Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

PubMed Central

Mittereder, N; March, K L; Trapnell, B C

1996-01-01

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy. PMID:8892868

A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function.

PubMed

Trubiroha, A; Gillotay, P; Giusti, N; Gacquer, D; Libert, F; Lefort, A; Haerlingen, B; De Deken, X; Opitz, R; Costagliola, S

2018-04-04

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor-Associated Genes in PDLSCs.

PubMed

Abd Rahman, Fazliny; Mohd Ali, Johari; Abdullah, Mariam; Abu Kasim, Noor Hayaty; Musa, Sabri

2016-07-01

This study investigates the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential, and expression of growth factor-associated genes in periodontal ligament stem cells (PDLSCs). Mesenchymal stem cells (MSCs) from PDL tissue were isolated from human premolars (n = 3). The MSCs' identity was confirmed by immunophenotyping and trilineage differentiation assays. Cell proliferation activity was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Polymerase chain reaction array was used to profile the expression of 84 growth factor-associated genes. Pathway analysis was used to identify the biologic functions and canonic pathways activated by ASA treatment. The osteogenic potential was evaluated through mineralization assay. ASA at 1,000 μM enhances osteogenic potential of PDLSCs. Using a fold change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and seven downregulated genes. Fibroblast growth factor 9 (FGF9), vascular endothelial growth factor A (VEGFA), interleukin-2, bone morphogenetic protein-10, VEGFC, and 2 (FGF2) were markedly upregulated (FC range, 6 to 15), whereas pleotropin, FGF5, brain-derived neurotrophic factor, and Dickkopf WNT signaling pathway inhibitor 1 were markedly downregulated (FC 32). Of the 84 growth factor-associated genes screened, 35 showed high cycle threshold values (≥35). ASA modulates the expression of growth factor-associated genes and enhances osteogenic potential in PDLSCs. ASA upregulated the expression of genes that could activate biologic functions and canonic pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration, and differentiation. These findings suggest that ASA enhances PDLSC function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay

PubMed Central

Quarello, Paola; Garelli, Emanuela; Brusco, Alfredo; Carando, Adriana; Mancini, Cecilia; Pappi, Patrizia; Vinti, Luciana; Svahn, Johanna; Dianzani, Irma; Ramenghi, Ugo

2012-01-01

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy. PMID:22689679

A comparative gene expression analysis of iron-limited cultures of Chaetoceros socialis and Pseudo-nitzschia arenysensis using newly developed iron assays

NASA Astrophysics Data System (ADS)

Abdala, Z. M.; Powell, K.; Cronin, D.; Chappell, D.

2016-02-01

A comparative gene expression analysis of iron-limited cultures of Chaetoceros socialis and Pseudo-nitzschia arenysensisusing newly developed iron assays Zuzanna M. Abdala, Kimberly Powell, Dylan P. Cronin, P. Dreux Chappell Diatoms, accounting for about 40% of the primary production in marine ecosystems, play a vital role in the dynamics of marine systems. Iron availability is understood to be a driving factor controlling productivity of many marine phytoplankton, including diatoms, as it functions as a cofactor for many proteins including several involved with photosynthetic processes. Previous work examining transcriptomes of diatoms of the Thalassiosira genus grown in controlled laboratory settings has identified genes whose expression can be used as sensitive markers of iron status. Data mining publically available diatom transcriptome data for these genes enables development of additional iron status assays for environmentally-relevant diatoms. For the present study, gene expression analysis of iron-limited laboratory cultures of Chaetoceros socialis and Pseudo-nitzschia arenysensis grown in continuous light was done using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). C. socialis and P. arenysensis serve as comparative models for analyzing gene expression in iron limitation in different ecological community assemblages. These data may ultimately assist to illuminate the function of iron in photosynthetic activity in diatoms.

Development of SNP Genotyping Assays for Seed Composition Traits in Soybean

PubMed Central

Patil, Gunvant; Chaudhary, Juhi; Vuong, Tri D.; Jenkins, Brian; Qiu, Dan; Kadam, Suhas; Shannon, Grover J.

2017-01-01

Seed composition is one of the most important determinants of the economic values in soybean. The quality and quantity of different seed components, such as oil, protein, and carbohydrates, are crucial ingredients in food, feed, and numerous industrial products. Soybean researchers have successfully developed and utilized a diverse set of molecular markers for seed trait improvement in soybean breeding programs. It is imperative to design and develop molecular assays that are accurate, robust, high-throughput, cost-effective, and available on a common genotyping platform. In the present study, we developed and validated KASP (Kompetitive allele-specific polymerase chain reaction) genotyping assays based on previously known functional mutant alleles for the seed composition traits, including fatty acids, oligosaccharides, trypsin inhibitor, and lipoxygenase. These assays were validated on mutant sources as well as mapping populations and precisely distinguish the homozygotes and heterozygotes of the mutant genes. With the obvious advantages, newly developed KASP assays in this study can substitute the genotyping assays that were previously developed for marker-assisted selection (MAS). The functional gene-based assay resource developed using common genotyping platform will be helpful to accelerate efforts to improve soybean seed composition traits. PMID:28630621

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

PubMed Central

Jin, Zhao; Di Rienzi, Sara C.; Janzon, Anders; Werner, Jeff J.; Angenent, Largus T.; Dangl, Jeffrey L.; Fowler, Douglas M.

2015-01-01

Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate. PMID:26637602

Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR

PubMed Central

Grüntzig, Verónica; Nold, Stephen C.; Zhou, Jizhong; Tiedje, James M.

2001-01-01

We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 106 gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier. PMID:11157241

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE PAGES

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; ...

2015-05-12

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

Improved Dual-Luciferase Reporter Assays for Nuclear Receptors

PubMed Central

Paguio, Aileen; Stecha, Pete; Wood, Keith V; Fan, Frank

2010-01-01

Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neoR fusion protein, which provides selection capability (G418 resistance, neoR) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains. PMID:21687560

Tobacco rattle virus (TRV) based silencing of cotton enoyl-CoA reductase (ECR) gene and the role of very long chain fatty acids in normal leaf development and resistance to wilt disease

USDA-ARS?s Scientific Manuscript database

A Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS) assay was employed as a reverse genetic approach to study gene function in cotton (Gossypium hirsutum). This approach was used to investigate the function of Enoyl-CoA reductase (GhECR) in pathogen defense. Amino acid sequence al...

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

PubMed

Lin, Shin-Jen; Ho, Hsin-Chiu; Lee, Yi-Fen; Liu, Ning-Chun; Liu, Su; Li, Gonghui; Shyr, Chih-Rong; Chang, Chawnshang

2012-06-07

Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice

PubMed Central

Badoni, Saurabh; Das, Sweta; Sayal, Yogesh K.; Gopalakrishnan, S.; Singh, Ashok K.; Rao, Atmakuri R.; Agarwal, Pinky; Parida, Swarup K.; Tyagi, Akhilesh K.

2016-01-01

We developed genome-wide 84634 ISM (intron-spanning marker) and 16510 InDel-fragment length polymorphism-based ILP (intron-length polymorphism) markers from genes physically mapped on 12 rice chromosomes. These genic markers revealed much higher amplification-efficiency (80%) and polymorphic-potential (66%) among rice accessions even by a cost-effective agarose gel-based assay. A wider level of functional molecular diversity (17–79%) and well-defined precise admixed genetic structure was assayed by 3052 genome-wide markers in a structured population of indica, japonica, aromatic and wild rice. Six major grain weight QTLs (11.9–21.6% phenotypic variation explained) were mapped on five rice chromosomes of a high-density (inter-marker distance: 0.98 cM) genetic linkage map (IR 64 x Sonasal) anchored with 2785 known/candidate gene-derived ISM and ILP markers. The designing of multiple ISM and ILP markers (2 to 4 markers/gene) in an individual gene will broaden the user-preference to select suitable primer combination for efficient assaying of functional allelic variation/diversity and realistic estimation of differential gene expression profiles among rice accessions. The genomic information generated in our study is made publicly accessible through a user-friendly web-resource, “Oryza ISM-ILP marker” database. The known/candidate gene-derived ISM and ILP markers can be enormously deployed to identify functionally relevant trait-associated molecular tags by optimal-resource expenses, leading towards genomics-assisted crop improvement in rice. PMID:27032371

Functional Assays to Screen and Dissect Genomic Hits: Doubling Down on the National Investment in Genomic Research.

PubMed

Musunuru, Kiran; Bernstein, Daniel; Cole, F Sessions; Khokha, Mustafa K; Lee, Frank S; Lin, Shin; McDonald, Thomas V; Moskowitz, Ivan P; Quertermous, Thomas; Sankaran, Vijay G; Schwartz, David A; Silverman, Edwin K; Zhou, Xiaobo; Hasan, Ahmed A K; Luo, Xiao-Zhong James

2018-04-01

The National Institutes of Health have made substantial investments in genomic studies and technologies to identify DNA sequence variants associated with human disease phenotypes. The National Heart, Lung, and Blood Institute has been at the forefront of these commitments to ascertain genetic variation associated with heart, lung, blood, and sleep diseases and related clinical traits. Genome-wide association studies, exome- and genome-sequencing studies, and exome-genotyping studies of the National Heart, Lung, and Blood Institute-funded epidemiological and clinical case-control studies are identifying large numbers of genetic variants associated with heart, lung, blood, and sleep phenotypes. However, investigators face challenges in identification of genomic variants that are functionally disruptive among the myriad of computationally implicated variants. Studies to define mechanisms of genetic disruption encoded by computationally identified genomic variants require reproducible, adaptable, and inexpensive methods to screen candidate variant and gene function. High-throughput strategies will permit a tiered variant discovery and genetic mechanism approach that begins with rapid functional screening of a large number of computationally implicated variants and genes for discovery of those that merit mechanistic investigation. As such, improved variant-to-gene and gene-to-function screens-and adequate support for such studies-are critical to accelerating the translation of genomic findings. In this White Paper, we outline the variety of novel technologies, assays, and model systems that are making such screens faster, cheaper, and more accurate, referencing published work and ongoing work supported by the National Heart, Lung, and Blood Institute's R21/R33 Functional Assays to Screen Genomic Hits program. We discuss priorities that can accelerate the impressive but incomplete progress represented by big data genomic research. © 2018 American Heart Association, Inc.

Inactivation of DNA mismatch repair by variants of uncertain significance in the PMS2 gene.

PubMed

Drost, Mark; Koppejan, Hester; de Wind, Niels

2013-11-01

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell-free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS. © 2013 WILEY PERIODICALS, INC.

Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae

PubMed Central

Wada, Ryuta; Maruyama, Jun-ichi; Yamaguchi, Haruka; Yamamoto, Nanase; Wagu, Yutaka; Paoletti, Mathieu; Archer, David B.; Dyer, Paul S.

2012-01-01

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. PMID:22327593

Characterization of BRCA2 Mutation in a Series of Functional Assays

DTIC Science & Technology

2005-05-01

9 Appendices .................................................................................... 10 Abstract Mutations in the BRCA2 gene account for...approximately 20% of all hereditary breast cancer. Many individuals undergo expensive clinical testing for mutations in the BRCA2 gene in order to...BRCA2 breast and ovarian cancer predisposition gene was identified in 1995. Mutations in the gene account for approximately 20% of all hereditary breast

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

PubMed

Palmieri, Ferdinando; Agrimi, Gennaro; Blanco, Emanuela; Castegna, Alessandra; Di Noia, Maria A; Iacobazzi, Vito; Lasorsa, Francesco M; Marobbio, Carlo M T; Palmieri, Luigi; Scarcia, Pasquale; Todisco, Simona; Vozza, Angelo; Walker, John

2006-01-01

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

A functional polymorphism of the TNF-{alpha} gene that is associated with type 2 DM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Susa, Shinji; Daimon, Makoto; Sakabe, Jun-Ichi

2008-05-09

To examine the association of the tumor necrosis factor-{alpha} (TNF-{alpha}) gene region with type 2 diabetes (DM), 11 single-nucleotide polymorphisms (SNPs) of the region were analyzed. The initial study using a sample set (148 cases vs. 227 controls) showed a significant association of the SNP IVS1G + 123A of the TNF-{alpha} gene with DM (p = 0.0056). Multiple logistic regression analysis using an enlarged sample set (225 vs. 716) revealed the significant association of the SNP with DM independently of any clinical traits examined (OR: 1.49, p = 0.014). The functional relevance of the SNP were examined by the electrophoreticmore » mobility shift assays using nuclear extracts from the U937 and NIH3T3 cells and luciferase assays in these cells with Simian virus 40 promoter- and TNF-{alpha} promoter-reporter gene constructs. The functional analyses showed that YY1 transcription factor bound allele-specifically to the SNP region and, the IVS1 + 123A allele had an increase in luciferase expression compared with the G allele.« less

Rrp1b, a New Candidate Susceptibility Gene for Breast Cancer Progression and Metastasis

PubMed Central

Crawford, Nigel P. S; Qian, Xiaolan; Ziogas, Argyrios; Papageorge, Alex G; Boersma, Brenda J; Walker, Renard C; Lukes, Luanne; Rowe, William L; Zhang, Jinghui; Ambs, Stefan; Lowy, Douglas R; Anton-Culver, Hoda; Hunter, Kent W

2007-01-01

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis. PMID:18081427

Loop-mediated isothermal amplification test for detection of Neisseria gonorrhoeae in urine samples and tolerance of the assay to the presence of urea.

PubMed

Edwards, Thomas; Burke, Patricia A; Smalley, Helen B; Gillies, Liz; Hobbs, Glyn

2014-06-01

A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤ 1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene

PubMed Central

Kim, Chun Sung; Choi, Hack Sun; Hwang, Cheol Kyu; Song, Kyu Young; Lee, Byung-Kwon; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2006-01-01

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N.Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464–46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator. PMID:17130167

Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.

PubMed

Loudig, Olivier; Brandwein-Gensler, Margaret; Kim, Ryung S; Lin, Juan; Isayeva, Tatyana; Liu, Christina; Segall, Jeffrey E; Kenny, Paraic A; Prystowsky, Michael B

2011-12-01

High-throughput gene expression profiling from formalin-fixed, paraffin-embedded tissues has become a reality, and several methods are now commercially available. The Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay (Illumina, Inc) is a full-transcriptome version of the original 512-gene complementary DNA-mediated annealing, selection, extension and ligation assay, allowing high-throughput profiling of 24,526 annotated genes from degraded and formalin-fixed, paraffin-embedded RNA. This assay has the potential to allow identification of novel gene signatures associated with clinical outcome using banked archival pathology specimen resources. We tested the reproducibility of the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay and its sensitivity for detecting differentially expressed genes in RNA extracted from matched fresh and formalin-fixed, paraffin-embedded cells, after 1 and 13 months of storage, using the human breast cell lines MCF7 and MCF10A. Then, using tumor worst pattern of invasion as a classifier, 1 component of the "risk model," we selected 12 formalin-fixed, paraffin-embedded oral squamous cell carcinomas for whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay analysis. We profiled 5 tumors with nonaggressive, nondispersed pattern of invasion, and 7 tumors with aggressive dispersed pattern of invasion and satellites scattered at least 1 mm apart. To minimize variability, the formalin-fixed, paraffin-embedded specimens were prepared from snap-frozen tissues, and RNA was obtained within 24 hours of fixation. One hundred four down-regulated genes and 72 up-regulated genes in tumors with aggressive dispersed pattern of invasion were identified. We performed quantitative reverse transcriptase polymerase chain reaction validation of 4 genes using Taqman assays and in situ protein detection of 1 gene by immunohistochemistry. Functional cluster analysis of genes up-regulated in tumors with aggressive pattern of invasion suggests presence of genes involved in cellular cytoarchitecture, some of which already associated with tumor invasion. Identification of these genes provides biologic rationale for our histologic classification, with regard to tumor invasion, and demonstrates that the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay is a powerful assay for profiling degraded RNA from archived specimens when combined with quantitative reverse transcriptase polymerase chain reaction validation. Copyright © 2011 Elsevier Inc. All rights reserved.

SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure

PubMed Central

2012-01-01

Background Single nucleotide polymorphism (SNP) validation and large-scale genotyping are required to maximize the use of DNA sequence variation and determine the functional relevance of candidate genes for complex stress tolerance traits through genetic association in rice. We used the bead array platform-based Illumina GoldenGate assay to validate and genotype SNPs in a select set of stress-responsive genes to understand their functional relevance and study the population structure in rice. Results Of the 384 putative SNPs assayed, we successfully validated and genotyped 362 (94.3%). Of these 325 (84.6%) showed polymorphism among the 91 rice genotypes examined. Physical distribution, degree of allele sharing, admixtures and introgression, and amino acid replacement of SNPs in 263 abiotic and 62 biotic stress-responsive genes provided clues for identification and targeted mapping of trait-associated genomic regions. We assessed the functional and adaptive significance of validated SNPs in a set of contrasting drought tolerant upland and sensitive lowland rice genotypes by correlating their allelic variation with amino acid sequence alterations in catalytic domains and three-dimensional secondary protein structure encoded by stress-responsive genes. We found a strong genetic association among SNPs in the nine stress-responsive genes with upland and lowland ecological adaptation. Higher nucleotide diversity was observed in indica accessions compared with other rice sub-populations based on different population genetic parameters. The inferred ancestry of 16% among rice genotypes was derived from admixed populations with the maximum between upland aus and wild Oryza species. Conclusions SNPs validated in biotic and abiotic stress-responsive rice genes can be used in association analyses to identify candidate genes and develop functional markers for stress tolerance in rice. PMID:22921105

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

PubMed Central

Wang, Xiaodan; Kültz, Dietmar

2017-01-01

Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of O. mossambicus. Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named “OSRE1.” Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1. Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation). PMID:28289196

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

PubMed Central

von Netzer, Frederick; Pilloni, Giovanni; Kleindienst, Sara; Krüger, Martin; Knittel, Katrin; Gründger, Friederike

2013-01-01

The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems. PMID:23124238

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

PubMed

Koko, Kiavash R; Chang, Shaohua; Hagaman, Ashleigh L; Fromer, Marc W; Nolan, Ryan S; Gaughan, John P; Zhang, Ping; Carpenter, Jeffrey P; Brown, Spencer A; Matthews, Martha; Bird, Dorothy

2017-06-01

Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 μM) or with low-dose paclitaxel (0.1 μM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles | Office of Cancer Genomics

Cancer.gov

Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic.

Identifying Functionally Linked Gene Modules Within Biological Pathways Assessed by ToxCast In Vitro Assays

EPA Science Inventory

The US EPA ToxCast program is using in vitro high-throughput screening assays to profile the bioactivity of environmental chemicals, with the ultimate goal of predicting in vivo toxicity. We hypothesize that in modeling toxicity it will be more constructive to understand the pert...

Genome-wide characterization of mammalian promoters with distal enhancer functions.

PubMed

Dao, Lan T M; Galindo-Albarrán, Ariel O; Castro-Mondragon, Jaime A; Andrieu-Soler, Charlotte; Medina-Rivera, Alejandra; Souaid, Charbel; Charbonnier, Guillaume; Griffon, Aurélien; Vanhille, Laurent; Stephen, Tharshana; Alomairi, Jaafar; Martin, David; Torres, Magali; Fernandez, Nicolas; Soler, Eric; van Helden, Jacques; Puthier, Denis; Spicuglia, Salvatore

2017-07-01

Gene expression in mammals is precisely regulated by the combination of promoters and gene-distal regulatory regions, known as enhancers. Several studies have suggested that some promoters might have enhancer functions. However, the extent of this type of promoters and whether they actually function to regulate the expression of distal genes have remained elusive. Here, by exploiting a high-throughput enhancer reporter assay, we unravel a set of mammalian promoters displaying enhancer activity. These promoters have distinct genomic and epigenomic features and frequently interact with other gene promoters. Extensive CRISPR-Cas9 genomic manipulation demonstrated the involvement of these promoters in the cis regulation of expression of distal genes in their natural loci. Our results have important implications for the understanding of complex gene regulation in normal development and disease.

The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.

PubMed

Fronza, G; Inga, A; Monti, P; Scott, G; Campomenosi, P; Menichini, P; Ottaggio, L; Viaggi, S; Burns, P A; Gold, B; Abbondandolo, A

2000-04-01

The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

PubMed

Kihara, Takahiro; Hiroe, Ayaka; Ishii-Hyakutake, Manami; Mizuno, Kouhei; Tsuge, Takeharu

2017-08-01

Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJ YB4 ) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJ YB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJ YB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJ YB4 , which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJ YB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

A functional variation in the hypocretin neuropeptide precursor gene may be associated with obstructive sleep apnea syndrome in Japan.

PubMed

Ahmed, Wael A; Tsutsumi, Makiko; Nakata, Seiichi; Mori, Terumi; Nishimura, Yoichi; Fujisawa, Toshiyuki; Kato, Ichiro; Nakashima, Mayuki; Kurahashi, Hiroki; Suzuki, Kenji

2012-04-01

To evaluate the association of hypocretin neuropeptide precursor gene (HCRT) variations with obstructive sleep apnea syndrome (OSAS) in a cohort of Japanese patients and to further evaluate whether the significant HCRT variations have potential functional consequences on HCRT expression. Case-control genetic association study. We studied the genetic variations within the HCRT gene. The study population consisted of 100 OSAS patients and 100 control subjects. The HCRT gene was amplified by polymerase chain reaction in all study subjects followed by direct sequencing and analysis of sequencing data. Two genetic variations within the HCRT intron, IVS1+16T>C (rs9902709) and IVS1-69G>C, were identified with significant differences between patients and controls (P < .05). A reporter gene assay using HeLa cells showed that the construct containing the C allele of the rs9902709 variation had significantly higher luciferase activity compared with the construct containing the T allele (P = .002). Furthermore, enzyme immunoassay revealed that subjects with T/C and C/C genotypes for rs9902709 had 1.4-fold and 1.5-fold increases in sera levels of orexin-A, respectively. Our genetic association study, followed by functional and quantitative phenotyping assays, demonstrated a functional locus within the HCRT gene, which may act to increase HCRT expression and lead to a protective effect against the development of OSAS. Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.

Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

PubMed Central

Sabbagh, Sébastien C.; Lepage, Christine; McClelland, Michael; Daigle, France

2012-01-01

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated. PMID:22574205

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

PubMed

Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Rui-Fen; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

2013-04-01

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

Inhibitory effect of cyanide on wastewater nitrification determined using SOUR and RNA-based gene-specific assays

EPA Science Inventory

The effect of CN- (CN-) on nitrification was examined with samples from nitrifying wastewater enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT-qPCR to quantify the transcripts of functional genes inv...

Targeted sequencing identifies 91 neurodevelopmental disorder risk genes with autism and developmental disability biases

PubMed Central

Stessman, Holly A. F.; Xiong, Bo; Coe, Bradley P.; Wang, Tianyun; Hoekzema, Kendra; Fenckova, Michaela; Kvarnung, Malin; Gerdts, Jennifer; Trinh, Sandy; Cosemans, Nele; Vives, Laura; Lin, Janice; Turner, Tychele N.; Santen, Gijs; Ruivenkamp, Claudia; Kriek, Marjolein; van Haeringen, Arie; Aten, Emmelien; Friend, Kathryn; Liebelt, Jan; Barnett, Christopher; Haan, Eric; Shaw, Marie; Gecz, Jozef; Anderlid, Britt-Marie; Nordgren, Ann; Lindstrand, Anna; Schwartz, Charles; Kooy, R. Frank; Vandeweyer, Geert; Helsmoortel, Celine; Romano, Corrado; Alberti, Antonino; Vinci, Mirella; Avola, Emanuela; Giusto, Stefania; Courchesne, Eric; Pramparo, Tiziano; Pierce, Karen; Nalabolu, Srinivasa; Amaral, David; Scheffer, Ingrid E.; Delatycki, Martin B.; Lockhart, Paul J.; Hormozdiari, Fereydoun; Harich, Benjamin; Castells-Nobau, Anna; Xia, Kun; Peeters, Hilde; Nordenskjöld, Magnus; Schenck, Annette; Bernier, Raphael A.; Eichler, Evan E.

2017-01-01

Gene-disruptive mutations contribute to the biology of neurodevelopmental disorders (NDDs), but most pathogenic genes are not known. We sequenced 208 candidate genes from >11,730 patients and >2,867 controls. We report 91 genes with an excess of de novo mutations or private disruptive mutations in 5.7% of patients, including 38 novel NDD genes. Drosophila functional assays of a subset bolster their involvement in NDDs. We identify 25 genes that show a bias for autism versus intellectual disability and highlight a network associated with high-functioning autism (FSIQ>100). Clinical follow-up for NAA15, KMT5B, and ASH1L reveals novel syndromic and non-syndromic forms of disease. PMID:28191889

Transcriptional regulation of podoplanin expression by Prox1 in lymphatic endothelial cells.

PubMed

Pan, Yanfang; Wang, Wen-di; Yago, Tadayuki

2014-07-01

Transcription factor prospero homeobox 1 (Prox-1) and podoplanin (PDPN), mucin-type transmembane protein, are both constantly expressed in lymphatic endothelial cells (LECs) and appear to function in an LEC-autonomous manner. Mice globally lacking PDPN (Pdpn(-/-)) develop abnormal and blood-filled lymphatic vessels that highly resemble those in inducible mice lacking Prox-1 (Prox1(-/-)). Prox1 has also been reported to induce PDPN expression in cultured ECs. Thus, we hypothesize that PDPN functions downstream of Prox1 and that its expression is regulated by Prox1 in LECs at the transcriptional level. We first identified four putative binding elements for Prox1 in the 5' upstream regulatory region of Pdpn gene and found that Prox1 directly binds to the 5' regulatory sequence of Pdpn gene in LECs by chromatin immunoprecipitation assay. DNA pull down assay confirmed that Prox1 binds to the putative binding element. In addition, luciferase reporter assay indicated that Prox1 binding to the 5' regulatory sequence of Pdpn regulates Pdpn gene expression. We are therefore the first to experimentally demonstrate that Prox1 regulates PDPN expression at the transcriptional level in the lymphatic vascular system. Copyright © 2014 Elsevier Inc. All rights reserved.

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

PubMed Central

Carver-Brown, Rachel K.; Reis, Arthur H.; Rice, Lisa M.; Czajka, John W.; Wangh, Lawrence J.

2012-01-01

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis. PMID:23326668

Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

PubMed

Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

2018-06-01

To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions.

PubMed

Skvortsova, Yulia V; Kondratieva, Sofia A; Zinovyeva, Marina V; Nikolaev, Lev G; Azhikina, Tatyana L; Gainetdinov, Ildar V

2016-01-01

Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression.

