10,335 arCOGs. Using this new set of arCOGs, we performed maximum likelihood reconstruction of the genome content of archaeal ancestral forms and gene gain and loss events in archaeal evolution. This reconstruction shows that the last Common Ancestor of the extant Archaea was an organism of greater complexity than most of the extant archaea, probably with over 2,500 protein-coding genes. The subsequent evolution of almost all archaeal lineages was apparently dominated by gene loss resulting in genome streamlining. Overall, in the evolution of Archaea as well as a representative set of bacteria that was similarly analyzed for comparison, gene losses are estimated to outnumber gene gains at least 4 to 1. Analysis of specific patterns of gene gain in Archaea shows that, although some groups, in particular Halobacteria, acquire substantially more genes than others, on the whole, gene exchange between major groups of Archaea appears to be largely random, with no major ‘highways’ of horizontal gene transfer. Conclusions The updated collection

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

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Eun Jin Cho

2012-03-01

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

Hepatitis C virus expressing reporter tagged NS5A protein

DEFF Research Database (Denmark)

2014-01-01

Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable...... growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5'UTR, or heterotypic 5'UTR (either of genotype 1a (strain H...

Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression.

Science.gov (United States)

Arnaiz, Olivier; Van Dijk, Erwin; Bétermier, Mireille; Lhuillier-Akakpo, Maoussi; de Vanssay, Augustin; Duharcourt, Sandra; Sallet, Erika; Gouzy, Jérôme; Sperling, Linda

2017-06-26

The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis

A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes.

Science.gov (United States)

Sahoo, Dipak K; Abeysekara, Nilwala S; Cianzio, Silvia R; Robertson, Alison E; Bhattacharyya, Madan K

2017-01-01

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes.

Directory of Open Access Journals (Sweden)

Dipak K Sahoo

Full Text Available Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs (F7 families were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

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Ruby Chandna

Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Downstream reporter gene imaging for signal transduction pathway of dopamine type 2 receptor

International Nuclear Information System (INIS)

Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung

2004-01-01

The Dopamine 2 receptor (D2R) signal pathway regulates gene expression by phosphorylation of proteins including cAMP reponse element-binding protein (CREB), a transcription factor. In this study, we developed a reporter strategy using the GAL4 fusion CREB to assess the phosphorylation of CREB, one of the targets of the D2R signal transduction pathway. We used three plasmids: GAL4 fusion transactivator (pCMV-CREB), firefly luciferase reporter with GAL4 binding sites (pG5-FLUC), and D2R plasmid (pCMV-D2R). Group 1 293T cells were transiently transfected with pCMV-CREB and pG5-FLUC, and group 2 cells were transfected with all three plasmids. Transfected cells were stimulated with different concentrations of dopamine (0-200 M). For animal studies, group 1 and 2 cells (1x10 6 ) were subcutaneously injected on the left and right thigh of six nude mice, respectively. Dopamine stimiulation was performed with intraperitoneal injection of L-DOPA incombination with carbidopa, a peripheral DOPA decarboxylase inhibitor. Bioluminescence optical imaging studies were performed before and after L-DOPA injection. In cell culture studies, group 1 cells showed strong luciferase activity which implies direct activation of the signaling pathway due to growth factors contained in culture medium. Group 2 cells showed strong luciferase activity and a further increase after administration of dopamine. In animal studies, group 1 and 2 cells showed bioluminescence signal before L-DOPA injection, but signal from group 2 cells significantly increased 12 h after L-DOPA injection. The signal from group 1 cells disappeared thereafter, but group 2 cells continued to show signal until 36 h of L-DOPA injection. This study demonstrates imaging of the D2R signal transduction pathway and should be useful for noninvasive imaging of downstream effects of G-coupled protein pathways

Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes

International Nuclear Information System (INIS)

Shibata, Toru; Akiyama, Nobutake; Noda, Makoto; Sasai, Keisuke; Hiraoka, Masahiro

1998-01-01

Purpose: Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy. Materials and Methods: Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed. Results: The construct pGL3/VEGF containing the 385 bp fragment of the 5' flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3-4, and 8-12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3' flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness. Conclusions: The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness

Methylated genes as new cancer biomarkers.

LENUS (Irish Health Repository)

Duffy, M J

2012-02-01

Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

HRGFish: A database of hypoxia responsive genes in fishes

Science.gov (United States)

Rashid, Iliyas; Nagpure, Naresh Sahebrao; Srivastava, Prachi; Kumar, Ravindra; Pathak, Ajey Kumar; Singh, Mahender; Kushwaha, Basdeo

2017-02-01

Several studies have highlighted the changes in the gene expression due to the hypoxia response in fishes, but the systematic organization of the information and the analytical platform for such genes are lacking. In the present study, an attempt was made to develop a database of hypoxia responsive genes in fishes (HRGFish), integrated with analytical tools, using LAMPP technology. Genes reported in hypoxia response for fishes were compiled through literature survey and the database presently covers 818 gene sequences and 35 gene types from 38 fishes. The upstream fragments (3,000 bp), covered in this database, enables to compute CG dinucleotides frequencies, motif finding of the hypoxia response element, identification of CpG island and mapping with the reference promoter of zebrafish. The database also includes functional annotation of genes and provides tools for analyzing sequences and designing primers for selected gene fragments. This may be the first database on the hypoxia response genes in fishes that provides a workbench to the scientific community involved in studying the evolution and ecological adaptation of the fish species in relation to hypoxia.

Characterization of MORE AXILLARY GROWTH genes in Populus.

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Olaf Czarnecki

Full Text Available Strigolactones are a new class of plant hormones that play a key role in regulating shoot branching. Studies of branching mutants in Arabidopsis, pea, rice and petunia have identified several key genes involved in strigolactone biosynthesis or signaling pathway. In the model plant Arabidopsis, MORE AXILLARY GROWTH1 (MAX1, MAX2, MAX3 and MAX4 are four founding members of strigolactone pathway genes. However, little is known about the strigolactone pathway genes in the woody perennial plants.Here we report the identification of MAX homologues in the woody model plant Populus trichocarpa. We identified the sequence homologues for each MAX protein in P. trichocarpa. Gene expression analysis revealed that Populus MAX paralogous genes are differentially expressed across various tissues and organs. Furthermore, we showed that Populus MAX genes could complement or partially complement the shoot branching phenotypes of the corresponding Arabidopsis max mutants.This study provides genetic evidence that strigolactone pathway genes are likely conserved in the woody perennial plants and lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.

Polycistronic gene expression in Aspergillus niger.

Science.gov (United States)

Schuetze, Tabea; Meyer, Vera

2017-09-25

Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

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Antonia Y. Tetteh

2014-01-01

Full Text Available Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0, but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3 treatment and then used quantitative real-time PCR (qRT-PCR to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

DDEC: Dragon database of genes implicated in esophageal cancer

KAUST Repository

Essack, Magbubah

2009-07-06

Background: Esophageal cancer ranks eighth in order of cancer occurrence. Its lethality primarily stems from inability to detect the disease during the early organ-confined stage and the lack of effective therapies for advanced-stage disease. Moreover, the understanding of molecular processes involved in esophageal cancer is not complete, hampering the development of efficient diagnostics and therapy. Efforts made by the scientific community to improve the survival rate of esophageal cancer have resulted in a wealth of scattered information that is difficult to find and not easily amendable to data-mining. To reduce this gap and to complement available cancer related bioinformatic resources, we have developed a comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer) with esophageal cancer related information, as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. Description: Manually curated 529 genes differentially expressed in EC are contained in the database. We extracted and analyzed the promoter regions of these genes and complemented gene-related information with transcription factors that potentially control them. We further, precompiled text-mined and data-mined reports about each of these genes to allow for easy exploration of information about associations of EC-implicated genes with other human genes and proteins, metabolites and enzymes, toxins, chemicals with pharmacological effects, disease concepts and human anatomy. The resulting database, DDEC, has a useful feature to display potential associations that are rarely reported and thus difficult to identify. Moreover, DDEC enables inspection of potentially new \\'association hypotheses\\' generated based on the precompiled reports. Conclusion: We hope that this resource will serve as a useful complement to the existing public resources and as a good starting point for researchers and physicians interested in EC genetics. DDEC is

Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: a performance overview.