Functional characterization of p53 pathway components in the ancient metazoan Trichoplax adhaerens

NASA Astrophysics Data System (ADS)

Siau, Jia Wei; Coffill, Cynthia R.; Zhang, Weiyun Villien; Tan, Yaw Sing; Hundt, Juliane; Lane, David; Verma, Chandra; Ghadessy, Farid

2016-09-01

The identification of genes encoding a p53 family member and an Mdm2 ortholog in the ancient placozoan Trichoplax adhaerens advocates for the evolutionary conservation of a pivotal stress-response pathway observed in all higher eukaryotes. Here, we recapitulate several key functionalities ascribed to this known interacting protein pair by analysis of the placozoan proteins (Tap53 and TaMdm2) using both in vitro and cellular assays. In addition to interacting with each other, the Tap53 and TaMdm2 proteins are also able to respectively bind human Mdm2 and p53, providing strong evidence for functional conservation. The key p53-degrading function of Mdm2 is also conserved in TaMdm2. Tap53 retained DNA binding associated with p53 transcription activation function. However, it lacked transactivation function in reporter genes assays using a heterologous cell line, suggesting a cofactor incompatibility. Overall, the data supports functional roles for TaMdm2 and Tap53, and further defines the p53 pathway as an evolutionary conserved fulcrum mediating cellular response to stress.

Comparison of the QuantiGene 2.0 Assay and Real-Time RT-PCR in the Detection of p53 Isoform mRNA Expression in Formalin-Fixed Paraffin-Embedded Tissues- A Preliminary Study

PubMed Central

Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.

2016-01-01

p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134

Exploiting Pre-rRNA Processing in Diamond Blackfan Anemia Gene Discovery and Diagnosis

PubMed Central

Farrar, Jason E.; Quarello, Paola; Fisher, Ross; O’Brien, Kelly A.; Aspesi, Anna; Parrella, Sara; Henson, Adrianna L.; Seidel, Nancy E.; Atsidaftos, Eva; Prakash, Supraja; Bari, Shahla; Garelli, Emanuela; Arceci, Robert J.; Dianzani, Irma; Ramenghi, Ugo; Vlachos, Adrianna; Lipton, Jeffrey M.; Bodine, David M.; Ellis, Steven R.

2014-01-01

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene. PMID:25042156

Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells.

PubMed

De Sutter, Valerie; Vanderhaeghen, Rudy; Tilleman, Sofie; Lammertyn, Freya; Vanhoutte, Isabelle; Karimi, Mansour; Inzé, Dirk; Goossens, Alain; Hilson, Pierre

2005-12-01

Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.

Identification of candidate MLO powdery mildew susceptibility genes in cultivated Solanaceae and functional characterization of tobacco NtMLO1.

PubMed

Appiano, Michela; Pavan, Stefano; Catalano, Domenico; Zheng, Zheng; Bracuto, Valentina; Lotti, Concetta; Visser, Richard G F; Ricciardi, Luigi; Bai, Yuling

2015-10-01

Specific homologs of the plant Mildew Locus O (MLO) gene family act as susceptibility factors towards the powdery mildew (PM) fungal disease, causing significant economic losses in agricultural settings. Thus, in order to obtain PM resistant phenotypes, a general breeding strategy has been proposed, based on the selective inactivation of MLO susceptibility genes across cultivated species. In this study, PCR-based methodologies were used in order to isolate MLO genes from cultivated solanaceous crops that are hosts for PM fungi, namely eggplant, potato and tobacco, which were named SmMLO1, StMLO1 and NtMLO1, respectively. Based on phylogenetic analysis and sequence alignment, these genes were predicted to be orthologs of tomato SlMLO1 and pepper CaMLO2, previously shown to be required for PM pathogenesis. Full-length sequence of the tobacco homolog NtMLO1 was used for a heterologous transgenic complementation assay, resulting in its characterization as a PM susceptibility gene. The same assay showed that a single nucleotide change in a mutated NtMLO1 allele leads to complete gene loss-of-function. Results here presented, also including a complete overview of the tobacco and potato MLO gene families, are valuable to study MLO gene evolution in Solanaceae and for molecular breeding approaches aimed at introducing PM resistance using strategies of reverse genetics.

ATM function and its relationship with ATM gene mutations in chronic lymphocytic leukemia with the recurrent deletion (11q22.3-23.2).

PubMed

Jiang, Y; Chen, H-C; Su, X; Thompson, P A; Liu, X; Do, K-A; Wierda, W; Keating, M J; Plunkett, W

2016-09-02

Approximately 10-20% of chronic lymphocytic leukemia (CLL) patients exhibit del(11q22-23) before treatment, this cohort increases to over 40% upon progression following chemoimmunotherapy. The coding sequence of the DNA damage response gene, ataxia-telangiectasia-mutated (ATM), is contained in this deletion. The residual ATM allele is frequently mutated, suggesting a relationship between gene function and clinical response. To investigate this possibility, we sought to develop and validate an assay for the function of ATM protein in these patients. SMC1 (structural maintenance of chromosomes 1) and KAP1 (KRAB-associated protein 1) were found to be unique substrates of ATM kinase by immunoblot detection following ionizing radiation. Using a pool of eight fluorescence in situ hybridization-negative CLL samples as a standard, the phosphorylation of SMC1 and KAP1 from 46 del (11q22-23) samples was analyzed using normal mixture model-based clustering. This identified 13 samples (28%) that were deficient in ATM function. Targeted sequencing of the ATM gene of these samples, with reference to genomic DNA, revealed 12 somatic mutations and 15 germline mutations in these samples. No strong correlation was observed between ATM mutation and function. Therefore, mutation status may not be taken as an indicator of ATM function. Rather, a direct assay of the kinase activity should be used in the development of therapies.

Establishment of an efficient virus-induced gene silencing (VIGS) assay in Arabidopsis by Agrobacterium-mediated rubbing infection.

PubMed

Manhães, Ana Marcia E de A; de Oliveira, Marcos V V; Shan, Libo

2015-01-01

Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.

Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

PubMed Central

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth

2015-01-01

ABSTRACT Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. PMID:25968644

A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

PubMed

Grassian, A R; Scales, T M E; Knutson, S K; Kuntz, K W; McCarthy, N J; Lowe, C E; Moore, J D; Copeland, R A; Keilhack, H; Smith, J J; Wickenden, J A; Ribich, S

2015-01-01

Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

A quantitative assay measuring the function of lipase maturation factor 1

PubMed Central

Yin, Fen; Doolittle, Mark H.; Péterfy, Miklós

2009-01-01

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression. PMID:19471043

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

PubMed Central

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

1999-01-01

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

An effector Peptide family required for Drosophila toll-mediated immunity.

PubMed

Clemmons, Alexa W; Lindsay, Scott A; Wasserman, Steven A

2015-04-01

In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes - the Bomanins (Boms) - that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire.

Identification of an Envelope Protein from the FRD Family of Human Endogenous Retroviruses (HERV-FRD) Conferring Infectivity and Functional Conservation among Simians

PubMed Central

Blaise, Sandra; Ruggieri, Alessia; Dewannieux, Marie; Cosset, François-Loic; Heidmann, Thierry

2004-01-01

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians—from New World monkeys to humans—are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation—located in the TM subunit—was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution. PMID:14694139

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians.

PubMed

Blaise, Sandra; Ruggieri, Alessia; Dewannieux, Marie; Cosset, François-Loic; Heidmann, Thierry

2004-01-01

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.

A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

PubMed

Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

2008-11-01

A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS).

PubMed

Krol, Kamil; Jendrysek, Justyna; Debski, Janusz; Skoneczny, Marek; Kurlandzka, Anna; Kaminska, Joanna; Dadlez, Michal; Skoneczna, Adrianna

2017-04-11

Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a "DNA cloud assay". We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP).

Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

PubMed

Zhang, Zhichun; Tian, Hua; Lv, Ping; Wang, Weiping; Jia, Zhuqing; Wang, Sainan; Zhou, Chunyan; Gao, Xuejun

2015-01-01

Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

Transcriptional Factor DLX3 Promotes the Gene Expression of Enamel Matrix Proteins during Amelogenesis

PubMed Central

Zhang, Zhichun; Tian, Hua; Lv, Ping; Wang, Weiping; Jia, Zhuqing; Wang, Sainan; Zhou, Chunyan; Gao, Xuejun

2015-01-01

Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease. PMID:25815730

ROLE OF GENETIC SUSCEPTIBILITY TO LATENT ADENOVIRAL INFECTION AND DECREASED LUNG FUNCTION

PubMed Central

Kasuga, Ikuma; Hogg, James C.; Paré, Peter D.; Hayashi, Shizu; Sedgwick, Edward G.; Ruan, Jian; Wallace, Alison M.; He, Jian-Qing; Zhang, Xiaozhu; Sandford, Andrew J.

2009-01-01

Background Latent adenoviral infection may amplify cigarette smoke-induced lung inflammation and therefore play an important role in the development of chronic obstructive pulmonary disease (COPD). Adenoviruses can evade the human immune response via their 19-kDa protein (19K) which delays the expression of class I human leukocyte antigen (HLA) proteins. The 19K protein shows higher affinity to HLA-B7 and A2 compared with HLA-A1 and A3. The receptor for adenovirus (CXADR) and integrin β5 (ITGB5) are host factors which might affect adenovirus infection. Therefore, we investigated the contribution of HLA, CXADR, and ITGB5 genetic variants to the presence of the E1A gene and to level of lung function. Methods Study subjects were assayed for HLA-B7, A1, A2 and A3 by PCR-based assays using allele-specific primers. Polymorphisms of the CXADR and ITGB5 genes were genotyped by PCR-based restriction fragment length polymorphism assays. Detection of adenoviral E1A gene was performed by a real-time PCR TaqMan assay. Results E1A positive individuals have a lower FEV1 compared with E1A negative individuals. However, there was no significant difference in E1A positivity rate between the high (HLA-B7 and A2) and low (HLA-A1 and A3) 19K affinity groups. There was also no significant difference in FEV1 level between each affinity group. There was no significant difference in E1A positivity rate or lung function among the CXADR and ITGB5 genotypes. Conclusions Genetic variants in HLA, CXADR and ITGB5 do not influence latent adenoviral infections and are not associated with COPD. PMID:19502044

Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making

PubMed Central

Thouvenot, Pierre; Ben Yamin, Barbara; Fourrière, Lou; Lescure, Aurianne; Boudier, Thomas; Del Nery, Elaine; Chauchereau, Anne; Goldgar, David E.; Stoppa-Lyonnet, Dominique; Nicolas, Alain; Millot, Gaël A.

2016-01-01

Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening. PMID:27272900

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

PubMed

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.

An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.

PubMed

Bhandari, Poonam; Kendler, Kenneth S; Bettinger, Jill C; Davies, Andrew G; Grotewiel, Mike

2009-10-01

Ethanol induces similar behavioral responses in mammals and the fruit fly, Drosophila melanogaster. By coupling assays for ethanol-related behavior to the genetic tools available in flies, a number of genes have been identified that influence physiological responses to ethanol. To enhance the utility of the Drosophila model for investigating genes involved in ethanol-related behavior, we explored the value of an assay that measures the sedative effects of ethanol on negative geotaxis, an evoked locomotor response. We established eRING (ethanol Rapid Iterative Negative Geotaxis) as an assay for quantitating the sedative effects of ethanol on negative geotaxis (i.e., startle-induced climbing). We validated the assay by assessing acute sensitivity to ethanol and rapid ethanol tolerance in several different control strains and in flies with mutations known to disrupt these behaviors. We also used eRING in a candidate screen to identify mutants with altered ethanol-related behaviors. Negative geotaxis measured in eRING assays was dose-dependently impaired by ethanol exposure. Flies developed tolerance to the intoxicating effects of ethanol when tested during a second exposure. Ethanol sensitivity and rapid ethanol tolerance varied across 4 control strains, but internal ethanol concentrations were indistinguishable in the 4 strains during a first and second challenge with ethanol. Ethanol sensitivity and rapid ethanol tolerance, respectively, were altered in flies with mutations in amnesiac and hangover, genes known to influence these traits. Additionally, mutations in the beta integrin gene myospheroid and the alpha integrin gene scab increased the initial sensitivity to ethanol and enhanced the development of rapid ethanol tolerance without altering internal ethanol concentrations. The eRING assay is suitable for investigating genetic mechanisms that influence ethanol sensitivity and rapid ethanol tolerance. Ethanol sensitivity and rapid ethanol tolerance depend on the function of alpha and beta integrins in flies.

Nano-sized calcium phosphate particles for periodontal gene therapy.

PubMed

Elangovan, Satheesh; Jain, Shardool; Tsai, Pei-Chin; Margolis, Henry C; Amiji, Mansoor

2013-01-01

Growth factors such as platelet-derived growth factor (PDGF) have significantly enhanced periodontal therapy outcomes with a high degree of variability, mostly due to the lack of continual supply for a required period of time. One method to overcome this barrier is gene therapy. The aim of this in vitro study is to evaluate PDGF-B gene delivery in fibroblasts using nano-sized calcium phosphate particles (NCaPP) as vectors. NCaPP incorporating green fluorescent protein (NCaPP-GFP) and PDGF-B (NCaPP-PDGF-B) plasmids were synthesized using an established precipitation system and characterized using transmission electron microscopy and 1.2% agarose gel electrophoresis. Biocompatibility and transfection of the nanoplexes in fibroblasts were evaluated using cytotoxicity assay and florescence microscopy, respectively. Polymerase chain reaction and enzyme-linked immunosorbent assay were performed to evaluate PDGF-B transfection after different time points of treatments, and the functionality of PDGF-B transfection was evaluated using the cell proliferation assay. Synthesized NCaPP nanoplexes incorporating the genes of GFP and PDGF-B were spherical in shape and measured about 30 to 50 nm in diameter. Gel electrophoresis confirmed DNA incorporation and stability within the nanoplexes, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reagent assay demonstrated their biocompatibility in fibroblasts. In vitro transfection studies revealed a higher and longer lasting transfection after NCaPP-PDGF-B treatment, which lasted up to 96 hours. Significantly enhanced fibroblast proliferation observed in NCaPP-PDGF-B-treated cells confirmed the functionality of these nanoplexes. NCaPP demonstrated higher levels of biocompatibility and efficiently transfected PDGF plasmids into fibroblasts under described in vitro conditions.

NMDA Receptors Regulate Genes Responsible for Major Immune Functions of Mononuclears in Human Peripheral Blood.

PubMed

Kuzmina, U Sh; Zainullina, L F; Sadovnikov, S V; Vakhitov, V A; Vakhitova, Yu V

2018-06-19

To determine the role of NMDA receptors in the functional regulation of immunocompetent cells, comparative assay was carried out for genes expressed in the mononuclears in peripheral blood of healthy persons under normal conditions and after blockade of these receptors. The genes, whose expression changed in response to blockade of NMDA receptors in mononuclears, encode the products involved in regulation of the major functions of immune cells, such as proliferation (IL4, VCAM1, and CDKN2A), apoptosis (BAX, MYC, CDKN2A, HSPB1, and CADD45A), activation (IL4R, IL4, VCAM1, and CDKN2A), and differentiation (IL4, VCAM1, and BAX).

Competitive Genomic Screens of Barcoded Yeast Libraries

PubMed Central

Urbanus, Malene; Proctor, Michael; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

2011-01-01

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment. PMID:21860376

Functional SNP associated with birth weight in independent populations identified with a permutation step added to GBLUP-GWAS

USDA-ARS?s Scientific Manuscript database

This study was conducted as an initial assessment of a newly available genotyping assay containing about 34,000 common SNP included on previous SNP chips, and 199,000 sequence variants predicted to affect gene function. Objectives were to identify functional variants associated with birth weight in...

Genome-wide identification of bacterial plant colonization genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cole, Benjamin J.; Feltcher, Meghan E.; Waters, Robert J.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44more » other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Here, our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.« less

Genome-wide identification of bacterial plant colonization genes

DOE PAGES

Cole, Benjamin J.; Feltcher, Meghan E.; Waters, Robert J.; ...

2017-09-22

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44more » other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Here, our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.« less

HER2 missense mutations have distinct effects on oncogenic signaling and migration

PubMed Central

Zabransky, Daniel J.; Yankaskas, Christopher L.; Cochran, Rory L.; Wong, Hong Yuen; Croessmann, Sarah; Chu, David; Kavuri, Shyam M.; Red Brewer, Monica; Rosen, D. Marc; Dalton, W. Brian; Cimino-Mathews, Ashley; Cravero, Karen; Button, Berry; Kyker-Snowman, Kelly; Cidado, Justin; Erlanger, Bracha; Parsons, Heather A.; Manto, Kristen M.; Bose, Ron; Lauring, Josh; Arteaga, Carlos L.; Konstantopoulos, Konstantinos; Park, Ben Ho

2015-01-01

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as “negative” by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them. PMID:26508629

A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

PubMed

Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

2015-10-01

During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Integration of biological data by kernels on graph nodes allows prediction of new genes involved in mitotic chromosome condensation

PubMed Central

Hériché, Jean-Karim; Lees, Jon G.; Morilla, Ian; Walter, Thomas; Petrova, Boryana; Roberti, M. Julia; Hossain, M. Julius; Adler, Priit; Fernández, José M.; Krallinger, Martin; Haering, Christian H.; Vilo, Jaak; Valencia, Alfonso; Ranea, Juan A.; Orengo, Christine; Ellenberg, Jan

2014-01-01

The advent of genome-wide RNA interference (RNAi)–based screens puts us in the position to identify genes for all functions human cells carry out. However, for many functions, assay complexity and cost make genome-scale knockdown experiments impossible. Methods to predict genes required for cell functions are therefore needed to focus RNAi screens from the whole genome on the most likely candidates. Although different bioinformatics tools for gene function prediction exist, they lack experimental validation and are therefore rarely used by experimentalists. To address this, we developed an effective computational gene selection strategy that represents public data about genes as graphs and then analyzes these graphs using kernels on graph nodes to predict functional relationships. To demonstrate its performance, we predicted human genes required for a poorly understood cellular function—mitotic chromosome condensation—and experimentally validated the top 100 candidates with a focused RNAi screen by automated microscopy. Quantitative analysis of the images demonstrated that the candidates were indeed strongly enriched in condensation genes, including the discovery of several new factors. By combining bioinformatics prediction with experimental validation, our study shows that kernels on graph nodes are powerful tools to integrate public biological data and predict genes involved in cellular functions of interest. PMID:24943848

Towards an informative mutant phenotype for every bacterial gene

DOE PAGES

Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; ...

2014-08-11

Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

Misregulation of Gene Expression and Sterility in Interspecies Hybrids: Causal Links and Alternative Hypotheses.

PubMed

Civetta, Alberto

2016-05-01

Understanding the origin of species is of interest to biologist in general and evolutionary biologist in particular. Hybrid male sterility (HMS) has been a focus in studies of speciation because sterility imposes a barrier to free gene flow between organisms, thus effectively isolating them as distinct species. In this review, I focus on the role of differential gene expression in HMS and speciation. Microarray and qPCR assays have established associations between misregulation of gene expression and sterility in hybrids between closely related species. These studies originally proposed disrupted expression of spermatogenesis genes as a causative of sterility. Alternatively, rapid genetic divergence of regulatory elements, particularly as they relate to the male sex (fast-male evolution), can drive the misregulation of sperm developmental genes in the absence of sterility. The use of fertile hybrids (both backcross and F1 progeny) as controls has lent support to this alternative explanation. Differences in gene expression between fertile and sterile hybrids can also be influenced by a pattern of faster evolution of the sex chromosome (fast-X evolution) than autosomes. In particular, it would be desirable to establish whether known X-chromosome sterility factors can act as trans-regulatory drivers of genome-wide patterns of misregulation. Genome-wide expression studies coupled with assays of proxies of sterility in F1 and BC progeny have identified candidate HMS genes but functional assays, and a better phenotypic characterization of sterility phenotypes, are needed to rigorously test how these genes might contribute to HMS.

Design and interpretation of microRNA-reporter gene activity.

PubMed

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

2013-06-15

MicroRNAs (miRNAs) are small noncoding RNA molecules that act as sequence specificity guides to direct post-transcriptional gene silencing. In doing so, miRNAs regulate many critical developmental processes, including cellular proliferation, differentiation, migration, and apoptosis, as well as more specialized biological functions such as dendritic spine development and synaptogenesis. Interactions between miRNAs and their miRNA recognition elements occur via partial complementarity, rendering tremendous redundancy in targeting such that miRNAs are predicted to regulate 60% of the genome, with each miRNA estimated to regulate more than 200 genes. Because these predictions are prone to false positives and false negatives, there is an ever present need to provide material support to these assertions to firmly establish the biological function of specific miRNAs in both normal and pathophysiological contexts. Using schizophrenia-associated miR-181b as an example, we present detailed guidelines and novel insights for the rapid establishment of a streamlined miRNA-reporter gene assay and explore various design concepts for miRNA-reporter gene applications, including bidirectional miRNA modulation. In exemplifying this approach, we report seven novel miR-181b target sites for five schizophrenia candidate genes (DISC1, BDNF, ENKUR, GRIA1, and GRIK1) and dissect a number of vital concepts regarding future developments for miRNA-reporter gene assays and the interpretation of their results. Copyright © 2013 Elsevier Inc. All rights reserved.

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability.

PubMed

Lovelock, Paul K; Wong, Ee Ming; Sprung, Carl N; Marsh, Anna; Hobson, Karen; French, Juliet D; Southey, Melissa; Sculley, Tom; Pandeya, Nirmala; Brown, Melissa A; Chenevix-Trench, Georgia; Spurdle, Amanda B; McKay, Michael J

2007-09-01

Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting. Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls. Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status. Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

Poly-γ-glutamic acid productivity of Bacillus subtilis BsE1 has positive function in motility and biocontrol against Fusarium graminearum.

PubMed

Wang, Luyao; Wang, Ning; Mi, Dandan; Luo, Yuming; Guo, Jianhua

2017-07-01

In this study, we investigate the relationship between γ-PGA productivity and biocontrol capacity of Bacillus subtilis BsE1; one bacterial isolate displayed 62.14% biocontrol efficacy against Fusarium root rot. The γ-PGA yield assay, motility assay, wheat root colonization assay, and biological control assay were analysed in different γ-PGA yield mutants of BsE1. The pgsB (PGA-synthase-CapB gene) deleted mutant of BsE1 reduced γ-PGA yield and exhibited apparent decline of in vitro motile ability. Deletion of pgsB impaired colonizing capacity of BsE1 on wheat root in 30 days, also lowered biocontrol efficacies from 62.08% (wild type BsE1) to 14.22% in greenhouse experiment against Fusarium root rot. The knockout of pgdS and ggt (genes relate to two γ-PGA degrading enzymes) on BsE1, leads to a considerable improvement in polymer yield and biocontrol efficacy, which attains higher level compared with wild type BsE1. Compared with ΔpgsB mutant, defense genes related to reactive oxygen species (ROS) and phytoalexin expressed changes by notable levels on wheat roots treated with BsE1, demonstrating the functional role γ-PGA plays in biocontrol against Fusarium root rot. γ-PGA is not only important to the motile and plant root colonization ability of BsE1, but also essential to the biological control performed by BsE1 against Fusarium root rot. Our goal in this study is to reveals a new perspective of BCAs screening on bacterial isolates, without good performance during pre-assays of antagonism ability.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.

Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study.

PubMed

Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

2016-11-10

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the functions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM.

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

PubMed

Looi, Kevin; Troy, Niamh M; Garratt, Luke W; Iosifidis, Thomas; Bosco, Anthony; Buckley, Alysia G; Ling, Kak-Ming; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Shaw, Nicole C; Sutanto, Erika N; Zosky, Graeme R; Rigby, Paul J; Larcombe, Alexander N; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2016-10-11

No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Assaying gene function by growth competition experiment.

PubMed

Merritt, Joshua; Edwards, Jeremy S

2004-07-01

High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

Integrated microfluidic devices for combinatorial cell-based assays.

PubMed

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert; Radu, Caius G; Witte, Owen N; Lee, Ki-Bum; Tseng, Hsian-Rong

2009-06-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-microChip), for parallel analyses of the effects of microenvironmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibroblast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-microChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

Integrated microfluidic devices for combinatorial cell-based assays

PubMed Central

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert

2010-01-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-μChip), for parallel analyses of the effects of microenvir-onmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibro-blast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-μChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology. PMID:19130244

Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1

PubMed Central

Sarzotti-Kelsoe, Marcella; Bailer, Robert T; Turk, Ellen; Lin, Chen-li; Bilska, Miroslawa; Greene, Kelli M.; Gao, Hongmei; Todd, Christopher A.; Ozaki, Daniel A.; Seaman, Michael S.; Mascola, John R.; Montefiori, David C.

2014-01-01

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. PMID:24291345

An extended data mining method for identifying differentially expressed assay-specific signatures in functional genomic studies.

PubMed

Rollins, Derrick K; Teh, Ailing

2010-12-17

Microarray data sets provide relative expression levels for thousands of genes for a small number, in comparison, of different experimental conditions called assays. Data mining techniques are used to extract specific information of genes as they relate to the assays. The multivariate statistical technique of principal component analysis (PCA) has proven useful in providing effective data mining methods. This article extends the PCA approach of Rollins et al. to the development of ranking genes of microarray data sets that express most differently between two biologically different grouping of assays. This method is evaluated on real and simulated data and compared to a current approach on the basis of false discovery rate (FDR) and statistical power (SP) which is the ability to correctly identify important genes. This work developed and evaluated two new test statistics based on PCA and compared them to a popular method that is not PCA based. Both test statistics were found to be effective as evaluated in three case studies: (i) exposing E. coli cells to two different ethanol levels; (ii) application of myostatin to two groups of mice; and (iii) a simulated data study derived from the properties of (ii). The proposed method (PM) effectively identified critical genes in these studies based on comparison with the current method (CM). The simulation study supports higher identification accuracy for PM over CM for both proposed test statistics when the gene variance is constant and for one of the test statistics when the gene variance is non-constant. PM compares quite favorably to CM in terms of lower FDR and much higher SP. Thus, PM can be quite effective in producing accurate signatures from large microarray data sets for differential expression between assays groups identified in a preliminary step of the PCA procedure and is, therefore, recommended for use in these applications.

Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription.

PubMed

Liu, Zhihui; Lam, Norris; Thiele, Carol J

2015-09-29

The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs.

Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing.