Science.gov (United States)

Deliolanis, Nikolaos C; Ale, Angelique; Morscher, Stefan; Burton, Neal C; Schaefer, Karin; Radrich, Karin; Razansky, Daniel; Ntziachristos, Vasilis

2014-10-01

A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents.

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

NARCIS (Netherlands)

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

2014-01-01

Previous research has shown that loneliness has a heritable component and that genes within the serotonin-, dopamine-, and oxytocin systems are related to loneliness in adolescence. In the present study, the relation between the BDNF Val66Met polymorphism and loneliness in adolescent boys and girls

GoGene: gene annotation in the fast lane.

Science.gov (United States)

Plake, Conrad; Royer, Loic; Winnenburg, Rainer; Hakenberg, Jörg; Schroeder, Michael

2009-07-01

High-throughput screens such as microarrays and RNAi screens produce huge amounts of data. They typically result in hundreds of genes, which are often further explored and clustered via enriched GeneOntology terms. The strength of such analyses is that they build on high-quality manual annotations provided with the GeneOntology. However, the weakness is that annotations are restricted to process, function and location and that they do not cover all known genes in model organisms. GoGene addresses this weakness by complementing high-quality manual annotation with high-throughput text mining extracting co-occurrences of genes and ontology terms from literature. GoGene contains over 4,000,000 associations between genes and gene-related terms for 10 model organisms extracted from more than 18,000,000 PubMed entries. It does not cover only process, function and location of genes, but also biomedical categories such as diseases, compounds, techniques and mutations. By bringing it all together, GoGene provides the most recent and most complete facts about genes and can rank them according to novelty and importance. GoGene accepts keywords, gene lists, gene sequences and protein sequences as input and supports search for genes in PubMed, EntrezGene and via BLAST. Since all associations of genes to terms are supported by evidence in the literature, the results are transparent and can be verified by the user. GoGene is available at http://gopubmed.org/gogene.

Inflammatory myofibroblastic tumors of the lung carrying a chimeric A2M-ALK gene: report of 2 infantile cases and review of the differential diagnosis of infantile pulmonary lesions.

Science.gov (United States)

Tanaka, Mio; Kohashi, Kenichi; Kushitani, Kei; Yoshida, Misa; Kurihara, Sho; Kawashima, Masumi; Ueda, Yuka; Souzaki, Ryota; Kinoshita, Yoshiaki; Oda, Yoshinao; Takeshima, Yukio; Hiyama, Eiso; Taguchi, Tomoaki; Tanaka, Yukichi

2017-08-01

We report 2 infantile cases of pulmonary tumor carrying a chimeric A2M-ALK gene. A2M-ALK is a newly identified anaplastic lymphoma kinase (ALK)-related chimeric gene from a tumor diagnosed as fetal lung interstitial tumor (FLIT). FLIT is a recently recognized infantile pulmonary lesion defined as a mass-like lesion that morphologically resembles the fetal lung. Grossly, FLIT characteristically appears as a well-circumscribed spongy mass, whereas the tumors in these patients were solid and firm. Histologically, the tumors showed intrapulmonary lesions composed of densely proliferating polygonal or spindle-shaped mesenchymal cells with diffuse and dense infiltrations of inflammatory cells forming microcystic or micropapillary structures lined by thyroid transcription factor 1-positive pneumocytes, favoring inflammatory myofibroblastic tumor rather than FLIT. The proliferating cells were immunoreactive for ALK, and A2M-ALK was identified in both tumors with reverse-transcription polymerase chain reaction. The dense infiltration of inflammatory cells, immunoreactivity for ALK, and identification of an ALK-related chimeric gene suggested a diagnosis of inflammatory myofibroblastic tumor. Histologically, most reported FLITs show sparse inflammatory infiltrates and a relatively low density of interstitial cells in the septa, although prominent infiltration of inflammatory cells and high cellularity of interstitial cells are seen in some FLITs. The present cases suggest that ALK rearrangements, including the chimeric A2M-ALK gene, may be present in these infantile pulmonary lesions, especially those with inflammatory cell infiltration. We propose that these infantile pulmonary lesions containing a chimeric A2M-ALK gene be categorized as a specific type of inflammatory myofibroblastic tumor that develops exclusively in neonates and infants. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging gene expression in human mesenchymal stem cells: from small to large animals