PubMed

Wen, Jianguo; Tao, Wenjing; Hao, Suyang; Zu, Youli

2017-06-13

Sickle cell disease (SCD) is a disorder of red blood cells (RBCs) expressing abnormal hemoglobin-S (HbS) due to genetic inheritance of homologous HbS gene. However, people with the sickle cell trait (SCT) carry a single allele of HbS and do not usually suffer from SCD symptoms, thus providing a rationale to treat SCD. To validate gene therapy potential, hematopoietic stem cells were isolated from the SCD patient blood and treated with CRISPR/Cas9 approach. To precisely dissect genome-editing effects, erythroid progenitor cells were cloned from single colonies of CRISPR-treated cells and then expanded for simultaneous gene, protein, and cellular function studies. Genotyping and sequencing analysis revealed that the genome-edited erythroid progenitor colonies were converted to SCT genotype from SCD genotype. HPLC protein assays confirmed reinstallation of normal hemoglobin at a similar level with HbS in the cloned genome-edited erythroid progenitor cells. For cell function evaluation, in vitro RBC differentiation of the cloned erythroid progenitor cells was induced. As expected, cell sickling assays indicated function reinstitution of the genome-edited offspring SCD RBCs, which became more resistant to sickling under hypoxia condition. This study is an exploration of genome editing of SCD HSPCs.

Functional characterization of putative cilia genes by high-content analysis

PubMed Central

Lai, Cary K.; Gupta, Nidhi; Wen, Xiaohui; Rangell, Linda; Chih, Ben; Peterson, Andrew S.; Bazan, J. Fernando; Li, Li; Scales, Suzie J.

2011-01-01

Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates. PMID:21289087

Over Expression of Wild Type or a Catalytically Dead Mutant of SIRTUIN 6 Does Not Influence NFκB Responses

PubMed Central

McKenary, Joanne; Hayes, Brian; Patel, Champa; Smith, Janet; Bridges, Angela; Fosberry, Andrew; Bhardwaja, Anshu; Mouzon, Bernadette; Chung, Chun-Wa; Barrett, Nathalie; Richmond, Nicola; Modha, Sundip; Solari, Roberto

2012-01-01

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways. PMID:22792191

FUN-L: gene prioritization for RNAi screens.

PubMed

Lees, Jonathan G; Hériché, Jean-Karim; Morilla, Ian; Fernández, José M; Adler, Priit; Krallinger, Martin; Vilo, Jaak; Valencia, Alfonso; Ellenberg, Jan; Ranea, Juan A; Orengo, Christine

2015-06-15

Most biological processes remain only partially characterized with many components still to be identified. Given that a whole genome can usually not be tested in a functional assay, identifying the genes most likely to be of interest is of critical importance to avoid wasting resources. Given a set of known functionally related genes and using a state-of-the-art approach to data integration and mining, our Functional Lists (FUN-L) method provides a ranked list of candidate genes for testing. Validation of predictions from FUN-L with independent RNAi screens confirms that FUN-L-produced lists are enriched in genes with the expected phenotypes. In this article, we describe a website front end to FUN-L. The website is freely available to use at http://funl.org © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Long-range evolutionary constraints reveal cis-regulatory interactions on the human X chromosome

PubMed Central

Naville, Magali; Ishibashi, Minaka; Ferg, Marco; Bengani, Hemant; Rinkwitz, Silke; Krecsmarik, Monika; Hawkins, Thomas A.; Wilson, Stephen W.; Manning, Elizabeth; Chilamakuri, Chandra S. R.; Wilson, David I.; Louis, Alexandra; Lucy Raymond, F.; Rastegar, Sepand; Strähle, Uwe; Lenhard, Boris; Bally-Cuif, Laure; van Heyningen, Veronica; FitzPatrick, David R.; Becker, Thomas S.; Roest Crollius, Hugues

2015-01-01

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression. PMID:25908307

Human Intellectual Disability Genes Form Conserved Functional Modules in Drosophila

PubMed Central

Oortveld, Merel A. W.; Keerthikumar, Shivakumar; Oti, Martin; Nijhof, Bonnie; Fernandes, Ana Clara; Kochinke, Korinna; Castells-Nobau, Anna; van Engelen, Eva; Ellenkamp, Thijs; Eshuis, Lilian; Galy, Anne; van Bokhoven, Hans; Habermann, Bianca; Brunner, Han G.; Zweier, Christiane; Verstreken, Patrik; Huynen, Martijn A.; Schenck, Annette

2013-01-01

Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules. PMID:24204314

Human intellectual disability genes form conserved functional modules in Drosophila.

PubMed

Oortveld, Merel A W; Keerthikumar, Shivakumar; Oti, Martin; Nijhof, Bonnie; Fernandes, Ana Clara; Kochinke, Korinna; Castells-Nobau, Anna; van Engelen, Eva; Ellenkamp, Thijs; Eshuis, Lilian; Galy, Anne; van Bokhoven, Hans; Habermann, Bianca; Brunner, Han G; Zweier, Christiane; Verstreken, Patrik; Huynen, Martijn A; Schenck, Annette

2013-10-01

Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences.

PubMed

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccas e 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like ( Sil1 ), scavenger receptor class C ( Src ), and scavenger receptor class B ( Srb3 and Srb4 ) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences

PubMed Central

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccase 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like (Sil1), scavenger receptor class C (Src), and scavenger receptor class B (Srb3 and Srb4) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean. PMID:29773992

Structural and Functional Assessment of APOBEC3G Macromolecular Complexes

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Bennett, Ryan P.; Salter, Jason D.; Smith, Harold C.

2016-01-01

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins. PMID:26988126

Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer

High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectiousmore » progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.« less

Association of vaspin gene polymorphisms with coronary artery disease in Chinese population and function study.

PubMed

Li, Hai Ling; Zhang, Hong Li; Jian, Wei Xia; Li, Qi; Peng, Wen Hui; Xu, Ya Wei

2013-01-16

Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a recently identified adipokine. Studies suggest it is involved in many diseases such as obesity, diabetes and coronary artery disease (CAD). This study is to investigate the association of single nucleotide polymorphisms (SNPs) in vaspin with CAD and its potential mechanisms. A total of 1570 consecutive patients undergoing coronary angiography were enrolled and the genotypes were determined by TaqMan allelic discrimination. Serum vaspin concentrations and mRNA expression levels were determined by ELISA and RT-PCR, respectively. Reporter gene assay was performed to investigate the effect of polymorphism on vaspin promoter function. After multivariate analysis, allele A of rs2236242 was found as an independent determinant of CAD (OR=1.32, p=0.004). Rs35262691 in vaspin promoter was associated with serum vaspin concentration and mRNA expression in peripheral blood mononuclear cells (PBMC) though no association had been found with CAD. Reporter gene assay further confirmed that CC genotype of rs35262691 had 2.1±0.4-fold higher activities than TT genotype in facilitating gene expression. Our results show that the variants of vaspin gene are associated with serum vaspin levels and risk for CAD in Chinese population. Copyright © 2012 Elsevier B.V. All rights reserved.

Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

PubMed Central

Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D.; Cashikar, Anil; King, Oliver D.; Auluck, Pavan K.; Geddie, Melissa L.; Valastyan, Julie S.; Karger, David R.; Lindquist, Susan; Fraenkel, Ernest

2009-01-01

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alpha-synuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures[OPEN

PubMed Central

Zhu, Bo; Zhang, Wenli; Jiang, Jiming

2015-01-01

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants. PMID:26373455

The soybean R2R3 MYB transcription factor GmMYB100 negatively regulates plant flavonoid biosynthesis.

PubMed

Yan, Junhui; Wang, Biao; Zhong, Yunpeng; Yao, Luming; Cheng, Linjing; Wu, Tianlong

2015-09-01

Soybean flavonoids, a group of important signaling molecules in plant-environment interaction, ubiquitously exist in soybean and are tightly regulated by many genes. Here we reported that GmMYB100, a gene encoding a R2R3 MYB transcription factor, is involved in soybean flavonoid biosynthesis. GmMYB100 is mainly expressed in flowers, leaves and immature embryo, and its level is decreased after pod ripening. Subcellular localization assay indicates that GmMYB100 is a nuclear protein. GmMYB100 has transactivation ability revealed by a yeast functional assay; whereas bioinformatic analysis suggests that GmMYB100 has a negative function in flavonoid biosynthesis. GmMYB100-overexpression represses the transcript levels of flavonoid-related genes in transgenic soybean hairy roots and Arabidopsis, and inhibits isoflavonoid (soybean) and flavonol (Arabidopsis) production in transgenic plants. Furthermore, the transcript levels of six flavonoid-related genes and flavonoid (isoflavonoid and flavone aglycones) accumulation are elevated in the GmMYB100-RNAi transgenic hairy roots. We also demonstrate that GmMYB100 protein depresses the promoter activities of soybean chalcone synthase and chalcone isomerase. These findings indicate that GmMYB100 is a negative regulator in soybean flavonoid biosynthesis pathway.

The Downregulation of MiR-182 Is Associated with the Growth and Invasion of Osteosarcoma Cells through the Regulation of TIAM1 Expression.

PubMed

Hu, Jun; Lv, Guohua; Zhou, Shuguang; Zhou, Yucheng; Nie, Bangxu; Duan, Hong; Zhang, Yunfeng; Yuan, Xiaofeng

2015-01-01

Osteosarcoma is the most common primary bone malignancy in children and young adults. Increasing results suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for osteosarcoma. MiR-182 expression level in osteosarcoma cell lines and tissues were assayed by qRT-PCR. MiRNA mimics or inhibitor were transfected for up-regulation or down-regulation of miR-182 expression. Cell function was assayed by CCK8, migration assay and invasion assay. The target genes of miR-182 were predicated by bioinformatics algorithm (TargetScan Human). MiR-182 was down-regulated in osteosarcoma tissues and cell lines. Overexpression of miR-182 inhibited tumor growth, migration and invasion. Subsequent investigation revealed that TIAM1 was a direct and functional target of miR-182 in osteosarcoma cells. Overexpression of miR-182 impaired TIAM1-induced inhibition of proliferation and invasion in osteosarcoma cells. Down-expression of miR-182 in osteosarcoma promoted tumor growth, migration and invasion by targeting TIAM1. MiR-182 might act as a tumor suppressor gene whose down-regulation contributes to the progression and metastasis of osteosarcoma, providing a potential therapy target for osteosarcoma patients.

In vitro perturbations of targets in cancer hallmark processes predict rodent chemical carcinogenesis.

PubMed

Kleinstreuer, Nicole C; Dix, David J; Houck, Keith A; Kavlock, Robert J; Knudsen, Thomas B; Martin, Matthew T; Paul, Katie B; Reif, David M; Crofton, Kevin M; Hamilton, Kerry; Hunter, Ronald; Shah, Imran; Judson, Richard S

2013-01-01

Thousands of untested chemicals in the environment require efficient characterization of carcinogenic potential in humans. A proposed solution is rapid testing of chemicals using in vitro high-throughput screening (HTS) assays for targets in pathways linked to disease processes to build models for priority setting and further testing. We describe a model for predicting rodent carcinogenicity based on HTS data from 292 chemicals tested in 672 assays mapping to 455 genes. All data come from the EPA ToxCast project. The model was trained on a subset of 232 chemicals with in vivo rodent carcinogenicity data in the Toxicity Reference Database (ToxRefDB). Individual HTS assays strongly associated with rodent cancers in ToxRefDB were linked to genes, pathways, and hallmark processes documented to be involved in tumor biology and cancer progression. Rodent liver cancer endpoints were linked to well-documented pathways such as peroxisome proliferator-activated receptor signaling and TP53 and novel targets such as PDE5A and PLAUR. Cancer hallmark genes associated with rodent thyroid tumors were found to be linked to human thyroid tumors and autoimmune thyroid disease. A model was developed in which these genes/pathways function as hypothetical enhancers or promoters of rat thyroid tumors, acting secondary to the key initiating event of thyroid hormone disruption. A simple scoring function was generated to identify chemicals with significant in vitro evidence that was predictive of in vivo carcinogenicity in different rat tissues and organs. This scoring function was applied to an external test set of 33 compounds with carcinogenicity classifications from the EPA's Office of Pesticide Programs and successfully (p = 0.024) differentiated between chemicals classified as "possible"/"probable"/"likely" carcinogens and those designated as "not likely" or with "evidence of noncarcinogenicity." This model represents a chemical carcinogenicity prioritization tool supporting targeted testing and functional validation of cancer pathways.

Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

PubMed

Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria S; Allende, Miguel L; Calcaterra, Nora B

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.

Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

PubMed Central

Mouguelar, Valeria S.; Allende, Miguel L.; Calcaterra, Nora B.

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates. PMID:23667590

Genome of the Asian longhorned beetle, Anoplophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle-plant interface

USDA-ARS?s Scientific Manuscript database

The Asian longhorned beetle (Anoplophora glabripennis; AGLAB) is a globally significant invasive species capable of inflicting severe feeding damage on many important orchard, ornamental and forest trees. Genome sequencing, annotation, gene expression assays, and functional and comparative genomic s...

Effect of heavy metals on nitrification activity as measured by RNA- and DNA-based function-specific assays

EPA Science Inventory

Heavy metals can inhibit nitrification, a key process for nitrogen removal in wastewater treatment. The transcriptional responses of functional genes (amoA, hao, nirK and norB) were measured in conjunction with specific oxygen uptake rate (sOUR) for nitrifying enrichment cultures...

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

PubMed Central

Schörg, Alexandra; Santambrogio, Sara; Platt, James L.; Schödel, Johannes; Lindenmeyer, Maja T.; Cohen, Clemens D.; Schrödter, Katrin; Mole, David R.; Wenger, Roland H.; Hoogewijs, David

2015-01-01

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. PMID:26007655

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

PubMed

Stockwell, B R; Haggarty, S J; Schreiber, S L

1999-02-01

Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

PubMed

Díaz-Magaña, Amada; Alva-Murillo, Nayeli; Chávez-Moctezuma, Martha P; López-Meza, Joel E; Ramírez-Díaz, Martha I; Cervantes, Carlos

2015-07-01

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA-  mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA-  mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA-  mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

Inhibitory effect of cyanide on wastewater nitrification determined using SOUR and RNA-based gene-specific assays.

PubMed

Kapoor, V; Elk, M; Li, X; Santo Domingo, J W

2016-08-01

The effect of cyanide (CN(-) ) on nitrification was examined with samples from nitrifying bacterial enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT-qPCR to quantify the transcripts of functional genes involved in nitrification. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to cyanide for a period of 12 h. The concentrations of CN(-) used in the batch assays were 0·03, 0·06, 0·1 and 1·0 mg l(-1) . There was considerable decrease in SOUR with increasing dosages of CN(-) . A decrease of more than 50% in nitrification activity was observed at 0·1 mg l(-1) CN(-) . Based on the RT-qPCR data, there was notable reduction in the transcript levels of amoA and hao for increasing CN(-) dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. The inhibitory effect of cyanide may be attributed to the affinity of cyanide to bind ferric haeme proteins, which disrupt protein structure and function. The correspondence between the relative expression of functional genes and SOUR shown in this study demonstrates the efficacy of RNA-based function-specific assays for better understanding of the effect of toxic compounds on nitrification activity in wastewater. The effect of cyanide on nitrifying bacteria was characterized by measuring physiological and transcriptional response. Cyanide was inhibitory to nitrification at concentrations that may be found in industrial waste. The RNA-based function-specific assays represent a mechanistic approach for better understanding the effect of toxic compounds on nitrification activity in wastewater. Moreover, the relative abundance of RNA transcripts can be used to closely track in situ nitrifying bacterial activity which can be used to predict inhibition events, thereby providing a metric to potentially improve performance of wastewater nitrifying systems. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

The pea END1 promoter drives anther-specific gene expression in different plant species.

PubMed

Gómez, María D; Beltrán, José-Pío; Cañas, Luis A

2004-10-01

END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (beta-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the -330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity.

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation

PubMed Central

Engel, Krysta L.; Mackiewicz, Mark; Hardigan, Andrew A.; Myers, Richard M.; Savic, Daniel

2016-01-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. PMID:27224938

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation.

PubMed

Engel, Krysta L; Mackiewicz, Mark; Hardigan, Andrew A; Myers, Richard M; Savic, Daniel

2016-09-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

PubMed

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

PubMed Central

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

Rapid genotyping assays for the 4-base pair deletion of canine MDR1/ABCB1 gene and low frequency of the mutant allele in Border Collie dogs.

PubMed

Mizukami, Keijiro; Chang, Hye-Sook; Yabuki, Akira; Kawamichi, Takuji; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Yamato, Osamu

2012-01-01

P-glycoprotein, encoded by the MDR1 or ABCB1 gene, is an integral component of the blood-brain barrier as an efflux pump for xenobiotics crucial in limiting drug uptake into the central nervous system. Dogs homozygous for a 4-base pair deletion of the canine MDR1 gene show altered expression or function of P-glycoprotein, resulting in neurotoxicosis after administration of the substrate drugs. In the present study, the usefulness of microchip electrophoresis for genotyping assays detecting this deletion mutation was evaluated. Mutagenically separated polymerase chain reaction (MS-PCR) and real-time PCR assays were newly developed and evaluated. Furthermore, a genotyping survey was carried out in a population of Border Collies dogs in Japan to determine the allele frequency in this breed. Microchip electrophoresis showed advantages in detection sensitivity and time saving over other modes of electrophoresis. The MS-PCR assay clearly discriminated all genotypes. Real-time PCR assay was most suitable for a large-scale survey due to its high throughput and rapidity. The genotyping survey demonstrated that the carrier and mutant allele frequencies were 0.49% and 0.25%, respectively, suggesting that the mutant allele frequency in Border Collies is markedly low compared to that in the susceptible dog breeds such as rough and smooth Collies.

Gene stage-specific expression in the microenvironment of pediatric myelodysplastic syndromes.

PubMed

Roela, Rosimeire A; Carraro, Dirce M; Brentani, Helena P; Kaiano, Jane H L; Simão, Daniel F; Guarnieiro, Roberto; Lopes, Luiz Fernando; Borojevic, Radovan; Brentani, M Mitzi

2007-05-01

Using cDNA microarray assays we have observed a clear difference in the gene expression pattern between bone marrow stromal cells obtained from healthy children (CT) and from pediatric patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) associated with MDS (MDS-AML). The global gene function profiling analysis indicated that in the pediatric MDS microenvironment the disease stages may be characterized mainly by underexpression of genes associated with biological processes such as transport. Furthermore, a subset of downregulated genes related to endocytosis and protein secretion was able to discriminate MDS from MDS-AML.

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery.

PubMed

Zhou, Yangbo; Tang, Zhaomin; Shi, Chunli; Shi, Shuai; Qian, Zhiyong; Zhou, Shaobing

2012-11-01

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (-M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.

Quantification of multiple gene expression in individual cells.

PubMed

Peixoto, António; Monteiro, Marta; Rocha, Benedita; Veiga-Fernandes, Henrique

2004-10-01

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.

c-Kit modifies the inflammatory status of smooth muscle cells

PubMed Central

Song, Lei; Martinez, Laisel; Zigmond, Zachary M.; Hernandez, Diana R.; Lassance-Soares, Roberta M.; Selman, Guillermo

2017-01-01

Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation. PMID:28626608

c-Kit modifies the inflammatory status of smooth muscle cells.

PubMed

Song, Lei; Martinez, Laisel; Zigmond, Zachary M; Hernandez, Diana R; Lassance-Soares, Roberta M; Selman, Guillermo; Vazquez-Padron, Roberto I

2017-01-01

c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (Kit W/W-v ) and control (Kit +/+ ) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.

Pancreatic Reference Set Application- Ann Killary-MD Anderson (2012) — EDRN Public Portal

Cancer.gov

ELISA assays. We propose to screen a two gene panel (TNC/TFPI) from functional genomic pathways approaches, by ELISA assays, in a collection of blinded plasma samples with the PCCG. In subsequent years, we will add to this panel additional candidates identified in the parent EDRN grant that are in the migration signature network or the 20q amplicon. For ELISA assays, we will utilize commercially available kits. Plasma levels of the predominant isoform of TNC, TNC-large variant (TNC-L) will be determined using a Human Tenascin-C Large (HMV) (FNIII-B) ELISA kit (IBL-America, Minneapolis, MN), which detects human TNC high molecular weight variant by sandwich ELISA. Plasma TFPI levels will be determined using a commercially available Quantikine Human TFPI ELISA kit (DTFP10) (R&D; Systems, Inc. Minneapolis, MN), which detects predominantly free TFPI and a very small percentage of LDL and HDL-bound TFPI by sandwich ELISA. Additional ELISA assays will be added in years 2-3 using ingenuity network analysis to identify secreted proteins that interact with migration signature proteins identified using functional genomic approaches. In addition, Dr. Sen’s lab has identified candidate biomarkers mapping with the 20q amplicon and which are both amplified and overexpressed as well as are secreted proteins. In years 2-3, we will examine all candidate markers by ELISA to determine 20q pathway markers increase the sensitivity and specificity of the 3p panel. Validation of a 4 miR Panel. For assaying the levels of miRs in plasma, qRT-PCR assays will be performed, as described (9). For the selected panel of miRs, we propose to develop a high throughput qRT-PCR assay by adapting our published method to a 96 well format where the critical steps of purification, such as LiCl precipitation and DNAse treatments will be done in successive steps in the same plates. In years 2-3, additional miRs that target both the 20q amplicon genes as well as miRs that target migration genes in Dr. Killary’

Functional Geno,ic Analysis of Breast Cancer Cell Tumorigenicity Using a Noval Gene Silencing Resource

DTIC Science & Technology

2006-04-01

Fig. 2B). In addition, luciferase assay on cells co-transfected with constructs expressing firefly and renilla luciferase genes showed a significant...positive cells. (C) BT474 cells were co-transfected with pGL3 plasmid expressing firefly luciferase, pRL plasmid expressing renilla luciferase, and...genes Per1 (A) and Bmal1 (B). BT474 cells were transfected with Per1 (A) and Bmal1 (B) firefly luciferase reporters, pRL plasmid expressing renilla

Some Like It Hot, Some Like It Warm: Phenotyping to Explore Thermotolerance Diversity

PubMed Central

Yeh, Ching-Hui; Kaplinsky, Nicholas J.; Hu, Catherine; Charng, Yee-yung

2012-01-01

Plants have evolved overlapping but distinct cellular responses to different aspects of high temperature stress. These responses include basal thermotolerance, short- and long-term acquired thermotolerance, and thermotolerance to moderately high temperatures. This thermotolerance diversity’ means that multiple phenotypic assays are essential for fully describing the functions of genes involved in heat stress responses. A large number of genes with potential roles in heat stress responses have been identified using genetic screens and genome wide expression studies. We examine the range of phenotypic assays that have been used to characterize thermotolerance phenotypes in both Arabidopsis and crop plants. Three major variables differentiate thermotolerance assays: 1) the heat stress regime used, 2) the developmental stage of the plants being studied, and 3) the actual phenotype which is scored. Consideration of these variables will be essential for deepening our understanding of the molecular genetics of plant thermotolerance. PMID:22920995

Genomic profiling of neutrophil transcripts in Asian Qigong practitioners: a pilot study in gene regulation by mind-body interaction.

PubMed

Li, Quan-Zhen; Li, Ping; Garcia, Gabriela E; Johnson, Richard J; Feng, Lili

2005-02-01

The great similarity of the genomes of humans and other species stimulated us to search for genes regulated by elements associated with human uniqueness, such as the mind-body interaction. DNA microarray technology offers the advantage of analyzing thousands of genes simultaneously, with the potential to determine healthy phenotypic changes in gene expression. The aim of this study was to determine the genomic profile and function of neutrophils in Falun Gong (FLG, an ancient Chinese Qigong) practitioners, with healthy subjects as controls. Six (6) Asian FLG practitioners and 6 Asian normal healthy controls were recruited for our study. The practitioners have practiced FLG for at least 1 year (range, 1-5 years). The practice includes daily reading of FLG books and daily practice of exercises lasting 1-2 hours. Selected normal healthy controls did not perform Qigong, yoga, t'ai chi, or any other type of mind-body practice, and had not followed any conventional physical exercise program for at least 1 year. Neutrophils were isolated from fresh blood and assayed for gene expression, using microarrays and RNase protection assay (RPA), as well as for function (phagocytosis) and survival (apoptosis). The changes in gene expression of FLG practitioners in contrast to normal healthy controls were characterized by enhanced immunity, downregulation of cellular metabolism, and alteration of apoptotic genes in favor of a rapid resolution of inflammation. The lifespan of normal neutrophils was prolonged, while the inflammatory neutrophils displayed accelerated cell death in FLG practitioners as determined by enzyme-linked immunosorbent assay. Correlating with enhanced immunity reflected by microarray data, neutrophil phagocytosis was significantly increased in Qigong practitioners. Some of the altered genes observed by microarray were confirmed by RPA. Qigong practice may regulate immunity, metabolic rate, and cell death, possibly at the transcriptional level. Our pilot study provides the first evidence that Qigong practice may exert transcriptional regulation at a genomic level. New approaches are needed to study how genes are regulated by elements associated with human uniqueness, such as consciousness, cognition, and spirituality.

Prunus transcription factors: breeding perspectives

PubMed Central

Bianchi, Valmor J.; Rubio, Manuel; Trainotti, Livio; Verde, Ignazio; Bonghi, Claudio; Martínez-Gómez, Pedro

2015-01-01

Many plant processes depend on differential gene expression, which is generally controlled by complex proteins called transcription factors (TFs). In peach, 1533 TFs have been identified, accounting for about 5.5% of the 27,852 protein-coding genes. These TFs are the reference for the rest of the Prunus species. TF studies in Prunus have been performed on the gene expression analysis of different agronomic traits, including control of the flowering process, fruit quality, and biotic and abiotic stress resistance. These studies, using quantitative RT-PCR, have mainly been performed in peach, and to a lesser extent in other species, including almond, apricot, black cherry, Fuji cherry, Japanese apricot, plum, and sour and sweet cherry. Other tools have also been used in TF studies, including cDNA-AFLP, LC-ESI-MS, RNA, and DNA blotting or mapping. More recently, new tools assayed include microarray and high-throughput DNA sequencing (DNA-Seq) and RNA sequencing (RNA-Seq). New functional genomics opportunities include genome resequencing and the well-known synteny among Prunus genomes and transcriptomes. These new functional studies should be applied in breeding programs in the development of molecular markers. With the genome sequences available, some strategies that have been used in model systems (such as SNP genotyping assays and genotyping-by-sequencing) may be applicable in the functional analysis of Prunus TFs as well. In addition, the knowledge of the gene functions and position in the peach reference genome of the TFs represents an additional advantage. These facts could greatly facilitate the isolation of genes via QTL (quantitative trait loci) map-based cloning in the different Prunus species, following the association of these TFs with the identified QTLs using the peach reference genome. PMID:26124770

Influence of cytochrome 2C19 allelic variants on on-treatment platelet reactivity evaluated by five different platelet function tests.

PubMed

Gremmel, Thomas; Kopp, Christoph W; Moertl, Deddo; Seidinger, Daniela; Koppensteiner, Renate; Panzer, Simon; Mannhalter, Christine; Steiner, Sabine

2012-05-01

The antiplatelet effect of clopidogrel has been linked to cytochrome P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of function variants were found to have a gene-dose effect on clopidogrel metabolism. However, genotyping is only one aspect of predicting response to clopidogrel and several platelet function tests are available to measure platelet response. Patients and methods We studied the influence of CYP2C19 allelic variants on on-treatment platelet reactivity as assessed by light transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay, multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined with the Infiniti® CYP450 2C19+ assay and categorized into four metabolizer states (ultrarapid, extensive, intermediate, poor). Platelet reactivity increased linearly from ultrarapid to poor metabolizers using the VerifyNow P2Y12 assay (P = 0.04), the VASP assay (P = 0.02) and the Impact-R (P = 0.04). The proportion of patients with high on-treatment residual platelet reactivity (HRPR) identified by LTA, the VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status decreased, while no such relationship could be identified for results of MEA and Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17) was an independent predictor of HRPR in LTA and the VASP assay while it did not reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the Impact-R. Depending on the type of platelet function test differences in the association of on-treatment platelet reactivity with CYP2C19 carrier status are observed. Copyright © 2011 Elsevier Ltd. All rights reserved.

A common variant of staphylococcal cassette chromosome mec type IVa in isolates from Copenhagen, Denmark, is not detected by the BD GeneOhm methicillin-resistant Staphylococcus aureus assay.

PubMed

Bartels, Mette Damkjaer; Boye, Kit; Rohde, Susanne Mie; Larsen, Anders Rhod; Torfs, Herbert; Bouchy, Peggy; Skov, Robert; Westh, Henrik

2009-05-01

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.

Discovering Functions of Unannotated Genes from a Transcriptome Survey of Wild Fungal Isolates

PubMed Central

Ellison, Christopher E.; Kowbel, David; Glass, N. Louise; Taylor, John W.

2014-01-01

ABSTRACT Most fungal genomes are poorly annotated, and many fungal traits of industrial and biomedical relevance are not well suited to classical genetic screens. Assigning genes to phenotypes on a genomic scale thus remains an urgent need in the field. We developed an approach to infer gene function from expression profiles of wild fungal isolates, and we applied our strategy to the filamentous fungus Neurospora crassa. Using transcriptome measurements in 70 strains from two well-defined clades of this microbe, we first identified 2,247 cases in which the expression of an unannotated gene rose and fell across N. crassa strains in parallel with the expression of well-characterized genes. We then used image analysis of hyphal morphologies, quantitative growth assays, and expression profiling to test the functions of four genes predicted from our population analyses. The results revealed two factors that influenced regulation of metabolism of nonpreferred carbon and nitrogen sources, a gene that governed hyphal architecture, and a gene that mediated amino acid starvation resistance. These findings validate the power of our population-transcriptomic approach for inference of novel gene function, and we suggest that this strategy will be of broad utility for genome-scale annotation in many fungal systems. PMID:24692637

Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

Lack of genetic diversity across diverse immune genes in an endangered mammal, the Tasmanian devil (Sarcophilus harrisii).

PubMed

Morris, Katrina M; Wright, Belinda; Grueber, Catherine E; Hogg, Carolyn; Belov, Katherine

2015-08-01

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll-like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome-level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole-genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29-220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long-term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad-scale immunogenetic diversity analysis in threatened species. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Functional Analysis of the Epidermal-Specific MYB Genes CAPRICE and WEREWOLF in Arabidopsis[W

PubMed Central

Tominaga, Rumi; Iwata, Mineko; Okada, Kiyotaka; Wada, Takuji

2007-01-01

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the developmental end state of plant cells. Two types of MYB transcription factors, R2R3-MYB and R3-MYB, are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of the R2R3-type MYB gene WEREWOLF (WER) and the R3-type MYB gene CAPRICE (CPC). Chimeric constructs made from the R3 MYB regions of WER and CPC used in reciprocal complementation experiments showed that the CPC R3 region cannot functionally substitute for the WER R3 region in the differentiation of hairless cells. However, WER R3 can substantially substitute for CPC R3. There are no differences in yeast interaction assays of WER or WER chimera proteins with GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). CPC and CPC chimera proteins also have similar activity in preventing GL3 WER and EGL3 WER interactions. Furthermore, we showed by gel mobility shift assays that WER chimera proteins do not bind to the GL2 promoter region. However, a CPC chimera protein, which harbors the WER R3 motif, still binds to the GL2 promoter region. PMID:17644729

Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis.

PubMed

Tominaga, Rumi; Iwata, Mineko; Okada, Kiyotaka; Wada, Takuji

2007-07-01

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the developmental end state of plant cells. Two types of MYB transcription factors, R2R3-MYB and R3-MYB, are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of the R2R3-type MYB gene WEREWOLF (WER) and the R3-type MYB gene CAPRICE (CPC). Chimeric constructs made from the R3 MYB regions of WER and CPC used in reciprocal complementation experiments showed that the CPC R3 region cannot functionally substitute for the WER R3 region in the differentiation of hairless cells. However, WER R3 can substantially substitute for CPC R3. There are no differences in yeast interaction assays of WER or WER chimera proteins with GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). CPC and CPC chimera proteins also have similar activity in preventing GL3 WER and EGL3 WER interactions. Furthermore, we showed by gel mobility shift assays that WER chimera proteins do not bind to the GL2 promoter region. However, a CPC chimera protein, which harbors the WER R3 motif, still binds to the GL2 promoter region.

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto

2009-12-18

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins aremore » known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.« less

Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1

PubMed Central

Dziuba, Natallia; Ferguson, Monique R.; O'Brien, William A.; Sanchez, Anthony; Prussia, Andrew J.; McDonald, Natalie J.; Friedrich, Brian M.; Li, Guangyu; Shaw, Michael W.; Sheng, Jinsong; Hodge, Thomas W.; Rubin, Donald H.

2012-01-01

Abstract Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation. PMID:22404213

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

PubMed

Dziuba, Natallia; Ferguson, Monique R; O'Brien, William A; Sanchez, Anthony; Prussia, Andrew J; McDonald, Natalie J; Friedrich, Brian M; Li, Guangyu; Shaw, Michael W; Sheng, Jinsong; Hodge, Thomas W; Rubin, Donald H; Murray, James L

2012-10-01

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2

PubMed Central

2011-01-01

Background The gene CHEK2 encodes a checkpoint kinase playing a key role in the DNA damage pathway. Though CHEK2 has been identified as an intermediate breast cancer susceptibility gene, only a small proportion of high-risk families have been explained by genetic variants located in its coding region. Alteration in gene expression regulation provides a potential mechanism for generating disease susceptibility. The detection of differential allelic expression (DAE) represents a sensitive assay to direct the search for a functional sequence variant within the transcriptional regulatory elements of a candidate gene. We aimed to assess whether CHEK2 was subject to DAE in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified. Methods We implemented an assay based on high-resolution melting (HRM) curve analysis and developed an analysis tool for DAE assessment. Results We observed allelic expression imbalance in 4 of the 41 LCLs examined. All four were carriers of the truncating mutation 1100delC. We confirmed previous findings that this mutation induces non-sense mediated mRNA decay. In our series, we ruled out the possibility of a functional sequence variant located in the promoter region or in a regulatory element of CHEK2 that would lead to DAE in the transcriptional regulatory milieu of freely proliferating LCLs. Conclusions Our results support that HRM is a sensitive and accurate method for DAE assessment. This approach would be of great interest for high-throughput mutation screening projects aiming to identify genes carrying functional regulatory polymorphisms. PMID:21569354

Successful In Vitro Expansion and Differentiation of Cord Blood Derived CD34+ Cells into Early Endothelial Progenitor Cells Reveals Highly Differential Gene Expression

PubMed Central

Topcic, Denijal; Haviv, Izhak; Merivirta, Ruusu-Maaria; Agrotis, Alexander; Leitner, Ephraem; Jowett, Jeremy B.; Bode, Christoph; Lappas, Martha; Peter, Karlheinz

2011-01-01

Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene. PMID:21858032

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

PubMed

Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

2014-08-18

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together with cryopreserved trout hepatocytes for screening estrogenic chemicals, resulting in a reduction of the time required to perform the assay and enabling greater access to the model system through the approach of cryopreservation.

Identifying Novel Helix–Loop–Helix Genes in Caenorhabditis elegans through a Classroom Demonstration of Functional Genomics

PubMed Central

Griffin, Vernetta; McMiller, Tracee; Jones, Erika; Johnson, Casonya M.

2003-01-01

A 14-week, undergraduate-level Genetics and Population Biology course at Morgan State University was modified to include a demonstration of functional genomics in the research laboratory. Students performed a rudimentary sequence analysis of the Caenorhabditis elegans genome and further characterized three sequences that were predicted to encode helix–loop–helix proteins. Students then used reverse transcription–polymerase chain reaction to determine which of the three genes is normally expressed in C. elegans. At the end of this laboratory activity, students were 1) to demonstrate a rudimentary knowledge of bioinformatics, including the ability to differentiate between “having” a gene and “expressing” a gene, and 2) to understand basic approaches to functional genomics, including one specific technique for assaying for gene expression. It was also anticipated that students would increase their skills at effectively communicating their research activities through written and/or oral presentation. This article describes the laboratory activity and the assessment of the effectiveness of the activity. PMID:12822036

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay.

PubMed

Frey, Amy B; Wilson, Deborah A; LaSalvia, Margaret M; Tan, Carmela D; Rodriguez, E Rene; Shrestha, Nabin K; Hall, Gerri S; Procop, Gary W

2011-11-01

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.

Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro.

PubMed

Lin, Sheyu; Zhang, Junyu; Xu, Jiawei; Wang, Honglian; Sang, Qing; Xing, Qinghe; He, Lin

2012-12-01

The SAM and SH3 domain containing 1 (SASH1) gene was originally identified as a potential tumor suppressor gene in breast cancer, mapped on chromosome 6q24.3. The expression of SASH1 plays a prognostic role in human colon cancer. Its expression is frequently downregulated in several human malignancies. However, the biological function of SASH1 in melanoma cells is yet to be determined. In this study, in order to investigate the tumor suppressive effects of the SASH1 gene, an A-375 stable melanoma cell line was established, overexpressing the SASH1 gene. The stable cell line was examined using proliferation assay, apoptosis assay, cell cycle analysis and real-time PCR. The results indicated that the tumor suppressive activity of SASH1 derived from G2/M arrest in A-375 cells, and that the phosphorylation of Cdc2 or the disruption of cyclin B-Cdc2 binding may be responsible for the G2/M arrest.

Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model.

PubMed

Li, Edward B; Truong, Dawn; Hallett, Shawn A; Mukherjee, Kusumika; Schutte, Brian C; Liao, Eric C

2017-09-01

Large-scale sequencing efforts have captured a rapidly growing catalogue of genetic variations. However, the accurate establishment of gene variant pathogenicity remains a central challenge in translating personal genomics information to clinical decisions. Interferon Regulatory Factor 6 (IRF6) gene variants are significant genetic contributors to orofacial clefts. Although approximately three hundred IRF6 gene variants have been documented, their effects on protein functions remain difficult to interpret. Here, we demonstrate the protein functions of human IRF6 missense gene variants could be rapidly assessed in detail by their abilities to rescue the irf6 -/- phenotype in zebrafish through variant mRNA microinjections at the one-cell stage. The results revealed many missense variants previously predicted by traditional statistical and computational tools to be loss-of-function and pathogenic retained partial or full protein function and rescued the zebrafish irf6 -/- periderm rupture phenotype. Through mRNA dosage titration and analysis of the Exome Aggregation Consortium (ExAC) database, IRF6 missense variants were grouped by their abilities to rescue at various dosages into three functional categories: wild type function, reduced function, and complete loss-of-function. This sensitive and specific biological assay was able to address the nuanced functional significances of IRF6 missense gene variants and overcome many limitations faced by current statistical and computational tools in assigning variant protein function and pathogenicity. Furthermore, it unlocked the possibility for characterizing yet undiscovered human IRF6 missense gene variants from orofacial cleft patients, and illustrated a generalizable functional genomics paradigm in personalized medicine.

Evidence-Based Annotation of Gene Function in Shewanella oneidensis MR-1 Using Genome-Wide Fitness Profiling across 121 Conditions

PubMed Central

Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Shao, Wenjun; Baumohl, Jason K.; Xu, Zhuchen; Nguyen, Michelle; Tamse, Raquel; Davis, Ronald W.; Arkin, Adam P.

2011-01-01

Most genes in bacteria are experimentally uncharacterized and cannot be annotated with a specific function. Given the great diversity of bacteria and the ease of genome sequencing, high-throughput approaches to identify gene function experimentally are needed. Here, we use pools of tagged transposon mutants in the metal-reducing bacterium Shewanella oneidensis MR-1 to probe the mutant fitness of 3,355 genes in 121 diverse conditions including different growth substrates, alternative electron acceptors, stresses, and motility. We find that 2,350 genes have a pattern of fitness that is significantly different from random and 1,230 of these genes (37% of our total assayed genes) have enough signal to show strong biological correlations. We find that genes in all functional categories have phenotypes, including hundreds of hypotheticals, and that potentially redundant genes (over 50% amino acid identity to another gene in the genome) are also likely to have distinct phenotypes. Using fitness patterns, we were able to propose specific molecular functions for 40 genes or operons that lacked specific annotations or had incomplete annotations. In one example, we demonstrate that the previously hypothetical gene SO_3749 encodes a functional acetylornithine deacetylase, thus filling a missing step in S. oneidensis metabolism. Additionally, we demonstrate that the orphan histidine kinase SO_2742 and orphan response regulator SO_2648 form a signal transduction pathway that activates expression of acetyl-CoA synthase and is required for S. oneidensis to grow on acetate as a carbon source. Lastly, we demonstrate that gene expression and mutant fitness are poorly correlated and that mutant fitness generates more confident predictions of gene function than does gene expression. The approach described here can be applied generally to create large-scale gene-phenotype maps for evidence-based annotation of gene function in prokaryotes. PMID:22125499

GIANT 2.0: genome-scale integrated analysis of gene networks in tissues.

PubMed

Wong, Aaron K; Krishnan, Arjun; Troyanskaya, Olga G

2018-05-25

GIANT2 (Genome-wide Integrated Analysis of gene Networks in Tissues) is an interactive web server that enables biomedical researchers to analyze their proteins and pathways of interest and generate hypotheses in the context of genome-scale functional maps of human tissues. The precise actions of genes are frequently dependent on their tissue context, yet direct assay of tissue-specific protein function and interactions remains infeasible in many normal human tissues and cell-types. With GIANT2, researchers can explore predicted tissue-specific functional roles of genes and reveal changes in those roles across tissues, all through interactive multi-network visualizations and analyses. Additionally, the NetWAS approach available through the server uses tissue-specific/cell-type networks predicted by GIANT2 to re-prioritize statistical associations from GWAS studies and identify disease-associated genes. GIANT2 predicts tissue-specific interactions by integrating diverse functional genomics data from now over 61 400 experiments for 283 diverse tissues and cell-types. GIANT2 does not require any registration or installation and is freely available for use at http://giant-v2.princeton.edu.

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kover, Karen, E-mail: kkover@cmh.edu; University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108; Yan, Yun

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up tomore » 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP expression in beta cells.« less

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

PubMed

Schörg, Alexandra; Santambrogio, Sara; Platt, James L; Schödel, Johannes; Lindenmeyer, Maja T; Cohen, Clemens D; Schrödter, Katrin; Mole, David R; Wenger, Roland H; Hoogewijs, David

2015-07-13

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the -82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the -82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the -82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

PubMed Central

Han, Xiao; Guo, Jinhai; Deng, Weiwei; Zhang, Chenying; Du, Peige; Shi, Taiping; Ma, Dalong

2008-01-01

Background Estrogen receptor α (ERα) is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE) with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131) as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE). In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells. PMID:18847501

Hybridization of Environmental Microbial Community Nucleic Acids by GeoChip.

PubMed

Van Nostrand, Joy D; Yin, Huaqin; Wu, Liyou; Yuan, Tong; Zhou, Jizhong

2016-01-01

Functional gene arrays, like the GeoChip, allow for the study of tens of thousands of genes in a single assay. The GeoChip array (5.0) contains probes for genes involved in geochemical cycling (N, C, S, and P), metal homeostasis, stress response, organic contaminant degradation, antibiotic resistance, secondary metabolism, and virulence factors as well as genes specific for fungi, protists, and viruses. Here, we briefly describe GeoChip design strategies (gene selection and probe design) and discuss minimum quantity and quality requirements for nucleic acids. We then provide detailed protocols for amplification, labeling, and hybridization of samples to the GeoChip.

Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

PubMed

Song, J; Doucette, C; Hanniford, D; Hunady, K; Wang, N; Sherf, B; Harrington, J J; Brunden, K R; Stricker-Krongrad, A

2005-06-01

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

Functional characterization of a prokaryotic Kir channel.

PubMed

Enkvetchakul, Decha; Bhattacharyya, Jaya; Jeliazkova, Iana; Groesbeck, Darcy K; Cukras, Catherine A; Nichols, Colin G

2004-11-05

The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.

Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)

PubMed Central

Krol, Kamil; Jendrysek, Justyna; Debski, Janusz; Skoneczny, Marek; Kurlandzka, Anna; Kaminska, Joanna; Dadlez, Michal; Skoneczna, Adrianna

2017-01-01

Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a “DNA cloud assay”. We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP). PMID:28212567

A PROP1-binding factor, AES cloned by yeast two-hybrid assay represses PROP1-induced Pit-1 gene expression.

PubMed

Sugiyama, Yuka; Ikeshita, Nobuko; Shibahara, Hiromi; Yamamoto, Daisuke; Kawagishi, Mayuko; Iguchi, Genzo; Iida, Keiji; Takahashi, Yutaka; Kaji, Hidesuke; Chihara, Kazuo; Okimura, Yasuhiko

2013-08-25

PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

ICE1 of Pyrus ussuriensis functions in cold tolerance by enhancing PuDREBa transcriptional levels through interacting with PuHHP1

NASA Astrophysics Data System (ADS)

Huang, Xiaosan; Li, Kongqing; Jin, Cong; Zhang, Shaoling

2015-12-01

ICE1 transcription factor plays an important role in plant cold stress via regulating the expression of stress-responsive genes. In this study, a PuICE1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. The expression levels of the PuICE1 were induced by cold, dehydration and salt, with the greatest induction under cold conditions. PuICE1 was localized in the nucleus and could bind specifically to the MYC element in the PuDREBa promoter. The PuICE1 fused to the GAL4 DNA-binding domain to have transcriptional activation activity. Ectopic expression of the PuICE1 in tomato conferred enhanced tolerance to cold stress at cold temperatures, less electrolyte leakage, less MDA content, higher chlorophyll content, higher survival rate, higher proline content, higher activities of enzymes. In additon, steady-state mRNA levels of six stress-responsive genes coding for either functional or regulatory genes were induced to higher levels in the transgenic lines by cold stress. Yeast two-hybrid, transient assay, split luciferase complementation and BiFC assays all revealed that PuHHP1 protein can physically interact with PuICE1. Taken together, these results demonstrated that PuICE1 plays a positive role in cold tolerance, which may be due to enhancement of PuDREBa transcriptional levels through interacting with the PuHHP1.

Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent.

PubMed Central

Murthy, S C; Bhat, G P; Thimmappaya, B

1985-01-01

A molecular dissection of the adenovirus EIIA early (E) promoter was undertaken to study the sequence elements required for transcription and to examine the nucleotide sequences, if any, specific for its trans-activation by the viral pre-early EIA gene product. A chimeric gene in which the EIIA-E promoter region fused to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene was used in transient assays to identify the transcriptional control regions. Deletion mapping studies revealed that the upstream DNA sequences up to -86 were sufficient for the optimal basal level transcription in HeLa cells and also for the EIA-induced transcription. A series of linker-scanning (LS) mutants were constructed to precisely identify the nucleotide sequences that control transcription. Analysis of these LS mutants allowed us to identify two regions of the promoter that are critical for the EIIA-E transcription. These regions are located between -29 and -21 (region I) and between -82 and -66 (region II). Mutations in region I affected initiation and appeared functionally similar to the "TATA" sequence of the commonly studied promoters. To examine whether or not the EIIA-E promoter contained DNA sequences specific for the trans-activation by the EIA, the LS mutants were analyzed in a cotransfection assay containing a plasmid carrying the EIA gene. CAT activity of all of the LS mutants was induced by the EIA gene in this assay, suggesting that the induction of transcription of the EIIA-E promoter by the EIA gene is not sequence-specific. Images PMID:3857577

Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality.

PubMed

Chebolu, S; Daniell, H

2009-01-01

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%-31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bio-reactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Expression, Folding, Assembly and Functionality

PubMed Central

Chebolu, S.; Daniell, H.

2009-01-01

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%–31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner. PMID:19401820

Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

PubMed

Stanley, Pamela; Sundaram, Subha

2014-06-03

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis. Copyright © 2014 John Wiley & Sons, Inc.

Translatomics combined with transcriptomics and proteomics reveals novel functional, recently evolved orphan genes in Escherichia coli O157:H7 (EHEC).

PubMed

Neuhaus, Klaus; Landstorfer, Richard; Fellner, Lea; Simon, Svenja; Schafferhans, Andrea; Goldberg, Tatyana; Marx, Harald; Ozoline, Olga N; Rost, Burkhard; Kuster, Bernhard; Keim, Daniel A; Scherer, Siegfried

2016-02-24

Genomes of E. coli, including that of the human pathogen Escherichia coli O157:H7 (EHEC) EDL933, still harbor undetected protein-coding genes which, apparently, have escaped annotation due to their small size and non-essential function. To find such genes, global gene expression of EHEC EDL933 was examined, using strand-specific RNAseq (transcriptome), ribosomal footprinting (translatome) and mass spectrometry (proteome). Using the above methods, 72 short, non-annotated protein-coding genes were detected. All of these showed signals in the ribosomal footprinting assay indicating mRNA translation. Seven were verified by mass spectrometry. Fifty-seven genes are annotated in other enterobacteriaceae, mainly as hypothetical genes; the remaining 15 genes constitute novel discoveries. In addition, protein structure and function were predicted computationally and compared between EHEC-encoded proteins and 100-times randomly shuffled proteins. Based on this comparison, 61 of the 72 novel proteins exhibit predicted structural and functional features similar to those of annotated proteins. Many of the novel genes show differential transcription when grown under eleven diverse growth conditions suggesting environmental regulation. Three genes were found to confer a phenotype in previous studies, e.g., decreased cattle colonization. These findings demonstrate that ribosomal footprinting can be used to detect novel protein coding genes, contributing to the growing body of evidence that hypothetical genes are not annotation artifacts and opening an additional way to study their functionality. All 72 genes are taxonomically restricted and, therefore, appear to have evolved relatively recently de novo.

Folate and Breast Cancer: Role of Intake, Blood Levels, and Metabolic Gene Polymorphisms

DTIC Science & Technology

2005-07-01

folate, and metabolic gene polymorphisms in relation to breast cancer risk: Months 1-19. b. Prepare blood samples for relevant assays: Months 1-19... gene polymorphism assays among the 184 cases and matched controls. The folate assays are on-going at this time. DNA assays will commence in the... methotrexate . Ann Oncol 13: 1915–1918, 2002 13. Toffoli G, Veronesi A, Boiocchi M, Crivellari D: MTHFR gene polymorphism and severe toxicity during

Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

PubMed Central

McSharry, James J.; McDonough, Ann C.; Olson, Betty A.; Drusano, George L.

2004-01-01

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses. PMID:14715540

The heptanucleotide motif GAGACGC is a key component of a cis-acting promoter element that is critical for SnSAG1 expression in Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Howe, Daniel K

2009-07-01

The apicomplexan parasite Sarcocystis neurona undergoes a complex process of intracellular development, during which many genes are temporally regulated. The described study was undertaken to begin identifying the basic promoter elements that control gene expression in S. neurona. Sequence analysis of the 5'-flanking region of five S. neurona genes revealed a conserved heptanucleotide motif GAGACGC that is similar to the WGAGACG motif described upstream of multiple genes in Toxoplasma gondii. The promoter region for the major surface antigen gene SnSAG1, which contains three heptanucleotide motifs within 135 bases of the transcription start site, was dissected by functional analysis using a dual luciferase reporter assay. These analyses revealed that a minimal promoter fragment containing all three motifs was sufficient to drive reporter molecule expression, with the presence and orientation of the 5'-most heptanucleotide motif being absolutely critical for promoter function. Further studies should help to identify additional sequence elements important for promoter function and for controlling gene expression during intracellular development by this apicomplexan pathogen.

TARGET Research Goals

Cancer.gov

TARGET researchers use various sequencing and array-based methods to examine the genomes, transcriptomes, and for some diseases epigenomes of select childhood cancers. This “multi-omic” approach generates a comprehensive profile of molecular alterations for each cancer type. Alterations are changes in DNA or RNA, such as rearrangements in chromosome structure or variations in gene expression, respectively. Through computational analyses and assays to validate biological function, TARGET researchers predict which alterations disrupt the function of a gene or pathway and promote cancer growth, progression, and/or survival. Researchers identify candidate therapeutic targets and/or prognostic markers from the cancer-associated alterations.

Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

PubMed

Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

2010-09-20

KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

PubMed

Nuñez, S B; Medin, J A; Braissant, O; Kemp, L; Wahli, W; Ozato, K; Segars, J H

1997-03-14

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

GA-DELLA pathway is involved in regulation of nitrogen deficiency-induced anthocyanin accumulation.