DEFF Research Database (Denmark)

Willmann, Jürgen K; Paulmurugan, Ramasamy; Rodriguez-Porcel, Martin

2009-01-01

To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.......To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning....

Nephrogenic diabetes insipidus in a patient with L1 syndrome: a new report of a contiguous gene deletion syndrome including L1CAM and AVPR2.

Science.gov (United States)

Knops, Noël B B; Bos, Krista K; Kerstjens, Mieke; van Dael, Karin; Vos, Yvonne J

2008-07-15

We report on an infant boy with congenital hydrocephalus due to L1 syndrome and polyuria due to diabetes insipidus. We initially believed his excessive urine loss was from central diabetes insipidus and that the cerebral malformation caused a secondary insufficient pituitary vasopressin release. However, he failed to respond to treatment with a vasopressin analogue, which pointed to nephrogenic diabetes insipidus (NDI). L1 syndrome and X-linked NDI are distinct clinical disorders caused by mutations in the L1CAM and AVPR2 genes, respectively, located in adjacent positions in Xq28. In this boy we found a deletion of 61,577 basepairs encompassing the entire L1CAM and AVPR2 genes and extending into intron 7 of the ARHGAP4 gene. To our knowledge this is the first description of a patient with a deletion of these three genes. He is the second patient to be described with L1 syndrome and NDI. During follow-up he manifested complications from the hydrocephalus and NDI including global developmental delay and growth failure with low IGF-1 and hypothyroidism. 2008 Wiley-Liss, Inc.

Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

International Nuclear Information System (INIS)

Pluskota, W.E.; Michalczyk, D.J.; Gorecki, R.J.

2005-01-01

The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5' regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS, LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5' end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV

Concomitant homozygosity for the prothrombin gene variant with mild deficiency of antithrombin III in a patient with multiple hepatic infarctions: a case report

Directory of Open Access Journals (Sweden)

Macheta M

2010-04-01

Full Text Available Abstract Introduction Hereditary causes of visceral thrombosis or thrombosis should be sought among young patients. We present a case of a young man presenting with multiple hepatic infarctions resulting in portal hypertension due to homozygosity of the prothrombin gene mutation not previously described in literature. Case presentation A 42-year-old Caucasian man with a previous history of idiopathic deep vein thrombosis 11 years earlier presented with vague abdominal pains and mildly abnormal liver function tests. An ultrasound and computed tomography scan showed evidence of hepatic infarction and portal hypertension (splenic varices. A thrombophilia screen confirmed a homozygous mutation for the prothrombin gene mutation, with mildly reduced levels of anti-thrombin III (AT III. Subsequent testing of his father and brother revealed heterozygosity for the same gene mutation. Conclusion Hepatic infarction is unusual due to the rich dual arterial and venous blood supply to the liver. In the absence of an arterial or haemodynamic insult causing hepatic infarction, a thrombophilia should be considered. To our knowledge, this is the first reported case of a hepatic infarction due to homozygosity of the prothrombin gene mutation. It is unclear whether homozygotes have a higher risk of thrombosis than heterozygotes. In someone presenting with a first thrombosis with this mutation, the case for life-long anticoagulation is unclear, but it may be necessary to prevent a second and more severe second thrombotic event, as occurred in this case.

The Tomato Terpene Synthase Gene Family1[W][OA

Science.gov (United States)

Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

2011-01-01

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

Aplicação de genes marcadores em estudos de ecologia microbiana com ênfase no sistema GUS Applications of markers genes on ecologic microbial studies with enphasis on GUS system

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