PubMed

Zhang, Yongqiang; Liu, Zhongjuan; Liu, Jianping; Lin, Sheng; Wang, Jianfeng; Lin, Wenxiong; Xu, Weifeng

2017-04-01

DELLA proteins positively regulate nitrogen deficiency-induced anthocyanin accumulation through directly interaction with PAP1 to enhance its transcriptional activity on anthocyanin biosynthetic gene expressions. Plants can survive a limiting nitrogen supply by undergoing adaptive responses, including induction of anthocyanin production. However, the detailed mechanism is still unclear. In this study, we found that this process was impaired and enhanced, respectively, by exogenous GA 3 (an active form of GAs) and paclobutrazol (PAC, a specific GA biosynthesis inhibitor) in Arabidopsis seedlings. Consistently, the nitrogen deficiency-induced transcript levels of several key genes involved in anthocyanin biosynthesis, including F3'H, DFR, LDOX, and UF3GT, were decreased and enhanced by exogenous GA 3 and PAC, respectively. Moreover, the nitrogen deficiency-induced anthocyanin accumulation and biosynthesis gene expressions were impaired in the loss-of-function mutant gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1 (della) but enhanced in the GA-insensitive mutant gai, suggesting that DELLA proteins, known as repressors of GA signaling, are necessary for fully induction of nitrogen deficiency-driven anthocyanin biosynthesis. Using yeast two-hybrid (Y2H) assay, pull-down assay, and luciferase complementation assay, it was found that RGA, a DELLA of Arabidopsis, could strongly interact with PAP1, a known regulatory transcription factor positively involved in anthocyanin biosynthesis. Furthermore, transient expression assays indicated that RGA and GAI could enhance the transcriptional activities of PAP1 on its downstream genes, including F3'H and DFR. Taken together, this study suggests that DELLAs are necessary regulators for nitrogen deficiency-induced anthocyanin accumulation through interaction with PAP1 and enhancement of PAP1's transcriptional activity on its target genes. GA-DELLA-involved anthocyanin accumulation is important for plant adaptation to nitrogen deficiency.

Functional modulation of human intestinal epithelial cell responses by Bifidobacterium infantis and Lactobacillus salivarius

PubMed Central

O'Hara, Ann M; O'Regan, Padraig; Fanning, Áine; O'Mahony, Caitlin; MacSharry, John; Lyons, Anne; Bienenstock, John; O'Mahony, Liam; Shanahan, Fergus

2006-01-01

Intestinal epithelial cells (IECs) and dendritic cells (DCs) play a pivotal role in antigen sampling and the maintenance of gut homeostasis. However, the interaction of commensal bacteria with the intestinal surface remains incompletely understood. Here we investigated immune cell responses to commensal and pathogenic bacteria. HT-29 human IECs were incubated with Bifidobacterium infantis 35624, Lactobacillus salivarius UCC118 or Salmonella typhimurium UK1 for varying times, or were pretreated with a probiotic for 2 hr prior to stimulation with S. typhimurium or flagellin. Gene arrays were used to examine inflammatory gene expression. Nuclear factor (NF)-κB activation, interleukin (IL)-8 secretion, pathogen adherence to IECs, and mucin-3 (MUC3) and E-cadherin gene expression were assayed by TransAM assay, enzyme-linked immunosorbent assay (ELISA), fluorescence, and real-time reverse transcriptase–polymerase chain reaction (RT-PCR), respectively. IL-10 and tumour necrosis factor (TNF)-α secretion by bacteria-treated peripheral blood-derived DCs were measured using ELISA. S. typhimurium increased expression of 36 of the 847 immune-related genes assayed, including NF-κB and IL-8. The commensal bacteria did not alter expression levels of any of the 847 genes. However, B. infantis and L. salivarius attenuated both IL-8 secretion at baseline and S. typhimurium-induced pro-inflammatory responses. B. infantis also limited flagellin-induced IL-8 protein secretion. The commensal bacteria did not increase MUC3 or E-cadherin expression, or interfere with pathogen binding to HT-29 cells, but they did stimulate IL-10 and TNF-α secretion by DCs. The data demonstrate that, although the intestinal epithelium is immunologically quiescent when it encounters B. infantis or L. salivarius, these commensal bacteria exert immunomodulatory effects on intestinal immune cells that mediate host responses to flagellin and enteric pathogens. PMID:16771855

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

PubMed

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Assay for mutagenesis in heterozygous diploid human lymphoblasts

DOEpatents

Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

1981-01-01

An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

Cloning and functional analysis of 5'-upstream region of the Pokemon gene.

PubMed

Yang, Yutao; Zhou, Xiaowei; Zhu, Xudong; Zhang, Chuanfu; Yang, Zhixin; Xu, Long; Huang, Peitang

2008-04-01

Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expression of the Pokemon gene in humans, its 5'-upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG-U and NEG-D elements were involved in negative regulation of the Pokemon promoter, whereas the POS-D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG-U, NEG-D and POS-D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG-U and NEG-D elements led to negative regulation of the Pokemon promoter. Moreover, the NEG-U and NEG-D elements needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy.

FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Gui-Fen; Chen, Shi-Yao, E-mail: shiyao_chen@163.com; Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai

Highlights: Black-Right-Pointing-Pointer The article revealed FoxP3 gene function in gastric cancer firstly. Black-Right-Pointing-Pointer Present the novel roles of FoxP3 in inhibiting proliferation and promoting apoptosis in gastric cancer cells. Black-Right-Pointing-Pointer Overexpression of FoxP3 increased proapoptotic molecules and repressed antiapoptotic molecules. Black-Right-Pointing-Pointer Silencing of FoxP3 reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Black-Right-Pointing-Pointer FoxP3 is sufficient for activating the apoptotic signaling pathway. -- Abstract: Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells.more » To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis in GC cells by regulating apoptotic signaling, which could be a promising therapeutic approach for gastric cancer.« less

Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density

PubMed Central

Richards, Neil; Parker, David S.; Johnson, Lisa A.; Allen, Benjamin L.; Barolo, Scott; Gumucio, Deborah L.

2015-01-01

The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci. PMID:26710299

Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study

PubMed Central

Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

2016-01-01

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the fprotein expression in unctions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM. PMID:27754828

Wnt Ligands Differentially Regulate Toxicity and Translocation of Graphene Oxide through Different Mechanisms in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Zhi, Lingtong; Ren, Mingxia; Qu, Man; Zhang, Hanyu; Wang, Dayong

2016-12-01

In this study, we investigated the possible involvement of Wnt signals in the control of graphene oxide (GO) toxicity using the in vivo assay system of Caenorhabditis elegans. In nematodes, the Wnt ligands, CWN-1, CWN-2, and LIN-44, were found to be involved in the control of GO toxicity. Mutation of cwn-1 or lin-44 gene induced a resistant property to GO toxicity and resulted in the decreased accumulation of GO in the body of nematodes, whereas mutation of cwn-2 gene induces a susceptible property to GO toxicity and an enhanced accumulation of GO in the body of nematodes. Genetic interaction assays demonstrated that mutation of cwn-1 or lin-44 was able to suppress the susceptibility to GO toxicity shown in the cwn-2 mutants. Loss-of-function mutations in all three of these Wnt ligand genes resulted in the resistance of nematodes to GO toxicity. Moreover, the Wnt ligands might differentially regulate the toxicity and translocation of GO through different mechanisms. These findings could be important in understanding the function of Wnt signals in the regulation of toxicity from environmental nanomaterials.

Microarray identification of novel genes downstream of Six1, a critical factor in cranial placode, somite and kidney development

PubMed Central

Yan, Bo; Neilson, Karen M.; Ranganathan, Ramya; Maynard, Thomas; Streit, Andrea; Moody, Sally A.

2014-01-01

Background Six1 plays an important role in the development of several vertebrate organs, including cranial sensory placodes, somites and kidney. Although Six1 mutations cause one form of Branchio-Otic Syndrome (BOS), the responsible gene in many patients has not been identified; genes that act downstream of Six1 are potential BOS candidates. Results We sought to identify novel genes expressed during placode, somite and kidney development by comparing gene expression between control and Six1-expressing ectodermal explants. The expression patterns of 19 of the significantly up-regulated and 11 of the significantly down-regulated genes were assayed from cleavage to larval stages. 28/30 genes are expressed in the otocyst, a structure that is functionally disrupted in BOS, and 26/30 genes are expressed in the nephric mesoderm, a structure that is functionally disrupted in the related Branchio-Otic-Renal (BOR) syndrome. We also identified the chick homologues of 5 genes and show that they have conserved expression patterns. Conclusions Of the 30 genes selected for expression analyses, all are expressed at many of the developmental times and appropriate tissues to be regulated by Six1. Many have the potential to play a role in the disruption of hearing and kidney function seen in BOS/BOR patients. PMID:25403746

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimelman, Aya; Levy, Asaf; Sberro, Hila

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes our assays revealed novel toxins and restriction enzymes, and new classes of smallmore » non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator dnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.« less

A qRT-PCR assay for the expression of all Mal d 1 isoallergen genes

PubMed Central

2013-01-01

Background A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars ‘Florina’ and ‘Gala’. Results We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent. Conclusion The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy. PMID:23522122

Studying Functions of All Yeast Genes Simultaneously

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Eason, Robert G.; Poumand, Nader; Herman, Zelek S.; Davis, Ronald W.; Anthony Kevin; Jejelowo, Olufisayo

2006-01-01

A method of studying the functions of all the genes of a given species of microorganism simultaneously has been developed in experiments on Saccharomyces cerevisiae (commonly known as baker's or brewer's yeast). It is already known that many yeast genes perform functions similar to those of corresponding human genes; therefore, by facilitating understanding of yeast genes, the method may ultimately also contribute to the knowledge needed to treat some diseases in humans. Because of the complexity of the method and the highly specialized nature of the underlying knowledge, it is possible to give only a brief and sketchy summary here. The method involves the use of unique synthetic deoxyribonucleic acid (DNA) sequences that are denoted as DNA bar codes because of their utility as molecular labels. The method also involves the disruption of gene functions through deletion of genes. Saccharomyces cerevisiae is a particularly powerful experimental system in that multiple deletion strains easily can be pooled for parallel growth assays. Individual deletion strains recently have been created for 5,918 open reading frames, representing nearly all of the estimated 6,000 genetic loci of Saccharomyces cerevisiae. Tagging of each deletion strain with one or two unique 20-nucleotide sequences enables identification of genes affected by specific growth conditions, without prior knowledge of gene functions. Hybridization of bar-code DNA to oligonucleotide arrays can be used to measure the growth rate of each strain over several cell-division generations. The growth rate thus measured serves as an index of the fitness of the strain.

Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens

PubMed Central

Dobrovolsky, Vasily N.

2013-01-01

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reticulocyte micronucleus (MN) assay, which provides a direct measurement of clastogenicity. Male F344 rats were treated acutely with X-rays, cyclophosphamide (CP) and Cis-platin (Cis-Pt), and the frequency of micronucleated reticulocytes (MN RETs) in peripheral blood was measured 1 or 2 days later. The frequencies of CD59-deficient Pig-a mutant erythrocytes and 6-thioguanine-resistant Hprt mutant T-lymphocytes were measured at several times up to 16 weeks after the exposure. All three clastogens induced strong increases in the frequency of MN RETs, with X-rays and Cis-Pt producing near linear dose responses. The three agents also were positive in the two gene mutation assays although the assays detected them with different efficiencies. The Pig-a assay was more efficient in detecting the effect of Cis-Pt treatment, whereas the Hprt assay was more efficient for X-rays and CP. The results indicate that the erythrocyte Pig-a assay can detect the in vivo mutagenicity of clastogens although its sensitivity is variable in comparison with the lymphocyte Hprt assay. PMID:23677247

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

PubMed Central

2011-01-01

Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests. PMID:21824406

An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

PubMed Central

Banerjee, Joydeep; Sahoo, Dipak Kumar; Dey, Nrisingha; Houtz, Robert L.; Maiti, Indu Bhushan

2013-01-01

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications. PMID:24260266

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

In planta functions of cytochrome P450 monooxygenase genes in the phytocassane biosynthetic gene cluster on rice chromosome 2.

PubMed

Ye, Zhongfeng; Yamazaki, Kohei; Minoda, Hiromi; Miyamoto, Koji; Miyazaki, Sho; Kawaide, Hiroshi; Yajima, Arata; Nojiri, Hideaki; Yamane, Hisakazu; Okada, Kazunori

2018-06-01

In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta.

A male-specific odorant receptor conserved through the evolution of sex pheromones in Ostrinia moth species

PubMed Central

Miura, Nami; Nakagawa, Tatsuro; Tatsuki, Sadahiro; Touhara, Kazushige; Ishikawa, Yukio

2009-01-01

In many moths, mate-finding communication is mediated by the female sex pheromones. Since differentiation of sex pheromones is often associated with speciation, it is intriguing to know how the changes in female sex pheromone have been tracked by the pheromone recognition system of the males. A male-specific odorant receptor was found to have been conserved through the evolution of sex pheromone communication systems in the genus Ostrinia (Lepidoptera: Crambidae). In an effort to characterize pheromone receptors of O. scapulalis, which uses a mixture of (E)-11- and (Z)-11-tetradecenyl acetates as a sex pheromone, we cloned a gene (OscaOR1) encoding a male-specific odorant receptor. In addition, we cloned a gene of the Or83b family (OscaOR2). Functional assays using Xenopus oocytes co-expressing OscaOR1 and OscaOR2 have shown that OscaOR1 is, unexpectedly, a receptor of (E)-11-tetradecenol (E11-14:OH), a single pheromone component of a congener O. latipennis. Subsequent studies on O. latipennis showed that this species indeed has a gene orthologous to OscaOR1 (OlatOR1), a functional assay of which confirmed it to be a gene encoding the receptor of E11-14:OH. Furthermore, investigations of six other Ostrinia species have revealed that all of them have a gene orthologous to OscaOR1, although none of these species, except O. ovalipennis, a species most closely related to O. latipennis, uses E11-14:OH as the pheromone component. The present findings suggest that the male-specific receptor of E11-14:OH was acquired before the divergence of the genus Ostrinia, and functionally retained through the evolution of this genus. PMID:19421342

The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation.

PubMed

Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

2013-07-01

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells.

The Cotton Transcription Factor TCP14 Functions in Auxin-Mediated Epidermal Cell Differentiation and Elongation1[C][W

PubMed Central

Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

2013-01-01

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells. PMID:23715527

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

PubMed Central

Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

2015-01-01

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression. PMID:26734018

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

PubMed

Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

2015-01-01

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

A functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged BCG or M.Tb (BCGlux/M.Tb lux).

PubMed

Newton, Sandra; Martineau, Adrian; Kampmann, Beate

2011-09-14

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays. Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date. Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model. Note: Definitions/Abbreviations BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability).

Downregulated PITX1 Modulated by MiR-19a-3p Promotes Cell Malignancy and Predicts a Poor Prognosis of Gastric Cancer by Affecting Transcriptionally Activated PDCD5.

PubMed

Qiao, Fengchang; Gong, Pihai; Song, Yunwei; Shen, Xiaohui; Su, Xianwei; Li, Yiping; Wu, Huazhang; Zhao, Zhujiang; Fan, Hong

2018-01-01

PITX1 has been identified as a potential tumor-suppressor gene in several malignant tumors. The molecular mechanism underlying PITX1, particularly its function as a transcription factor regulating gene expression during tumorigenesis, is still poorly understood. The expression level and location of PITX1 were determined by quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical staining in gastric cancer (GC). The effect of PITX1 on the GC cell proliferation and tumorigenesis was analyzed in vitro and in vivo. To explore how PITX1 suppresses cell proliferation, we used PITX1-ChIP-sequencing to measure genome-wide binding sites of PITX1 and assessed global function associations based on its putative target genes. ChIP-PCR, electrophoretic mobility shift assay, and promoter reporter assays examined whether PITX1 bound to PDCD5 and regulated its expression. The function of PDCD5 in GC cell apoptosis was further examined in vitro and in vivo. The relationship between the PITX1 protein level and GC patient prognosis was evaluated by the Kaplan-Meier estimator. Meanwhile, the expression level of miR-19a-3p, which is related to PITX1, was also detected by luciferase reporter assay, qRT-PCR, and western blotting. The expression level of PITX1 was decreased in GC tissues and cell lines. Elevated PITX1 expression significantly suppressed the cell proliferation of GC cells and tumorigenesis in vitro and in vivo. PITX1 knockdown blocked its inhibition of GC cell proliferation. PITX1 bound to whole genome-wide sites, with these targets enriched on genes with functions mainly related to cell growth and apoptosis. PITX1 bound to PDCD5, an apoptosis-related gene, during tumorigenesis, and cis-regulated PDCD5 expression. Increased PDCD5 expression in GC cells not only induced GC cell apoptosis, but also suppressed GC cell growth in vitro and in vivo. Moreover, PITX1 expression was regulated by miR-19a-3p. More importantly, a decreased level of PITX1 protein was correlated with poor GC patient prognosis. Decreased expression of PITX1 predicts shorter overall survival in GC patients. As a transcriptional activator, PITX1 regulates apoptosis-related genes, including PDCD5, during gastric carcinogenesis. These data indicate PDCD5 to be a novel and feasible therapeutic target for GC. © 2018 The Author(s). Published by S. Karger AG, Basel.

The R2R3 MYB transcription factor PavMYB10.1 involves in anthocyanin biosynthesis and determines fruit skin colour in sweet cherry (Prunus avium L.).

PubMed

Jin, Wanmei; Wang, Hua; Li, Maofu; Wang, Jing; Yang, Yuan; Zhang, Xiaoming; Yan, Guohua; Zhang, Hong; Liu, Jiashen; Zhang, Kaichun

2016-11-01

Sweet cherry is a diploid tree species and its fruit skin has rich colours from yellow to blush to dark red. The colour is closely related to anthocyanin biosynthesis and is mainly regulated at the transcriptional level by transcription factors that regulate the expression of multiple structural genes. However, the genetic and molecular bases of how these genes ultimately determine the fruit skin colour traits remain poorly understood. Here, our genetic and molecular evidences identified the R2R3 MYB transcription factor PavMYB10.1 that is involved in anthocyanin biosynthesis pathway and determines fruit skin colour in sweet cherry. Interestingly, we identified three functional alleles of the gene causally leading to the different colours at mature stage. Meanwhile, our experimental results of yeast two-hybrid assays and chromatin immunoprecipitation assays revealed that PavMYB10.1 might interact with proteins PavbHLH and PavWD40, and bind to the promoter regions of the anthocyanin biosynthesis genes PavANS and PavUFGT; these findings provided to a certain extent mechanistic insight into the gene's functions. Additionally, genetic and molecular evidences confirmed that PavMYB10.1 is a reliable DNA molecular marker to select fruit skin colour in sweet cherry. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.

PubMed

Singh, Chandra K; Ojha, Abhishek; Kachru, Devendra N

2007-01-01

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.

Human transcription factor hTAF(II)150 (CIF150) is involved in transcriptional regulation of cell cycle progression.

PubMed

Martin, J; Halenbeck, R; Kaufmann, J

1999-08-01

Here we present evidence that CIF150 (hTAF(II)150), the human homolog of Drosophila TAF(II)150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAF(II)150) seems to be less tightly associated with human transcription factor IID than hTAF(II)130 is associated with hTAF(II)250. The transient functional knockout of CIF150 (hTAF(II)150) protein led to cell cycle arrest at the G(2)/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAF(II)150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAF(II)150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAF(II)150) function. We defined a CIF150 (hTAF(II)150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAF(II)150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression.

Human Transcription Factor hTAFII150 (CIF150) Is Involved in Transcriptional Regulation of Cell Cycle Progression

PubMed Central

Martin, Jay; Halenbeck, Robert; Kaufmann, Jörg

1999-01-01

Here we present evidence that CIF150 (hTAFII150), the human homolog of Drosophila TAFII150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAFII150) seems to be less tightly associated with human transcription factor IID than hTAFII130 is associated with hTAFII250. The transient functional knockout of CIF150 (hTAFII150) protein led to cell cycle arrest at the G2/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAFII150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAFII150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAFII150) function. We defined a CIF150 (hTAFII150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAFII150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression. PMID:10409744

Characterization of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Mutations in Familial Isolated Pituitary Adenoma Families

PubMed Central

Igreja, Susana; Chahal, Harvinder S; King, Peter; Bolger, Graeme B; Srirangalingam, Umasuthan; Guasti, Leonardo; Chapple, J Paul; Trivellin, Giampaolo; Gueorguiev, Maria; Guegan, Katie; Stals, Karen; Khoo, Bernard; Kumar, Ajith V; Ellard, Sian; Grossman, Ashley B; Korbonits, Márta

2010-01-01

Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation-negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc. PMID:20506337

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

PubMed

Gupta, Kartik; Pilli, Vijaya Satish Sekhar; Aradhyam, Gopala Krishna

2016-10-28

Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

PubMed Central

Cherry, Jonathan J.; Kobayashi, Dione T.; Lynes, Maureen M.; Naryshkin, Nikolai N.; Tiziano, Francesco Danilo; Zaworski, Phillip G.; Rubin, Lee L.

2014-01-01

Abstract Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ∼1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials. PMID:25147906

Simultaneous detection of multiple biomarkers by means of SERS on polymer nanopillar gold arrays

NASA Astrophysics Data System (ADS)

Morasso, Carlo; Picciolini, Silvia; Mehn, Dora; Pellacani, Paola; Frangolho, Ana; Marchesini, Gerardo; Vanna, Renzo; Gualerzi, Alice; Bedoni, Marzia; Marabelli, Franco; Gramatica, Furio

2016-03-01

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.

Activity screening of carrier domains within nonribosomal peptide synthetases using complex substrate mixtures and large molecule mass spectrometry.

PubMed

Dorrestein, Pieter C; Blackhall, Jonathan; Straight, Paul D; Fischbach, Michael A; Garneau-Tsodikova, Sylvie; Edwards, Daniel J; McLaughlin, Shaun; Lin, Myat; Gerwick, William H; Kolter, Roberto; Walsh, Christopher T; Kelleher, Neil L

2006-02-14

For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

Functional Profiling Using the Saccharomyces Genome Deletion Project Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

The ability to measure and quantify the fitness of an entire organism requires considerably more complex approaches than simply using traditional "omic" methods that examine, for example, the abundance of RNA transcripts, proteins, or metabolites. The yeast deletion collections represent the only systematic, comprehensive set of null alleles for any organism in which such fitness measurements can be assayed. Generated by the Saccharomyces Genome Deletion Project, these collections allow the systematic and parallel analysis of gene functions using any measurable phenotype. The unique 20-bp molecular barcodes engineered into the genome of each deletion strain facilitate the massively parallel analysis of individual fitness. Here, we present functional genomic protocols for use with the yeast deletion collections. We describe how to maintain, propagate, and store the deletion collections and how to perform growth fitness assays on single and parallel screening platforms. Phenotypic fitness analyses of the yeast mutants, described in brief here, provide important insights into biological functions, mechanisms of drug action, and response to environmental stresses. It is important to bear in mind that the specific assays described in this protocol represent some of the many ways in which these collections can be assayed, and in this description particular attention is paid to maximizing throughput using growth as the phenotypic measure. © 2016 Cold Spring Harbor Laboratory Press.

Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons.

PubMed

Nie, Jianhui; Huang, Weijin; Wu, Xueling; Wang, Youchun

2014-09-01

The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera > human sera > mouse sera, P < 0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation. © 2014 Wiley Periodicals, Inc.

A Synthetic DNA-Binding Domain Guides Distinct Chromatin-Modifying Small Molecules to Activate an Identical Gene Network.

PubMed

Han, Le; Pandian, Ganesh N; Chandran, Anandhakumar; Sato, Shinsuke; Taniguchi, Junichi; Kashiwazaki, Gengo; Sawatani, Yoshito; Hashiya, Kaori; Bando, Toshikazu; Xu, Yufang; Qian, Xuhong; Sugiyama, Hiroshi

2015-07-20

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tumor-suppressive microRNA-497 targets IKKβ to regulate NF-κB signaling pathway in human prostate cancer cells.

PubMed

Kong, Xiang-Jie; Duan, Liu-Jian; Qian, Xiao-Qiang; Xu, Ding; Liu, Hai-Long; Zhu, Ying-Jian; Qi, Jun

2015-01-01

Prostate cancer (PCa) is one of the most prevalent malignant tumors, PCa-related death is mainly due to the high probability of metastasis. MicroRNAs (miRNAs) play an important role in cancer initiation, progression and metastasis by regulating their target genes. real-time PCR was used to detected the expression of microRNA-497. The molecular biological function was investigated by using cell proliferation assays, cell cycle assay, and migration and invasion assay. We used several Algorithms and confirmed that IKKβ is directly regulated by miR-497. Here, we found miR-497 is downregulated in human prostate cancer (PCa) and inhibites the proliferation activity, migration and invasion of PC3-AR cells. Subsequently, IKKβ is confi rmed as a target of miR-497. Furthermore, knockdown of IKKβ expression resulted in decreased proliferation activity, migration and invasion. Finally, similar results was found after treatment with a novel IKK-β inhibitor (IMD-0354) in PC3-AR cells. CDK8, MMP-9, and PSA were involved in all these process. Taken together, our results show evidence that miR-497 may function as a tumor suppressor genes by regulating IKK-β in PCa, and may provide a strategy for blocking PCa metastasis.

MDA-9/syntenin and IGFBP-2 promote angiogenesis in human melanoma.

PubMed

Das, Swadesh K; Bhutia, Sujit K; Azab, Belal; Kegelman, Timothy P; Peachy, Leyla; Santhekadur, Prasanna K; Dasgupta, Santanu; Dash, Rupesh; Dent, Paul; Grant, Steven; Emdad, Luni; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B

2013-01-15

Melanoma differentiation-associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacologic approaches were used to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma, and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chorioallantoic membrane (CAM) assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several proangiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the extracellular matrix (ECM), activating Src and FAK resulting in activation by phosphorylation of Akt, which induces hypoxia inducible factor 1-α (HIF-1α). The HIF-1α activates transcription of insulin growth factor-binding protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell nonautonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (nonautonomous).

MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma

PubMed Central

Das, Swadesh K.; Bhutia, Sujit K.; Azab, Belal; Kegelman, Timothy P.; Peachy, Leyla; Santhekadur, Prasanna K.; Dasgupta, Santanu; Dash, Rupesh; Dent, Paul; Grant, Steven; Emdad, Luni; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B.

2012-01-01

Melanoma differentiation associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacological approaches were employed to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, CAM assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several pro-angiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the ECM activating Src and FAK resulting in activation by phosphorylation of Akt, which induces HIF-1α. The HIF-1α activates transcription of Insulin Growth Factor Binding Protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell non-autonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (non-autonomous). PMID:23233738

Genome-Wide SNP Genotyping to Infer the Effects on Gene Functions in Tomato

PubMed Central

Hirakawa, Hideki; Shirasawa, Kenta; Ohyama, Akio; Fukuoka, Hiroyuki; Aoki, Koh; Rothan, Christophe; Sato, Shusei; Isobe, Sachiko; Tabata, Satoshi

2013-01-01

The genotype data of 7054 single nucleotide polymorphism (SNP) loci in 40 tomato lines, including inbred lines, F1 hybrids, and wild relatives, were collected using Illumina's Infinium and GoldenGate assay platforms, the latter of which was utilized in our previous study. The dendrogram based on the genotype data corresponded well to the breeding types of tomato and wild relatives. The SNPs were classified into six categories according to their positions in the genes predicted on the tomato genome sequence. The genes with SNPs were annotated by homology searches against the nucleotide and protein databases, as well as by domain searches, and they were classified into the functional categories defined by the NCBI's eukaryotic orthologous groups (KOG). To infer the SNPs' effects on the gene functions, the three-dimensional structures of the 843 proteins that were encoded by the genes with SNPs causing missense mutations were constructed by homology modelling, and 200 of these proteins were considered to carry non-synonymous amino acid substitutions in the predicted functional sites. The SNP information obtained in this study is available at the Kazusa Tomato Genomics Database (http://plant1.kazusa.or.jp/tomato/). PMID:23482505

Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis

PubMed Central

Zhang, Huimin; Wang, Qingjing; Fisher, Derek J.; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O.; Feng, Youjun

2016-01-01

Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake 3H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis. PMID:27161258

Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis.

PubMed

Zhang, Huimin; Wang, Qingjing; Fisher, Derek J; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O; Feng, Youjun

2016-05-10

Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake (3)H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis.

Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter.

PubMed

Hayashi, Keiko; Yoshida, Hitoshi

2009-02-01

The plant genome contains a large number of disease resistance (R) genes that have evolved through diverse mechanisms. Here, we report that a long terminal repeat (LTR) retrotransposon contributed to the evolution of the rice blast resistance gene Pit. Pit confers race-specific resistance against the fungal pathogen Magnaporthe grisea, and is a member of the nucleotide-binding site leucine-rich repeat (NBS-LRR) family of R genes. Compared with the non-functional allele Pit(Npb), the functional allele Pit(K59) contains four amino acid substitutions, and has the LTR retrotransposon Renovator inserted upstream. Pathogenesis assays using chimeric constructs carrying the various regions of Pit(K59) and Pit(Npb) suggest that amino acid substitutions might have a potential effect in Pit resistance; more importantly, the upregulated promoter activity conferred by the Renovator sequence is essential for Pit function. Our data suggest that transposon-mediated transcriptional activation may play an important role in the refunctionalization of additional 'sleeping' R genes in the plant genome.

Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

NASA Astrophysics Data System (ADS)

Das, Debobrato

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Maternally expressed gene 3, an imprinted noncoding RNA gene, is associated with meningioma pathogenesis and progression.

PubMed

Zhang, Xun; Gejman, Roger; Mahta, Ali; Zhong, Ying; Rice, Kimberley A; Zhou, Yunli; Cheunsuchon, Pornsuk; Louis, David N; Klibanski, Anne

2010-03-15

Meningiomas are common tumors, representing 15% to 25% of all central nervous system tumors. NF2 gene inactivation on chromosome 22 has been shown as an early event in tumorigenesis; however, few factors underlying tumor growth and progression have been identified. The chromosomal abnormalities of 14q32 are often associated with meningioma pathogenesis and progression; therefore, it has been proposed that an as yet unidentified tumor suppressor is present at this locus. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 which encodes a noncoding RNA with an antiproliferative function. We found that MEG3 mRNA is highly expressed in normal arachnoidal cells. However, MEG3 is not expressed in the majority of human meningiomas or the human meningioma cell lines IOMM-Lee and CH157-MN. There is a strong association between loss of MEG3 expression and tumor grade. Allelic loss at the MEG3 locus is also observed in meningiomas, with increasing prevalence in higher grade tumors. In addition, there is an increase in CpG methylation within the promoter and the imprinting control region of MEG3 gene in meningiomas. Functionally, MEG3 suppresses DNA synthesis in both IOMM-Lee and CH157-MN cells by approximately 60% in bromodeoxyuridine incorporation assays. Colony-forming efficiency assays show that MEG3 inhibits colony formation in CH157-MN cells by approximately 80%. Furthermore, MEG3 stimulates p53-mediated transactivation in these cell lines. Therefore, these data are consistent with the hypothesis that MEG3, which encodes a noncoding RNA, may be a tumor suppressor gene at chromosome 14q32 involved in meningioma progression via a novel mechanism.

Identification of transcriptional regulatory nodes in soybean defense networks using transient co-transactivation assays

PubMed Central

Wang, Yongli; Wang, Hui; Ma, Yujie; Du, Haiping; Yang, Qing; Yu, Deyue

2015-01-01

Plant responses to major environmental stressors, such as insect feeding, not only occur via the functions of defense genes but also involve a series of regulatory factors. Our previous transcriptome studies proposed that, in addition to two defense-related genes, GmVSPβ and GmN:IFR, a high proportion of transcription factors (TFs) participate in the incompatible soybean-common cutworm interaction networks. However, the regulatory mechanisms and effects of these TFs on those induced defense-related genes remain unknown. In the present work, we isolated and identified 12 genes encoding MYB, WRKY, NAC, bZIP, and DREB TFs from a common cutworm-induced cDNA library of a resistant soybean line. Sequence analysis of the promoters of three co-expressed genes, including GmVSPα, GmVSPβ, and GmN:IFR, revealed the enrichment of various TF-binding sites for defense and stress responses. To further identify the regulatory nodes composed of these TFs and defense gene promoters, we performed extensive transient co-transactivation assays to directly test the transcriptional activity of the 12 TFs binding at different levels to the three co-expressed gene promoters. The results showed that all 12 TFs were able to transactivate the GmVSPβ and GmN:IFR promoters. GmbZIP110 and GmMYB75 functioned as distinct regulators of GmVSPα/β and GmN:IFR expression, respectively, while GmWRKY39 acted as a common central regulator of GmVSPα/β and GmN:IFR expression. These corresponding TFs play crucial roles in coordinated plant defense regulation, which provides valuable information for understanding the molecular mechanisms involved in insect-induced transcriptional regulation in soybean. More importantly, the identified TFs and suitable promoters can be used to engineer insect-resistant plants in molecular breeding studies. PMID:26579162

Genome-wide screen for modulation of hepatic apolipoprotein A-I (ApoA-I) secretion.

PubMed

Miles, Rebecca R; Perry, William; Haas, Joseph V; Mosior, Marian K; N'Cho, Mathias; Wang, Jian W J; Yu, Peng; Calley, John; Yue, Yong; Carter, Quincy; Han, Bomie; Foxworthy, Patricia; Kowala, Mark C; Ryan, Timothy P; Solenberg, Patricia J; Michael, Laura F

2013-03-01

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

Multi-targeted priming for genome-wide gene expression assays.

PubMed

Adomas, Aleksandra B; Lopez-Giraldez, Francesc; Clark, Travis A; Wang, Zheng; Townsend, Jeffrey P

2010-08-17

Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

PubMed

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Long noncoding RNAs as enhancers of gene expression.

PubMed

Ørom, U A; Derrien, T; Guigo, R; Shiekhattar, R

2010-01-01

The human genome contains thousands of long noncoding RNAs (ncRNAs) transcribed from diverse genomic locations. A large set of long ncRNAs is transcribed independent of protein-coding genes. We have used the GENCODE annotation of the human genome to identify 3019 long ncRNAs expressed in various human cell lines and tissue. This set of long ncRNAs responds to differentiation signals in primary human keratinocytes and is coexpressed with important regulators of keratinocyte development. Depletion of a number of these long ncRNAs leads to the repression of specific genes in their surrounding locus, supportive of an activating function for ncRNAs. Using reporter assays, we confirmed such activating function and show that such transcriptional enhancement is mediated through the long ncRNA transcripts. Our studies show that long ncRNAs exhibit functions similar to classically defined enhancers, through an RNA-dependent mechanism.

Novel strategies to enforce an epithelial phenotype in mesenchymal cells

PubMed Central

Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

2014-01-01

E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae)

PubMed Central

Michel, Joanna L; Chen, Yegao; Zhang, Hongjie; Huang, Yue; Krunic, Alecjev; Orjala, Jimmy; Veliz, Mario; Soni, Kapil K.; Soejarto, Djaja Doel; Caceres, Armando; Perez, Alice; Mahady, Gail B

2010-01-01

Ethnopharmacological relevance Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q’eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors. Aim of the study To investigate the estrogenic and serotonergic activities of P. hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7. Materials and methods The effects of the P. hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC-MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays. Results Three butenolides including one new compound (1) were isolated from the leaves of P. hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT7 with IC50 values of 16.1 and 8.3 μM, respectively, and using GTP shift assays, compound 1 was found to be a partial agonist of the 5-HT7 receptor. The P. hispidum leaf extracts, as well as compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects. Conclusions Extracts of P. hispidum act as agonists of the ER and 5-HT7 receptors. Compound 1, a new natural product, identified as 9, 10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT7 agonist. Compounds 2 and 3 are reported for the first time in P. hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 μM. These activities are consistent with the Q’eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle. PMID:20304039

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae).

PubMed

Michel, Joanna L; Chen, Yegao; Zhang, Hongjie; Huang, Yue; Krunic, Aleksej; Orjala, Jimmy; Veliz, Mario; Soni, Kapil K; Soejarto, Djaja Doel; Caceres, Armando; Perez, Alice; Mahady, Gail B

2010-05-27

Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q'eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors. To investigate the estrogenic and serotonergic activities of Piper hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7. The effects of the Piper hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC-MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays. Three butenolides including one new compound (1) were isolated from the leaves of Piper hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT(7) with IC(50) values of 16.1 and 8.3 microM, respectively, and using GTP shift assays, Compound 1 was found to be a partial agonist of the 5-HT(7) receptor. The Piper hispidum leaf extracts, as well as Compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects. Extracts of Piper hispidum act as agonists of the ER and 5-HT(7) receptors. Compound 1, a new natural product, identified as 9,10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT(7) agonist. Compounds 2 and 3 are reported for the first time in Piper hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 microM. These activities are consistent with the Q'eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

High-throughput single-cell PCR using microfluidic emulsions

NASA Astrophysics Data System (ADS)

Guo, Mira; Mazutis, Linas; Agresti, Jeremy; Sommer, Morten; Dantas, Gautam; Church, George; Turnbaugh, Peter; Weitz, David

2012-02-01

The human gut and other environmental samples contain large populations of diverse bacteria that are poorly characterized and unculturable, yet have many functions relevant to human health. Our goal is to identify exactly which species carry some gene of interest, such as a carbohydrate metabolism gene. Conventional metagenomic assays sequence DNA extracted in bulk from populations of mixed cell types, and are therefore unable to associate a gene of interest with a species-identifying 16S gene, to determine that the two genes originated from the same cell. We solve this problem by microfluidically encapsulating single bacteria cells in drops, using PCR to amplify the two genes inside any drop whose encapsulated cell contains both genes, and sequencing the DNA from those drops that contain both amplification products.

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

PubMed

Findley, Seth D; Mormile, Melanie R; Sommer-Hurley, Andrea; Zhang, Xue-Cheng; Tipton, Peter; Arnett, Krista; Porter, James H; Kerley, Monty; Stacey, Gary

2011-11-01

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

The Molecular Biology of Nitroamine Degradation in Soils

DTIC Science & Technology

2015-07-26

analysis and activity assays .............................................................................. 28 Determination of a putative...81 Figure 52: Specific XplA activity in cells treated with different nitrogen sources. .......... 83 Figure 53: Effect of... activity . Our efforts to develop a functional screen for genes from the soil metagenome were unsuccessful. We developed efficient methods of

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

Reactivation of Embryonic Nodal Signaling is Associated with Tumor Progression and Promotes the Growth of Prostate Cancer Cells

PubMed Central

Lawrence, Mitchell G.; Margaryan, Naira V.; Loessner, Daniela; Collins, Angus; Kerr, Kris M.; Turner, Megan; Seftor, Elisabeth A.; Stephens, Carson R.; Lai, John; BioResource, APC; Postovit, Lynne-Marie; Clements, Judith A.; Hendrix, Mary J.C.

2011-01-01

Background Nodal is a member of the Transforming Growth Factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity. Methods Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes. Results Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal’s co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes. Conclusions An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells. PMID:21656830

Initial experience with GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.

PubMed

Patil, Naveen; Saba, Hamida; Marco, Asween; Samant, Rohan; Mukasa, Leonard

2014-01-01

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. Rapid and accurate diagnosis of pulmonary and extra-pulmonary tuberculosis (TB) is still a great challenge. The GeneXpert MTB/RIF assay is a novel integrated diagnostic system for the diagnosis of tuberculosis and rapid detection of Rifampin (RIF) resistance in clinical specimens. In 2012, the Arkansas Tuberculosis Control Program introduced GeneXpert MTB/RIF assay to replace the labour-intensive Mycobacterium Tuberculosis Direct (MTD) assay. To rapidly diagnose TB within two hours and to simultaneously detect RIF resistance. Describe the procedure used to introduce GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.Characterise the current gap in rapid M. tuberculosis diagnosis in Arkansas.Assess factors that predict acid fast bacilli (AFB) smearnegative but culture-positive cases in Arkansas.Illustrate, with two case reports, the role of GeneXpert MTB/RIF assay in reduction of time to confirmation of M. tuberculosis diagnosis in the first year of implementation. Between June 2012 and June 2013, all AFB sputum smearpositive cases and any others, on request by the physician, had GeneXpert MTB/RIF assay performed as well as traditional M. tuberculosis culture and susceptibilities using Mycobacteria Growth Indicator Tube (MGIT) 960 and Löwenstein-Jensen (LJ) slants. Surveillance data for January 2009-June 2013 was analysed to characterise sputum smear-negative but culture-positive cases. Seventy-one TB cases were reported from June 2012- June 2013. GeneXpert MTB/RIF assay identified all culture-positive cases as well as three cases that were negative on culture. Also, this rapid assay identified all six smear-negative but M. tuberculosis culture-positive cases; two of these cases are described as case reports. GeneXpert MTB/RIF assay has made rapid TB diagnosis possible, with tremendous potential in determining isolation of TB suspects on one hand, and quickly ruling out TB whenever suspected.

Cost-effectiveness of the 21-gene assay for guiding adjuvant chemotherapy decisions in early breast cancer.

PubMed

Paulden, Mike; Franek, Jacob; Pham, Ba'; Bedard, Philippe L; Trudeau, Maureen; Krahn, Murray

2013-01-01

Adjuvant chemotherapy decisions in early breast cancer are complex. The 21-gene assay can potentially aid such decisions, but costs US $4175 per patient. Adjuvant! Online is a freely available decision aid. We evaluate the cost-effectiveness of using the 21-gene assay in conjunction with Adjuvant! Online, and of providing adjuvant chemotherapy conditional upon risk classification. A probabilistic Markov decision model simulated risk classification, treatment, and the natural history of breast cancer in a hypothetical cohort of 50-year-old women with lymph node-negative, estrogen receptor- and/or progesterone receptor-positive, human epidermal growth factor receptor 2/neu-negative early breast cancer. Cost-effectiveness was considered from an Ontario public-payer perspective by deriving the lifetime incremental cost (2012 Canadian dollars) per quality-adjusted life-year (QALY) for each strategy, and the probability each strategy is cost-effective, assuming a willingness-to-pay of $50,000 per QALY. The 21-gene assay has an incremental cost per QALY in patients at low, intermediate, or high Adjuvant Online! risk of $22,440 (probability cost-effective 78.46%), $2,526 (99.40%), or $1,111 (99.82%), respectively. In patients at low (high) 21-gene assay risk, adjuvant chemotherapy increases (reduces) costs and worsens (improves) health outcomes. For patients at intermediate 21-gene assay risk and low, intermediate, or high Adjuvant! Online risk, chemotherapy has an incremental cost per QALY of $44,088 (50.59%), $1,776 (77.65%), or $1,778 (82.31%), respectively. The 21-gene assay appears cost-effective, regardless of Adjuvant! Online risk. Adjuvant chemotherapy appears cost-effective for patients at intermediate or high 21-gene assay risk, although this finding is uncertain in patients at intermediate 21-gene assay and low Adjuvant! Online risk. Copyright © 2013 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

PubMed Central

2009-01-01

Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. Conclusion This automated process allows laboratories to discover DNA variations in a short time and at low cost. PMID:19835634

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes.

PubMed

Bennett, Richard R; Schneider, Hal E; Estrella, Elicia; Burgess, Stephanie; Cheng, Andrew S; Barrett, Caitlin; Lip, Va; Lai, Poh San; Shen, Yiping; Wu, Bai-Lin; Darras, Basil T; Beggs, Alan H; Kunkel, Louis M

2009-10-18

One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. This automated process allows laboratories to discover DNA variations in a short time and at low cost.

Knockdown of long noncoding RNA 00152 (LINC00152) inhibits human retinoblastoma progression.

PubMed

Li, Songhe; Wen, Dacheng; Che, Songtian; Cui, Zhihua; Sun, Yabin; Ren, Hua; Hao, Jilong

2018-01-01

A growing body of evidence supports the involvement of long noncoding RNA 00152 (LINC00152) in the progression and metastasis of multiple cancers. However, the exact roles of LINC00152 in the progression of human retinoblastoma (RB) remain unknown. We explored the expression and biological function of human RB. The expression level of LINC00152 in RB tissues and cells was analyzed using quantitative real-time PCR. The function of LINC00152 was determined using a series of in vitro assays. In vivo, a nude mouse model was established to analyze the function of LINC00152. Gene and protein expressions were detected using quantitative real-time PCR and Western blot assays, respectively. The expression of LINC00152 mRNA was upregulated in RB tissues and cell lines. Knockdown of LINC00152 significantly inhibited cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and caspase-3 and caspase-8 activities in vitro, as well as suppressing tumorigenesis in vivo. We identified several genes related to proliferation, apoptosis, and invasion including Ki-67, Bcl-2, and MMP-9 that were transcriptionally inactivated by LINC00152. Taken together, these data implicate LINC00152 as a therapeutic target in RB.

[Cloning,expression and functional identification of secoisolariciresinol dehydrogenase gene from Dysosma versipellis callus].

PubMed

Shen, Yun; Chen, Ri-Dao; Xie, Ke-Bo; Zou, Jian-Hua; Dai, Jun-Gui

2016-12-01

Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin. Copyright© by the Chinese Pharmaceutical Association.

GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2016-01-01

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

MoSfl1 Is Important for Virulence and Heat Tolerance in Magnaporthe oryzae

PubMed Central

Li, Guotian; Zhou, Xiaoying; Kong, Lingan; Wang, Yuling; Zhang, Haifeng; Zhu, Heng; Mitchell, Thomas K.; Dean, Ralph A.; Xu, Jin-Rong

2011-01-01

The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae. PMID:21625508

Idiopathic Hypogonadotropic Hypogonadism Caused by Inactivating Mutations in SRA1

PubMed Central

Kotan, Leman Damla; Cooper, Charlton; Darcan, Şükran; Carr, Ian M.; Özen, Samim; Yan, Yi; Hamedani, Mohammad K.; Gürbüz, Fatih; Mengen, Eda; Turan, İhsan; Ulubay, Ayça; Akkuş, Gamze; Yüksel, Bilgin; Topaloğlu, A. Kemal; Leygue, Etienne

2016-01-01

Objective: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). Methods: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. Results: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. Conclusion: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process. PMID:27086651

Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

PubMed Central

2010-01-01

Background Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions. Results We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation. Conclusions The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family. PMID:20964856

Analysis of the role of Arabidopsis class I TCP genes AtTCP7, AtTCP8, AtTCP22, and AtTCP23 in leaf development

PubMed Central

Aguilar-Martínez, José A.; Sinha, Neelima

2013-01-01

TCP family of plant-specific transcription factors regulates plant form through control of cell proliferation and differentiation. This gene family is comprised of two groups, class I and class II. While the role of class II TCP genes in plant development is well known, data about the function of some class I TCP genes is lacking. We studied a group of phylogenetically related class I TCP genes: AtTCP7, AtTCP8, AtTCP22, and AtTCP23. The similar expression pattern in young growing leaves found for this group suggests similarity in gene function. Gene redundancy is characteristic in this group, as also seen in the class II TCP genes. We generated a pentuple mutant tcp8 tcp15 tcp21 tcp22 tcp23 and show that loss of function of these genes results in changes in leaf developmental traits. We also determined that these factors are able to mutually interact in a yeast two-hybrid assay and regulate the expression of KNOX1 genes. To circumvent the issue of genetic redundancy, dominant negative forms with SRDX repressor domain were used. Analysis of transgenic plants expressing AtTCP7-SRDX and AtTCP23-SRDX indicate a role of these factors in the control of cell proliferation. PMID:24137171

Analysis of the role of Arabidopsis class I TCP genes AtTCP7, AtTCP8, AtTCP22, and AtTCP23 in leaf development.

PubMed

Aguilar-Martínez, José A; Sinha, Neelima

2013-01-01

TCP family of plant-specific transcription factors regulates plant form through control of cell proliferation and differentiation. This gene family is comprised of two groups, class I and class II. While the role of class II TCP genes in plant development is well known, data about the function of some class I TCP genes is lacking. We studied a group of phylogenetically related class I TCP genes: AtTCP7, AtTCP8, AtTCP22, and AtTCP23. The similar expression pattern in young growing leaves found for this group suggests similarity in gene function. Gene redundancy is characteristic in this group, as also seen in the class II TCP genes. We generated a pentuple mutant tcp8 tcp15 tcp21 tcp22 tcp23 and show that loss of function of these genes results in changes in leaf developmental traits. We also determined that these factors are able to mutually interact in a yeast two-hybrid assay and regulate the expression of KNOX1 genes. To circumvent the issue of genetic redundancy, dominant negative forms with SRDX repressor domain were used. Analysis of transgenic plants expressing AtTCP7-SRDX and AtTCP23-SRDX indicate a role of these factors in the control of cell proliferation.

Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

PubMed

Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J

2009-10-01

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens.

PubMed

Chen, Junfeng; Yang, Chingyuan; Tizioto, Polyana C; Huang, Huan; Lee, Mi O K; Payne, Harold R; Lawhon, Sara D; Schroeder, Friedhelm; Taylor, Jeremy F; Womack, James E

2016-01-01

Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease.

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

PubMed

Klangnurak, Wanlada; Fukuyo, Taketo; Rezanujjaman, M D; Seki, Masahide; Sugano, Sumio; Suzuki, Yutaka; Tokumoto, Toshinobu

2018-01-01

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.

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dimopoulou, Myrto, E-mail: myrto.dimopoulou@wur.nl

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effectivemore » dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic acid gene set identifies early embryotoxic responses to azoles. • Toxic versus pharmacologic potency determines functional efficacy.« less

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

NF1 Neuronal Genotype Phenotype Relationships

DTIC Science & Technology

2017-06-01

neurofibromin protein that are required for correct function in Drosophila. We will use these insights for subsequent CRISPR /Cas9 gene editing of humnan induced...selecting the most informative of these to develop isogenic human iPSC models using CRISPR /Cas9 gene editing. These NF1 mutant iPSC lines will then be used...Together, the results of these assays will help us to focus on specific missense mutations, which we plan to engineer into hiPSCs using CRISPR /Cas9

Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

DTIC Science & Technology

2013-10-01

dilution transplantation functional assays, we estimated the fMaSC population to be 10-20% pure. Therefore, we inferred that its gene expression...measured by the gold standard of in vivo transplantation . This approach will not only enable us to identify biomarkers useful for prospectively...image of two of the 96 Fluidigm-C1 capture wells containing candidate fMaSC cells. green=live (Calcein-AM), red= dead (Ethidium Bromide). (B) RT-PCR

P53 Mutation Analysis to Predict Tumor Response in Patients Undergoing Neoadjuvant Treatment for Locally Advanced Breast Cancer

DTIC Science & Technology

2006-10-01

then sequenced (for GeneChip- positiv SSCP (for GeneChip-negative). We have received a total of 43 core breast biopsy DNA samples from the UNC... quantitative luciferase reporter. Both reporters exploit a “rheostatable” promoter for p53 expression and utilize the “delitto perfetto” in vivo... quantitative luciferase-based assay is also being used to characterize the altered function sistent an tion T mutants in greater detail. Preliminary

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation

PubMed Central

Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

2014-01-01

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-l-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., 1994), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis. PMID:24286468

2-Aminoimidazole facilitates efficient gene delivery in a low molecular weight poly(amidoamine) dendrimer.

PubMed

Wang, Jing; Hu, Xuefeng; Wang, Dongli; Xie, Cao; Lu, Weiyue; Song, Jie; Wang, Ruifeng; Gao, Chunli; Liu, Min

2018-06-20

Functional groups have shown great potential in gene delivery. However, a number of the reported functional groups can only overcome one certain physiological barrier, resulting in limited transfection efficiencies. Based on the structure-activity relationships of both imidazolyl and guanidyl, we designed a novel multifunctional group, 2-aminoimidazole (AM), for gene delivery. On modifying with the AM group, the transfection efficiency of low molecular weight poly(amidoamine) (G2) was 200 times greater than the parent dendrimer in vitro. In contrast, the transfection efficiency of G2 showed a decreasing trend when it was grafted with imidazole. Assays revealed that the AM group played multiple roles in gene delivery, including condensing DNA into monodisperse nanoparticles of 80-90 nm in diameter, achieving nearly ten times higher cellular-uptake efficacy, and enhancing the abilities of endosome/lysosome escape and nuclear localization. What's more, AM showed low toxicity. These results demonstrate that the AM group could be a promising tool in non-viral gene delivery.

Isolation and Functional Analyses of a Putative Floral Homeotic C-Function Gene in a Basal Eudicot London Plane Tree (Platanus acerifolia)

PubMed Central

Liu, Guofeng; Bao, Manzhu

2013-01-01

The identification of mutants in model plant species has led to the isolation of the floral homeotic function genes that play crucial roles in flower organ specification. However, floral homeotic C-function genes are rarely studied in basal eudicots. Here, we report the isolation and characterization of the AGAMOUS (AG) orthologous gene (PaAG) from a basal eudicot London plane tree (Platanus acerifolia Willd). Phylogenetic analysis showed that PaAG belongs to the C- clade AG group of genes. PaAG was found to be expressed predominantly in the later developmental stages of male and female inflorescences. Ectopic expression of PaAG-1 in tobacco (Nicotiana tabacum) resulted in morphological alterations of the outer two flower whorls, as well as some defects in vegetative growth. Scanning electron micrographs (SEMs) confirmed homeotic sepal-to-carpel transformation in the transgenic plants. Protein interaction assays in yeast cells indicated that PaAG could interact directly with PaAP3 (a B-class MADS-box protein in P. acerifolia), and also PaSEP1 and PaSEP3 (E-class MADS-box proteins in P. acerifolia). This study performed the functional analysis of AG orthologous genes outside core eudicots and monocots. Our findings demonstrate a conserved functional role of AG homolog in London plane tree, which also represent a contribution towards understanding the molecular mechanisms of flower development in this monoecious tree species. PMID:23691041

A Dual-Color Reporter Assay of Cohesin-Mediated Gene Regulation in Budding Yeast Meiosis.

PubMed

Fan, Jinbo; Jin, Hui; Yu, Hong-Guo

2017-01-01

In this chapter, we describe a quantitative fluorescence-based assay of gene expression using the ratio of the reporter green fluorescence protein (GFP) to the internal red fluorescence protein (RFP) control. With this dual-color heterologous reporter assay, we have revealed cohesin-regulated genes and discovered a cis-acting DNA element, the Ty1-LTR, which interacts with cohesin and regulates gene expression during yeast meiosis. The method described here provides an effective cytological approach for quantitative analysis of global gene expression in budding yeast meiosis.

Germline Missense Changes in the APC Gene and Their Relationship to Disease.

PubMed

Scott, Rodney J; Crooks, Renee; Rose, Lindy; Attia, John; Thakkinstian, Ammarin; Thomas, Lesley; Spigelman, Allan D; Meldrum, Cliff J

2004-05-15

Familial adenomatous polyposis (FAP) is characterized by the presence of hundreds to thousands of adenomas that carpet the entire colon and rectum. Nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene account for the majority of mutations identified to date and predispose primarily to the typical disease phenotype. Some APC mutations are associated with a milder form of the disease known as attenuated FAP. Virtually all mutations that have been described in the APC gene result in the formation of a premature stop codon and very little is known about missense mutations apart from a common Ashkenazi Jewish mutation (1307 K) and a British E1317Q missense change. The incidence of missense mutations in the APC gene has been underreported since the APC gene lends itself to analysis using an artificial transcription and translation assay known as the Protein Truncation Test (PTT) or the In Vitro Synthetic Protein assay (IVSP).In this report we have used denaturing high performance liquid chromatography to analyse the entire coding sequence of the APC gene to determine if a cohort of patients adhering to the diagnostic criteria of FAP to assess the frequency of missense mutations in the APC gene. Altogether 112 patients were studied and 22 missense mutations were identified. From the total of 22 missense changes, 13 were silent changes and the remaining 9 resulted in amino acid substitutions. One or more of these changes were identified multiple times in 62.5% of the population under study.The results reveal that missense mutations in the APC gene appear not to radically alter protein function but may be associated with more subtle processing of RNA transcripts which in turn could result in the expression of differentially spliced forms of the APC gene which may interfere with the functional activity of the APC protein.

A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1

PubMed Central

Lattka, E.; Eggers, S.; Moeller, G.; Heim, K.; Weber, M.; Mehta, D.; Prokisch, H.; Illig, T.; Adamski, J.

2010-01-01

Fatty acid desaturases (FADS) play an important role in the formation of omega-6 and omega-3 highly unsaturated fatty acids (HUFAs). The composition of HUFAs in the human metabolome is important for membrane fluidity and for the modulation of essential physiological functions such as inflammation processes and brain development. Several recent studies reported significant associations of single nucleotide polymorphisms (SNPs) in the human FADS gene cluster with HUFA levels and composition. The presence of the minor allele correlated with a decrease of desaturase reaction products and an accumulation of substrates. We performed functional studies with two of the associated polymorphisms (rs3834458 and rs968567) and showed an influence of polymorphism rs968567 on FADS2 promoter activity by luciferase reporter gene assays. Electrophoretic mobility shift assays proved allele-dependent DNA-binding ability of at least two protein complexes to the region containing SNP rs968567. One of the proteins binding to this region in an allele-specific manner was shown to be the transcription factor ELK1 (a member of ETS domain transcription factor family). These results indicate that rs968567 influences FADS2 transcription and offer first insights into the modulation of complex regulation mechanisms of FADS2 gene transcription by SNPs. PMID:19546342

A common FADS2 promoter polymorphism increases promoter activity and facilitates binding of transcription factor ELK1.

PubMed

Lattka, E; Eggers, S; Moeller, G; Heim, K; Weber, M; Mehta, D; Prokisch, H; Illig, T; Adamski, J

2010-01-01

Fatty acid desaturases (FADS) play an important role in the formation of omega-6 and omega-3 highly unsaturated fatty acids (HUFAs). The composition of HUFAs in the human metabolome is important for membrane fluidity and for the modulation of essential physiological functions such as inflammation processes and brain development. Several recent studies reported significant associations of single nucleotide polymorphisms (SNPs) in the human FADS gene cluster with HUFA levels and composition. The presence of the minor allele correlated with a decrease of desaturase reaction products and an accumulation of substrates. We performed functional studies with two of the associated polymorphisms (rs3834458 and rs968567) and showed an influence of polymorphism rs968567 on FADS2 promoter activity by luciferase reporter gene assays. Electrophoretic mobility shift assays proved allele-dependent DNA-binding ability of at least two protein complexes to the region containing SNP rs968567. One of the proteins binding to this region in an allele-specific manner was shown to be the transcription factor ELK1 (a member of ETS domain transcription factor family). These results indicate that rs968567 influences FADS2 transcription and offer first insights into the modulation of complex regulation mechanisms of FADS2 gene transcription by SNPs.

The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells.

PubMed

Ugolini, S; Bruschi, C V

1996-12-01

In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.

Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

PubMed Central

Lombardo, Fabrizio; Ghani, Yasmeen; Kafatos, Fotis C.; Christophides, George K.

2013-01-01

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens. PMID:23382679

NF-κB Participates in the Stem Cell Phenotype of Ovarian Cancer Cells.

PubMed

Gonzalez-Torres, Carolina; Gaytan-Cervantes, Javier; Vazquez-Santillan, Karla; Mandujano-Tinoco, Edna Ayerim; Ceballos-Cancino, Gisela; Garcia-Venzor, Alfredo; Zampedri, Cecilia; Sanchez-Maldonado, Paulina; Mojica-Espinosa, Raul; Jimenez-Hernandez, Luis Enrique; Maldonado, Vilma

2017-05-01

NF-κB is a transcription factor involved in cancer stem cells maintenance of many tumors. Little is known about the specific stem-associated upstream regulators of this pathway in ovarian cancer. The Aim of the study was to analyze the role of the canonical and non-canonical NF-κB pathways in stem cells of ovarian cancer cell lines. Stem cells were isolated using sorting cytometry. Western blot and RT-PCR were used to quantify protein and messenger RNA levels. Loss and gain of function assays were performed using siRNAs and dominant-negative proteins, respectively. NF-κB binding activity was measured with a reporter gene assay. The stem phenotype was estimated with clonogenic assays using soft agar, colony formation, ovospheres formation and in vivo tumorigenicity assays. The CD44+ subpopulation of SKOV3 ovarian cancer cell line presented higher mRNA levels of key stemness genes, an increased tumorigenic capacity and higher expression of the RelA, RelB and IKKα. When the canonical pathway was inhibited by means of a dominant-negative version of IkBα, the stem cell population was reduced, as shown by a reduced CD44+ subpopulation, a decrease in the expression of the stemness genes and a reduction of the stem phenotype. In addition, IKKα, the main upstream non-canonical kinase, was highly expressed in the CSC population. Accordingly, when IKKα was inhibited using shRNAs, the expression of the stemness genes was reduced. This report is the first to show the importance of several elements of both NF-κB pathway in maintaining the ovarian cancer stem cell population. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci

PubMed Central

2009-01-01

Background The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes. Results Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences. Conclusion The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods. Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission. PMID:19719847

A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food.

PubMed

Lee, Nari; Kwon, Kyung Yoon; Oh, Su Kyung; Chang, Hyun-Joo; Chun, Hyang Sook; Choi, Sung-Wook

2014-07-01

A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

Identification of Location and Kinetically Defined Mechanism of Cofactors and Reporter Genes in the Cascade of Steroid-regulated Transactivation*

PubMed Central

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

2012-01-01

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

QuASAR-MPRA: accurate allele-specific analysis for massively parallel reporter assays.

PubMed

Kalita, Cynthia A; Moyerbrailean, Gregory A; Brown, Christopher; Wen, Xiaoquan; Luca, Francesca; Pique-Regi, Roger

2018-03-01

The majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRAs), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets. We have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data, we found 602 SNPs with significant (false discovery rate 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high-throughput reporter assays. http://github.com/piquelab/QuASAR/tree/master/mpra. fluca@wayne.edu or rpique@wayne.edu. Supplementary data are available online at Bioinformatics. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Hypoxia-Response Element (HRE)–Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium

PubMed Central

Li, Wande

2013-01-01

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5′-ACGTG-3′) exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10–100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)–treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at −387/−383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation. PMID:23161664

Hypoxia-response element (HRE)-directed transcriptional regulation of the rat lysyl oxidase gene in response to cobalt and cadmium.

PubMed

Gao, Song; Zhou, Jing; Zhao, Yinzhi; Toselli, Paul; Li, Wande

2013-04-01

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

Maternally Expressed Gene 3, an imprinted non-coding RNA gene, is associated with meningioma pathogenesis and progression

PubMed Central

Zhang, Xun; Gejman, Roger; Mahta, Ali; Zhong, Ying; Rice, Kimberley A.; Zhou, Yunli; Cheunsuchon, Pornsuk; Louis, David N.; Klibanski, Anne

2010-01-01

Meningiomas are common tumors, representing 15-25% of all central nervous system tumors. NF2 gene inactivation on chromosome 22 has been shown as an early event in tumorigenesis; however, few factors underlying tumor growth and progression have been identified. Chromosomal abnormalities of 14q32 are often associated with meningioma pathogenesis and progression; therefore it has been proposed that an as yet unidentified tumor suppressor is present at this locus. MEG3 is an imprinted gene located at 14q32 that encodes a non-coding RNA with an anti-proliferative function. We found that MEG3 mRNA is highly expressed in normal arachnoidal cells. However, MEG3 is not expressed in the majority of human meningiomas or the human meningioma cell lines IOMM-Lee and CH157-MN. There is a strong association between loss of MEG3 expression and tumor grade. Allelic loss at the MEG3 locus is also observed in meningiomas, with increasing prevalence in higher grade tumors. In addition, there is an increase in CpG methylation within the promoter and the imprinting control region of MEG3 gene in meningiomas. Functionally, MEG3 suppresses DNA synthesis in both IOMM-Lee and CH157-MN cells by approximately 60% in BrdU incorporation assays. Colony-forming efficiency assays show that MEG3 inhibits colony formation in CH157-MN cells by approximately 80%. Furthermore, MEG3 stimulates p53-mediated transactivation in these cell lines. Therefore, these data are consistent with the hypothesis that MEG3, which encodes a non-coding RNA, may be a tumor suppressor gene at chromosome 14q32 involved in meningioma progression via a novel mechanism. PMID:20179190

Selenium effect on selenoprotein transcriptome in chondrocytes.

PubMed

Yan, Jidong; Zheng, Yuewen; Min, Zixin; Ning, Qilan; Lu, Shemin

2013-04-01

Selenium is an essential micronutrient and exerts its biological functions predominantly through selenoproteins. Selenium deficiency is associated with cartilage function. This study demonstrated that all 24 selenoprotein transcripts in mouse genome were detectable in ATDC5 chondrocytes except deiodinase 1 (DIO1), DIO2, and selenoprotein V (Sel V), while all 25 selenoprotein transcripts in human genome were detectable in C28/I2 chondrocytes except glutathione peroxidase 6 (GPx6) and DIO1. In addition, gene expression of five selenoproteins (GPx1, Sel H, Sel N, Sel P, and Sel W) was up-regulated and two selenoproteins (SPS2 and Sel O) was down-regulated by sodium selenite (Se) in both ATDC5 and C28/I2 cells. Gene expression of six selenoproteins (TrxR1, Sel I, Sel M, Sel R, Sel S, Sel T) and one selenoprotein (GPx3) was up-regulated by Se in ATDC5 and C28/I2 cells, respectively. Gene expression of one selenoprotein (TrxR2) was down-regulated by Se only in ATDC5 cells. Further transcription inhibition assay showed that both transcriptional and posttranscriptional mechanisms involved in Se-regulated gene expression of GPx1, TrxR1, TrxR2, SPS2, Sel O, and Sel S. However, Se-regulated gene expression of Sel H, Sel I, Sel M, Sel N, Sel P, Sel R, Sel T, and Sel W mainly at posttranscriptional level. Moreover, new protein synthesis inhibition assay indicated that Se-mediated new protein synthesis also played roles in Se-regulated gene expression of GPx1, TrxR1, TrxR2, Sel H, Sel O, Sel P, Sel R, and Sel W. In summary, this study described the selenoprotein transcriptome, Se-regulated selenoproteins and possible mechanisms involved in chondrocytes.

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.

PubMed

Mayrhofer, Severine; Pöggeler, Stefanie

2005-04-01

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

PubMed Central

Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

2009-01-01

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

PubMed

Smiljanic, M; Kaase, M; Ahmad-Nejad, P; Ghebremedhin, B

2017-07-10

Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. Among the carbapenem-resistant isolates the genes bla KPC , bla VIM , bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC , bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

PubMed

Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

2009-01-01

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

PubMed

Winters, Jennifer L; Davila, Jaime I; McDonald, Amber M; Nair, Asha A; Fadra, Numrah; Wehrs, Rebecca N; Thomas, Brittany C; Balcom, Jessica R; Jin, Long; Wu, Xianglin; Voss, Jesse S; Klee, Eric W; Oliver, Gavin R; Graham, Rondell P; Neff, Jadee L; Rumilla, Kandelaria M; Aypar, Umut; Kipp, Benjamin R; Jenkins, Robert B; Jen, Jin; Halling, Kevin C

2018-06-13

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Candida albicans CHT3 encodes the functional homolog of the Cts1 chitinase of Saccharomyces cerevisiae.

PubMed

Dünkler, Alexander; Walther, Andrea; Specht, Charles A; Wendland, Jürgen

2005-11-01

Chitin synthesis and chitin degradation play an important role in cellular morphogenesis and influence the cell shape of fungal organisms. The Candida albicans genome contains four chitinase genes, CHT1, CHT2, and CHT3, which are homologous to the Saccharomyces cerevisiae CTS1 gene and C. albicans CHT4, which is homologous to S. cerevisiae CTS2. To determine which of the C. albicans CHT genes represents the functional homolog of the S. cerevisiae CTS1 gene we constructed mutants of these genes and characterized the resulting phenotypes using morphological assays such as in vivo time lapse microscopy and enzymatic assays to determine the chitinase activity. Deletion of CaCHT1 and CaCHT2 provided no phenotypic alterations in liquid culture but resulted in increased hyphal growth on solid media. Deletion of CaCHT3 generated chains of unseparated cells in the yeast growth phase strongly resembling the cts1 deletion phenotype of S. cerevisiae cells. Expression of CHT3 under control of the regulatable MAL2-promoter in C. albicans resulted in the reversion of the cell separation defect when cells were grown in maltose. Cht3, but not Cht2 when expressed in S. cerevisiae was also able to reverse the cell separation defect of the S. cerevisiae c ts1 deletion strain. Measurements of chitinase activity from yeast cells of C. albicans showed that Cht2 is bound to cells, consistent with it being GPI-anchored while Cht3 is secreted into growth medium; Cht3 is also the principal, observed activity.

The NB-LRR gene Pm60 confers powdery mildew resistance in wheat.

PubMed

Zou, Shenghao; Wang, Huan; Li, Yiwen; Kong, Zhaosheng; Tang, Dingzhong

2018-04-01

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T. urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucine-rich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiled-coil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

HOXB2, an adverse prognostic indicator for stage I lung adenocarcinomas, promotes invasion by transcriptional regulation of metastasis-related genes in HOP-62 non-small cell lung cancer cells.

PubMed

Inamura, Kentaro; Togashi, Yuki; Ninomiya, Hironori; Shimoji, Takashi; Noda, Tetsuo; Ishikawa, Yuichi

2008-01-01

Previously, using microarray and real-time RT-PCR analysis, we established that HOXB2 is an adverse prognostic indicator for Stage I lung adenocarcinomas. HOXB2 is one of the homeobox master development-controlling genes regulating morphogenesis and cell differentiation. The molecular functions of HOXB2 were analyzed with a small interfering RNA (siRNA) approach in HOP-62 human non-small cell lung cancer (NSCLC) cells featuring high HOXB2 expression. Matrigel invasion assays and microarray gene expression analysis were compared between the HOXB2-siRNA cells and the control cells. The Matrigel invasion assays showed attenuation of HOXB2 expression by siRNA to result in a significant decrease of invasiveness compared to the control cells (p = 0.0013, paired t-test). On microarray gene expression analysis, up-regulation of many metastasis-related genes and others correlating with HOXB2 expression was observed in the control case. With attenuation of HOXB2 expression, downregulation was noted for laminins alpha 4 and 5, involved in enriched signaling, and for Mac-2BP (Mac-2 binding protein) and integrin beta 4 amongst the genes having an enriched glycoprotein ontology. HOXB2 promotes invasion of lung cancer cells through the regulation of metastasis-related genes.

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

PubMed

Kristof, Jessica; Sakrison, Kellen; Jin, Xiaoping; Nakamaru, Kenji; Schneider, Matthias; Beckman, Robert A; Freeman, Daniel; Spittle, Cindy; Feng, Wenqin

2017-01-01

In preclinical studies, heregulin ( HRG ) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8 , and EIF2B1 ), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

Physical Mapping of bchG, orf427, and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides: Functional Assignment of the Bacteriochlorophyll Synthetase Gene

PubMed Central

Addlesee, Hugh A.; Fiedor, Leszek; Hunter, C. Neil

2000-01-01

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248–7255, 1999). An operonal arrangement of the genes is proposed. PMID:10809697

Identifying Neurofibromin-Specific Regulatory Nodes for Therapeutic Targeting in NF1

DTIC Science & Technology

2016-10-01

adapter protein SPRED1, to function, and we are utilizing the latest technical innovations including CRISPR technology to find genes that regulate...neurofibromin depends on the adapter protein SPRED1, to function, and we are utilizing the latest technical innovations including CRISPR technology...signaling NF1-Null HEK 293T cells have been generated using CRISPR /Cas9 and single clones have been expanded for biochemical assays (Figure 2). NF1-Null

Discovering functions of unannotated genes from a transcriptome survey of wild fungal isolates.

PubMed

Ellison, Christopher E; Kowbel, David; Glass, N Louise; Taylor, John W; Brem, Rachel B

2014-04-01

Most fungal genomes are poorly annotated, and many fungal traits of industrial and biomedical relevance are not well suited to classical genetic screens. Assigning genes to phenotypes on a genomic scale thus remains an urgent need in the field. We developed an approach to infer gene function from expression profiles of wild fungal isolates, and we applied our strategy to the filamentous fungus Neurospora crassa. Using transcriptome measurements in 70 strains from two well-defined clades of this microbe, we first identified 2,247 cases in which the expression of an unannotated gene rose and fell across N. crassa strains in parallel with the expression of well-characterized genes. We then used image analysis of hyphal morphologies, quantitative growth assays, and expression profiling to test the functions of four genes predicted from our population analyses. The results revealed two factors that influenced regulation of metabolism of nonpreferred carbon and nitrogen sources, a gene that governed hyphal architecture, and a gene that mediated amino acid starvation resistance. These findings validate the power of our population-transcriptomic approach for inference of novel gene function, and we suggest that this strategy will be of broad utility for genome-scale annotation in many fungal systems. IMPORTANCE Some fungal species cause deadly infections in humans or crop plants, and other fungi are workhorses of industrial chemistry, including the production of biofuels. Advances in medical and industrial mycology require an understanding of the genes that control fungal traits. We developed a method to infer functions of uncharacterized genes by observing correlated expression of their mRNAs with those of known genes across wild fungal isolates. We applied this strategy to a filamentous fungus and predicted functions for thousands of unknown genes. In four cases, we experimentally validated the predictions from our method, discovering novel genes involved in the metabolism of nutrient sources relevant for biofuel production, as well as colony morphology and starvation resistance. Our strategy is straightforward, inexpensive, and applicable for predicting gene function in many fungal species.

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain Stag's Leap.

PubMed

Chen, J; Wu, F; Zheng, Z; Deng, X; Burbank, L P; Stenger, D C

2016-04-21

ITALIC! Xylella fastidiosasubsp. ITALIC! fastidiosacauses Pierce's disease of grapevine. Presented here is the draft genome sequence of the Stag's Leap strain, previously used in pathogenicity/virulence assays to evaluate grapevine germplasm bearing Pierce's disease resistance and a phenotypic assessment of knockout mutants to determine gene function. Copyright © 2016 Chen et al.

Deletion of ultraconserved elements yields viable mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahituv, Nadav; Zhu, Yiwen; Visel, Axel

2007-07-15

Ultraconserved elements have been suggested to retainextended perfect sequence identity between the human, mouse, and ratgenomes due to essential functional properties. To investigate thenecessities of these elements in vivo, we removed four non-codingultraconserved elements (ranging in length from 222 to 731 base pairs)from the mouse genome. To maximize the likelihood of observing aphenotype, we chose to delete elements that function as enhancers in amouse transgenic assay and that are near genes that exhibit markedphenotypes both when completely inactivated in the mouse as well as whentheir expression is altered due to other genomic modifications.Remarkably, all four resulting lines of mice lackingmore » these ultraconservedelements were viable and fertile, and failed to reveal any criticalabnormalities when assayed for a variety of phenotypes including growth,longevity, pathology and metabolism. In addition more targeted screens,informed by the abnormalities observed in mice where genes in proximityto the investigated elements had been altered, also failed to revealnotable abnormalities. These results, while not inclusive of all thepossible phenotypic impact of the deleted sequences, indicate thatextreme sequence constraint does not necessarily reflect crucialfunctions required for viability.« less

Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues.

PubMed

Knudsen, Beatrice S; Allen, April N; McLerran, Dale F; Vessella, Robert L; Karademos, Jonathan; Davies, Joan E; Maqsodi, Botoul; McMaster, Gary K; Kristal, Alan R

2008-03-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR(FROZEN), the gold standard, and bDNA(FFPE) ranged from 0.60 to 0.94, similar to those from qPCR(FROZEN) and qPCR(FFPE). Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.

Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues

PubMed Central

Knudsen, Beatrice S.; Allen, April N.; McLerran, Dale F.; Vessella, Robert L.; Karademos, Jonathan; Davies, Joan E.; Maqsodi, Botoul; McMaster, Gary K.; Kristal, Alan R.

2008-01-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93–100%) than for qPCR (82.4–95%). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, similar to those from qPCRFROZEN and qPCRFFPE. Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management. PMID:18276773

integIRTy: a method to identify genes altered in cancer by accounting for multiple mechanisms of regulation using item response theory.

PubMed

Tong, Pan; Coombes, Kevin R

2012-11-15

Identifying genes altered in cancer plays a crucial role in both understanding the mechanism of carcinogenesis and developing novel therapeutics. It is known that there are various mechanisms of regulation that can lead to gene dysfunction, including copy number change, methylation, abnormal expression, mutation and so on. Nowadays, all these types of alterations can be simultaneously interrogated by different types of assays. Although many methods have been proposed to identify altered genes from a single assay, there is no method that can deal with multiple assays accounting for different alteration types systematically. In this article, we propose a novel method, integration using item response theory (integIRTy), to identify altered genes by using item response theory that allows integrated analysis of multiple high-throughput assays. When applied to a single assay, the proposed method is more robust and reliable than conventional methods such as Student's t-test or the Wilcoxon rank-sum test. When used to integrate multiple assays, integIRTy can identify novel-altered genes that cannot be found by looking at individual assay separately. We applied integIRTy to three public cancer datasets (ovarian carcinoma, breast cancer, glioblastoma) for cross-assay type integration which all show encouraging results. The R package integIRTy is available at the web site http://bioinformatics.mdanderson.org/main/OOMPA:Overview. kcoombes@mdanderson.org. Supplementary data are available at Bioinformatics online.

Assessment of the potential pathogenicity of missense mutations identified in the GTPase-activating protein (GAP)-related domain of the neurofibromatosis type-1 (NF1) gene.

PubMed

Thomas, Laura; Richards, Mark; Mort, Matthew; Dunlop, Elaine; Cooper, David N; Upadhyaya, Meena

2012-12-01

Neurofibromatosis type-1 (NF1) is caused by constitutional mutations of the NF1 tumor-suppressor gene. Although ∼85% of inherited NF1 microlesions constitute truncating mutations, the remaining ∼15% are missense mutations whose pathological relevance is often unclear. The GTPase-activating protein-related domain (GRD) of the NF1-encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1-GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1-GRD expression vector and activated Ras was assayed by an enzyme-linked immunosorbent assay (ELISA). Ten NF1-GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP-bound Ras in comparison to wild-type NF1 protein. The remaining five NF1-GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild-type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1-GRD structure and function. © 2012 Wiley Periodicals, Inc.

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

PubMed

Sørensen, M S; Duch, M; Paludan, K; Jørgensen, P; Pedersen, F S

1992-03-15

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

PubMed

Williamson, Charles H D; Vazquez, Adam J; Hill, Karen; Smith, Theresa J; Nottingham, Roxanne; Stone, Nathan E; Sobek, Colin J; Cocking, Jill H; Fernández, Rafael A; Caballero, Patricia A; Leiser, Owen P; Keim, Paul; Sahl, Jason W

2017-09-15

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( ha positive [ ha + ] or orfX + ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene ( bont ) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type ( ha + or orfX + ) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest. Copyright © 2017 Williamson et al.

Evaluation of the GeneXpert MTB/RIF assay on extrapulmonary and respiratory samples other than sputum: a low burden country experience.

PubMed

Pandey, Sushil; Congdon, Jacob; McInnes, Bradley; Pop, Alina; Coulter, Christopher

2017-01-01

The aim of this study was to assess the performance of the GeneXpert MTB/RIF assay on extrapulmonary (EP) and respiratory (non-sputum) clinical samples of patients suspected of having tuberculosis (TB) from Queensland, Australia. A total of 269 EP and respiratory (non-sputum) clinical samples collected from Qld patients who were suspected of having TB were subjected to the GeneXpert MTB/RIF analysis, Ziehl-Neelsen (ZN) staining, Mycobacterium tuberculosis (MTB) culture and drug susceptibility testing. Phenotypic and genotypic data were compared. The overall performance analysis of the GeneXpert MTB/RIF assay for detection of MTB complex demonstrated sensitivity of 89%, specificity of 95%, PPV of 89% and NPV of 95% using culture as a reference standard. The GeneXpert MTB/RIF analysis of acid-fast bacilli (AFB) smear positive samples and AFB smear negative samples showed sensitivities of 100% and 77%, respectively. Looking at individual EP and respiratory (non-sputum) sample types, the sensitivity ranged from 60% to 100% although the specificity ranged from 33% to 100% with the specificity of lymph node tissue biopsy being the lowest. The GeneXpert MTB/RIF assay detected 11% more TB cases than culture and 27% more cases than ZN microscopy. Due to insufficient numbers of presenting rifampicin resistance cases, performance analysis of the GeneXpert MTB/RIF assay on rifampicin resistance could not be carried out. The GeneXpert MTB/RIF assay is potentially valuable for TB diagnosis in the majority of the EP and respiratory (other than sputum) samples in our setting. Although the GeneXpert MTB/RIF assay provides rapid diagnostic results, the overall sensitivity to rule out the disease is suboptimal for some specimen types. Performance varied according to specimen type and AFB smear status. The sensitivity and specificity of lymph node tissue was 63% and 33%. Care must be taken when using the GeneXpert MTB/RIF assay for detection of MTB in lymph node tissue samples. All samples should be cultured regardless of the GeneXpert MTB/RIF assay result. Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

PubMed Central

van der Klift, Heleen M; Jansen, Anne M L; van der Steenstraten, Niki; Bik, Elsa C; Tops, Carli M J; Devilee, Peter; Wijnen, Juul T

2015-01-01

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants. PMID:26247049

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

PubMed

Revollo, Javier; Pearce, Mason G; Petibone, Dayton M; Mittelstaedt, Roberta A; Dobrovolsky, Vasily N

2015-05-01

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

PubMed

Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

2015-04-20

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus

PubMed Central

Elek, Zsuzsanna; Németh, Nóra; Nagy, Géza; Németh, Helga; Somogyi, Anikó; Hosszufalusi, Nóra; Sasvári-Székely, Mária; Rónai, Zsolt

2015-01-01

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ 2-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function. PMID:26426397

[Effects of intrasplenic transplantation of IL-18 gene-modified fetal hepatocytes on mouse immune function

PubMed

Yao, Hang-Ping; Zhang, Li-Huang; Sun, Wen-Ji; Leng, Jian-Hang

2002-04-01

OBJECTIVE: To investigate the effects of IL-18 gene-modified fetal hepatocytes (AdmIL-18/MNL.CL2) intrasplenic transplantation on mouse immune function. METHODS: Forty mice were evenly divided into 4 groups of 10. Each group received an intrasplenic transplantation one of the following: AdmIL-18/BNL.CL2, Ad-LacZ/BNL.CL2 (virus control), BNL.CL2 (cell control) and PBS (blank control). After two weeks, the mice were sacrificed. Serum cytokine levels, Mpsi and splenic cell culture supernatant and liver tissue extracts supernatants were measured using ELISA. Hepatic cytokines mRNA expression were determined by RT-PCR. THe cytotoxicity of peritoneal Mpsi and NK activity of spienocytes were detected by LDH release assay. The proliferation of splenic lymphocytes was determined by MTT assay. RESULTS: The IL-18, IL-2,IFN-gamma, TNF-alpha levels of serum, Mpsi and splenocyte culture supernatant, liver tissue extracts supernatants in mice transplanted with AdmIL-18/BNL.CL2 were higher and the IL-4, IL-10 levels were lower compared to their levels in other 3 groups. The highest IL-18, IL-2, IFN-gamma, TNF-alpha and the lowest IL-4, IL-10 mRNA expressions in the liver were observed in mice transplanted with AdmIL-18/BNL.CL2. The mice transplanted with AdmIL-18/BNL.Cl2 showed significantly increases cytotoxicity of Mpsi, NK activity and splenic cell proliferation compared with the other 3 groups. CONCLUSION: AdmIL-18 can be effectively transfected into mice fetal heptocytes which subsequently IL-18. Intransplenic transplantation of IL-18 gene-modified fetal hepatocytes may augment mouse immune function and provide an useful basis for targeted gene therapy of liver disease.

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus.

PubMed

Elek, Zsuzsanna; Németh, Nóra; Nagy, Géza; Németh, Helga; Somogyi, Anikó; Hosszufalusi, Nóra; Sasvári-Székely, Mária; Rónai, Zsolt

2015-01-01

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ2-test in combination with correction for multiple testing. For functional analysis, the entire 3' UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity.

PubMed

Tsang-A-Sjoe, M W P; Bultink, I E M; Korswagen, L A; van der Horst, A; Rensink, I; de Boer, M; Hamann, D; Voskuyl, A E; Wouters, D

2017-12-01

Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi-organ auto-immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls. Gene copy number (GCN) variation, silencing CT-insertion, and the retroviral HERV-K(C4) insertion) were analyzed with multiplex ligation-dependent probe amplification. Increased susceptibility to SLE was found for low GCN (≪2) of C4A. Serositis was the only clinical manifestation associated with low C4A GCN. One additional novel silencing mutation in the C4A gene was found by Sanger sequencing. This mutation causes a premature stop codon in exon 11. Protein concentrations of C4 isoforms C4A and C4B were determined with ELISA and were significantly lower in SLE patients compared to healthy controls. To study C4 isotypes on a functional level, a new C4 assay was developed, which distinguishes C4A from C4B by its binding capacity to amino or hydroxyl groups, respectively. This assay showed high correlation with ELISA and detected crossing over of Rodgers and Chido antigens in 3.2% (8/244) of individuals. The binding capacity of available C4 to its substrates was unaffected in SLE. Our study provides, for the first time, a complete overview of C4 in SLE from genetic variation to binding capacity using a novel test. As this test detects crossing over of Rodgers and Chido antigens, it will allow for more accurate measurement of C4 in future studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural, functional and evolutionary characterization of major drought transcription factors families in maize

NASA Astrophysics Data System (ADS)

Mittal, Shikha; Banduni, Pooja; Mallikarjuna, Mallana G.; Rao, Atmakuri R.; Jain, Prashant A.; Dash, Prasanta K.; Thirunavukkarasu, Nepolean

2018-05-01

Drought is one of the major threats to maize production. In order to improve the production and to breed tolerant hybrids, understanding the genes and regulatory mechanisms during drought stress is important. Transcription factors (TFs) play a major role in gene regulation and many TFs have been identified in response to drought stress. In our experiment, a set of 15 major TF families comprising 1436 genes was structurally and functionally characterized using in-silico tools and a gene expression assay. All 1436 genes were mapped on 10 chromosome of maize. The functional annotation indicated the involvement of these genes in ABA signaling, ROS scavenging, photosynthesis, stomatal regulation, and sucrose metabolism. Duplication was identified as the primary force in divergence and expansion of TF families. Phylogenetic relationship was developed individually for each TF family as well as combined TF families. Phylogenetic analysis grouped the TF family of genes into TF-specific and mixed groups. Phylogenetic analysis of genes belonging to various TF families suggested that the origin of TFs occurred in the lineage of maize evolution. Gene structure analysis revealed that more number of genes were intron-rich as compared to intronless genes. Drought-responsive CRE’s such as ABREA, ABREB, DRE1 and DRECRTCOREAT have been identified. Expression and interaction analyses identified leaf-specific bZIP TF, GRMZM2G140355, as a potential contributor toward drought tolerance in maize. We also analyzed protein-protein interaction network of 269 drought-responsive genes belonging to different drought-related TFs. The information generated on structural and functional characteristics, expression and interaction of the drought-related TF families will be useful to decipher the drought tolerance mechanisms and to derive drought-tolerant genotypes in maize.

A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence1[OPEN

PubMed Central

Leng, Yujia; Yang, Yaolong; Ren, Deyong; Dai, Liping; Wang, Yuqiong; Chen, Long; Tu, Zhengjun; Gao, Yihong; Zhu, Li; Hu, Jiang; Gao, Zhenyu; Guo, Longbiao; Lin, Yongjun

2017-01-01

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes. PMID:28455404

The homeodomain transcription factor TaHDZipI-2 from wheat regulates frost tolerance, flowering time and spike development in transgenic barley.

PubMed

Kovalchuk, Nataliya; Chew, William; Sornaraj, Pradeep; Borisjuk, Nikolai; Yang, Nannan; Singh, Rohan; Bazanova, Natalia; Shavrukov, Yuri; Guendel, Andre; Munz, Eberhard; Borisjuk, Ljudmilla; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

2016-07-01

Homeodomain leucine zipper class I (HD-Zip I) transcription factors (TFs) play key roles in the regulation of plant growth and development under stresses. Functions of the TaHDZipI-2 gene isolated from the endosperm of developing wheat grain were revealed. Molecular characterization of TaHDZipI-2 protein included studies of its dimerisation, protein-DNA interactions and gene activation properties using pull-down assays, in-yeast methods and transient expression assays in wheat cells. The analysis of TaHDZipI-2 gene functions was performed using transgenic barley plants. It included comparison of developmental phenotypes, yield components, grain quality, frost tolerance and the levels of expression of potential target genes in transgenic and control plants. Transgenic TaHDZipI-2 lines showed characteristic phenotypic features that included reduced growth rates, reduced biomass, early flowering, light-coloured leaves and narrowly elongated spikes. Transgenic lines produced 25-40% more seeds per spike than control plants, but with 50-60% smaller grain size. In vivo lipid imaging exposed changes in the distribution of lipids between the embryo and endosperm in transgenic seeds. Transgenic lines were significantly more tolerant to frost than control plants. Our data suggest the role of TaHDZipI-2 in controlling several key processes underlying frost tolerance, transition to flowering and spike development. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence.

PubMed

Leng, Yujia; Yang, Yaolong; Ren, Deyong; Huang, Lichao; Dai, Liping; Wang, Yuqiong; Chen, Long; Tu, Zhengjun; Gao, Yihong; Li, Xueyong; Zhu, Li; Hu, Jiang; Zhang, Guangheng; Gao, Zhenyu; Guo, Longbiao; Kong, Zhaosheng; Lin, Yongjun; Qian, Qian; Zeng, Dali

2017-06-01

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 ( del1 ). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes. © 2017 American Society of Plant Biologists. All Rights Reserved.

Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2014-08-15

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of a toxicogenomic approach to the local lymph node assay (LLNA).

PubMed

Boverhof, Darrell R; Gollapudi, B Bhaskar; Hotchkiss, Jon A; Osterloh-Quiroz, Mandy; Woolhiser, Michael R

2009-02-01

Genomic technologies have the potential to enhance and complement existing toxicology endpoints; however, assessment of these approaches requires a systematic evaluation including a robust experimental design with genomic endpoints anchored to traditional toxicology endpoints. The present study was conducted to assess the sensitivity of genomic responses when compared with the traditional local lymph node assay (LLNA) endpoint of lymph node cell proliferation and to evaluate the responses for their ability to provide insights into mode of action. Female BALB/c mice were treated with the sensitizer trimellitic anhydride (TMA), following the standard LLNA dosing regimen, at doses of 0.1, 1, or 10% and traditional tritiated thymidine ((3)HTdR) incorporation and gene expression responses were monitored in the auricular lymph nodes. Additional mice dosed with either vehicle or 10% TMA and sacrificed on day 4 or 10, were also included to examine temporal effects on gene expression. Analysis of (3)HTdR incorporation revealed TMA-induced stimulation indices of 2.8, 22.9, and 61.0 relative to vehicle with an EC(3) of 0.11%. Examination of the dose-response gene expression responses identified 9, 833, and 2122 differentially expressed genes relative to vehicle for the 0.1, 1, and 10% TMA dose groups, respectively. Calculation of EC(3) values for differentially expressed genes did not identify a response that was more sensitive than the (3)HTdR value, although a number of genes displayed comparable sensitivity. Examination of temporal responses revealed 1760, 1870, and 953 differentially expressed genes at the 4-, 6-, and 10-day time points respectively. Functional analysis revealed many responses displayed dose- and time-specific induction patterns within the functional categories of cellular proliferation and immune response, including numerous immunoglobin genes which were highly induced at the day 10 time point. Overall, these experiments have systematically illustrated the potential utility of genomic endpoints to enhance the LLNA and support further exploration of this approach through examination of a more diverse array of chemicals.

Transrepressive function of TLX requires the histone demethylase LSD1.

PubMed

Yokoyama, Atsushi; Takezawa, Shinichiro; Schüle, Roland; Kitagawa, Hirochika; Kato, Shigeaki

2008-06-01

TLX is an orphan nuclear receptor (also called NR2E1) that regulates the expression of target genes by functioning as a constitutive transrepressor. The physiological significance of TLX in the cytodifferentiation of neural cells in the brain is known. However, the corepressors supporting the transrepressive function of TLX have yet to be identified. In this report, Y79 retinoblastoma cells were subjected to biochemical techniques to purify proteins that interact with TLX, and we identified LSD1 (also called KDM1), which appears to form a complex with CoREST and histone deacetylase 1. LSD1 interacted with TLX directly through its SWIRM and amine oxidase domains. LSD1 potentiated the transrepressive function of TLX through its histone demethylase activity as determined by a luciferase assay using a genomically integrated reporter gene. LSD1 and TLX were recruited to a TLX-binding site in the PTEN gene promoter, accompanied by the demethylation of H3K4me2 and deacetylation of H3. Knockdown of either TLX or LSD1 derepressed expression of the endogenous PTEN gene and inhibited cell proliferation of Y79 cells. Thus, the present study suggests that LSD1 is a prime corepressor for TLX.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

PubMed Central

Vizeacoumar, Franco J; Arnold, Roland; Vizeacoumar, Frederick S; Chandrashekhar, Megha; Buzina, Alla; Young, Jordan T F; Kwan, Julian H M; Sayad, Azin; Mero, Patricia; Lawo, Steffen; Tanaka, Hiromasa; Brown, Kevin R; Baryshnikova, Anastasia; Mak, Anthony B; Fedyshyn, Yaroslav; Wang, Yadong; Brito, Glauber C; Kasimer, Dahlia; Makhnevych, Taras; Ketela, Troy; Datti, Alessandro; Babu, Mohan; Emili, Andrew; Pelletier, Laurence; Wrana, Jeff; Wainberg, Zev; Kim, Philip M; Rottapel, Robert; O'Brien, Catherine A; Andrews, Brenda; Boone, Charles; Moffat, Jason

2013-01-01

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model. PMID:24104479

Activity of etv5a and etv5b genes in the hypothalamus of fasted zebrafish is influenced by serotonin.

PubMed

Mechaly, Alejandro S; Richardson, Ebony; Rinkwitz, Silke

2017-05-15

Serotonin has been implicated in the inhibition of food intake in vertebrates. However, the mechanisms through which serotonin acts has yet to be elucidated. Recently, ETV5 (ets variant gene 5) has been associated with obesity and food intake control mechanisms in mammals. We have analyzed a putative physiological function of the two etv5 paralogous genes (etv5a and etv5b) in neuronal food intake control in adult zebrafish that have been exposed to different nutritional conditions. A feeding assay was established and fluoxetine, a selective serotonin re-uptake inhibitor (SSRI), was applied. Gene expression changes in the hypothalamus were determined using real-time PCR. Fasting induced an up-regulation of etv5a and etv5b in the hypothalamus, whereas increased serotonin levels in the fasted fish counteracted the increase in expression. To investigate potential mechanisms the expression of further food intake control genes was determined. The results show that an increase of serotonin in fasting fish causes a reduction in the activity of genes stimulating food intake. This is in line with a previously demonstrated anorexigenic function of serotonin. Our results suggest that obesity-associated ETV5 has a food intake stimulating function and that this function is modulated through serotonin. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical-genetic profile analysis of five inhibitory compounds in yeast.

PubMed

Alamgir, Md; Erukova, Veronika; Jessulat, Matthew; Azizi, Ali; Golshani, Ashkan

2010-08-06

Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s). Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays. Chemical-genetic profiles provide insight into the molecular mechanism(s) of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.

Drug transporter expression profiling in a three-dimensional kidney proximal tubule in vitro nephrotoxicity model.

PubMed

Diekjürgen, Dorina; Grainger, David W

2018-05-09

Given currently poor toxicity translational predictions for drug candidates, improved mechanistic understanding underlying nephrotoxicity and drug renal clearance is needed to improve drug development and safety screening. Therefore, better relevant and well-characterized in vitro screening models are required to reliably predict human nephrotoxicity. Because kidney proximal tubules are central to active drug uptake and secretion processes and therefore to nephrotoxicity, this study acquired regio-specific expression data from recently reported primary proximal tubule three-dimensional (3D) hyaluronic acid gel culture and non-gel embedded cultured murine proximal tubule suspensions used in nephrotoxicity assays. Quantitative assessment of the mRNA expression of 21 known kidney tubule markers and important proximal tubule transporters with known roles in drug transport was obtained. Asserting superior gene expression levels over current commonly used two-dimensional (2D) kidney cell culture lines was the study objective. Hence, we compare previously published gel-based 3D proximal tubule fragment culture and their non-gel suspensions for up to 1 week. We demonstrate that 3D tubule culture exhibits superior gene expression levels and profiles compared to published commonly used 2D kidney cell lines (Caki-1 and HK-2) in plastic plate monocultures. Additionally, nearly all tested genes retain mRNA expression after 7 days in both proximal tubule cultures, a limitation of 2D cell culture lines. Importantly, gel presence is shown not to interfere with the gene expression assay. Western blots confirm protein expression of OAT1 and 3 and OCT2. Functional transport assays confirm their respective transporter functions in vitro. Overall, results validate retention of essential toxicity-relevant transporters in this published 3D proximal tubule model over conventional 2D kidney cell cultures, producing opportunities for more reliable, sensitive, and comprehensive drug toxicity studies relevant to drug development and nephrotoxicity goals.

A 3'-untranslated region (3'UTR) induces organ adhesion by regulating miR-199a* functions.

PubMed

Lee, Daniel Y; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Deng, Zhaoqun; Yang, Burton B

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18-24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3'UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3'UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3'UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3'UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3'UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3'UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3'UTR may be an approach in the development of gene therapy.

Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system.

PubMed

Baoutina, A; Coldham, T; Bains, G S; Emslie, K R

2010-08-01

As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3.

PubMed

Loya, Travis J; O'Rourke, Thomas W; Reines, Daniel

2012-08-01

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 'tail' forms an α-helical multimerization domain that helps assemble it onto an RNA substrate.

Evaluation of the BD GeneOhm assay using the rotor-gene 6000 platform for rapid detection of methicillin-resistant Staphylococcus aureus from pooled screening swabs.

PubMed

Smith, Melvyn Howard; Hodgson, Julian; Eltringham, Ian Joseph

2010-12-01

As health services move toward universal methicillin-resistant Staphylococcus aureus (MRSA) screening for hospital admissions, the most cost-effective approach is yet to be defined. In this study, one of the largest to date, we evaluated the performance of the BD GeneOhm MRSA assay on the Rotor-Gene 6000 thermal cycler, using samples taken directly from pooled MRSA screens. Results were compared with the same assay performed on the Smart-Cycler II platform and overnight broth culture. Samples yielding discrepant results were subjected to detailed analysis with an in-house PCR and patient note review. A total of 1,428 pooled MRSA screens were tested. Sensitivities and specificities of 85.3% and 95.8% for the Rotor-Gene and 81% and 95.7% for the Smart-Cycler were obtained, compared with broth enrichment. The sensitivity of the BD GeneOhm assay was increased to 100% when the results of in-house PCR and patient note review were taken into account. This study demonstrates that the Rotor-Gene 6000 thermal cycler is a reliable platform for use with the BD GeneOhm assay. It also proves that commercial PCR can be performed direct on pooled samples in selective broth, without the need for overnight incubation.

Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements

PubMed Central

Cushman-Vokoun, Allison M.; Connealy, Solomon; Greiner, Timothy C.

2010-01-01

Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. PMID:20959612

Brief Report: A mass spectrometry assay to simultaneously analyze ROS1 and RET fusion gene expression in non-small cell lung cancer

PubMed Central

Wijesinghe, Priyanga; Bepler, Gerold

2014-01-01

Introduction ROS1 and RET gene fusions were recently discovered in non-small cell lung cancer (NSCLC) as potential therapeutic targets with small molecule kinase inhibitors. The conventional screening methods of these fusions are time consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing PCR and the sensitivity of mass spectrometry. Methods The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false negative results. To flag false positives, the system also comprises two independent assays for each fusion gene junction. Results The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using cDNA from lung tissue of healthy individuals. The system was further validated using cDNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors. Conclusion The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover. PMID:25384172

Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria.

PubMed

Zhang, Yijing; Yao, Yi; Du, Weixing; Wu, Kai; Xu, Wenyue; Lin, Min; Tan, Huabing; Li, Jian

2017-07-01

In order to achieve better outcomes for treatment and in the prophylaxis of malaria, it is imperative to develop a sensitive, specific, and accurate assay for early diagnosis of Plasmodium falciparum infection, which is the major cause of malaria. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay with P. falciparum unique genes for sensitive, specific, and accurate detection of P. falciparum infection. The unique genes of P. falciparum were randomly selected from PlasmoDB. The LAMP primers of the unique genes were designed using PrimerExplorer V4. LAMP assays with primers from unique genes of P. falciparum and conserved 18S rRNA gene were developed and their sensitivity was assessed. The specificity of the most sensitive LAMP assay was further examined using genomic DNA from Plasmodium vivax, Plasmodium yoelii and Toxoplasma gondii. Finally, the unique gene-based LAMP assay was validated using clinical samples of P. falciparum infection cases. A total of 31 sets of top-scored LAMP primers from nine unique genes were selected from the pools of designed primers. The LAMP assay with PF3D7_1253300-5 was the most sensitive with the detection limit 5 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. vivax, P. yoelii, and T. gondii. The LAMP assay with PF3D7_0112300 (18S rRNA) was less sensitive with the detection limit 50 parasites/μl, and it displayed negative LAMP assay with the genomic DNA samples of P. yoelii and T. gondii, but displayed positive LAMP detection with P. vivax. The positive detection rate of the LAMP assay with PF3D7_1253300-5 was 90% (27/30), higher than that (80%, 24/30) of the positive rate of PF3D7_0112300 (18S rRNA) in examining clinical samples of P. falciparum infection cases. The LAMP assay with the primer set PF3D7_1253300-5 was more sensitive, specific, and accurate than those with PF3D7_0112300 (18S rRNA) in examining P. falciparum infection, and therefore it is a promising tool for diagnosis of P. falciparum infection.

Eos Negatively Regulates Human γ-globin Gene Transcription during Erythroid Differentiation

PubMed Central

Yu, Hai-Chuan; Zhao, Hua-Lu; Wu, Zhi-Kui; Zhang, Jun-Wu

2011-01-01

Background Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4), a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. Methodology/Principal Findings Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs). DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3) of the β-globin locus control region (LCR), the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C) assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. Conclusions/Significance Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation. PMID:21829552

Genetic Control of Plant Root Colonization by the Biocontrol agent, Pseudomonas fluorescens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cole, Benjamin J.; Fletcher, Meghan; Waters, Jordan

Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguouslymore » identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity and pathogen resistance.« less

Discovery of mutations in homologous recombination genes in African-American women with breast cancer.

PubMed

Ding, Yuan Chun; Adamson, Aaron W; Steele, Linda; Bailis, Adam M; John, Esther M; Tomlinson, Gail; Neuhausen, Susan L

2018-04-01

African-American women are more likely to develop aggressive breast cancer at younger ages and experience poorer cancer prognoses than non-Hispanic Caucasians. Deficiency in repair of DNA by homologous recombination (HR) is associated with cancer development, suggesting that mutations in genes that affect this process may cause breast cancer. Inherited pathogenic mutations have been identified in genes involved in repairing DNA damage, but few studies have focused on African-Americans. We screened for germline mutations in seven HR repair pathway genes in DNA of 181 African-American women with breast cancer, evaluated the potential effects of identified missense variants using in silico prediction software, and functionally characterized a set of missense variants by yeast two-hybrid assays. We identified five likely-damaging variants, including two PALB2 truncating variants (Q151X and W1038X) and three novel missense variants (RAD51C C135R, and XRCC3 L297P and V337E) that abolish protein-protein interactions in yeast two-hybrid assays. Our results add to evidence that HR gene mutations account for a proportion of the genetic risk for developing breast cancer in African-Americans. Identifying additional mutations that diminish HR may provide a tool for better assessing breast cancer risk and improving approaches for targeted treatment.

Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

EPA Science Inventory

Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

EPA Science Inventory

Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

[Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro].

PubMed

Wang, Kai; Peng, Jian-Qiang; Chen, Fang-Ping; Wu, Xiao-Bin

2006-04-01

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.

Profiling neuronal ion channelopathies with non-invasive brain imaging and dynamic causal models: Case studies of single gene mutations

PubMed Central

Gilbert, Jessica R.; Symmonds, Mkael; Hanna, Michael G.; Dolan, Raymond J.; Friston, Karl J.; Moran, Rosalyn J.

2016-01-01

Clinical assessments of brain function rely upon visual inspection of electroencephalographic waveform abnormalities in tandem with functional magnetic resonance imaging. However, no current technology proffers in vivo assessments of activity at synapses, receptors and ion-channels, the basis of neuronal communication. Using dynamic causal modeling we compared electrophysiological responses from two patients with distinct monogenic ion channelopathies and a large cohort of healthy controls to demonstrate the feasibility of assaying synaptic-level channel communication non-invasively. Synaptic channel abnormality was identified in both patients (100% sensitivity) with assay specificity above 89%, furnishing estimates of neurotransmitter and voltage-gated ion throughput of sodium, calcium, chloride and potassium. This performance indicates a potential novel application as an adjunct for clinical assessments in neurological and psychiatric settings. More broadly, these findings indicate that biophysical models of synaptic channels can be estimated non-invasively, having important implications for advancing human neuroimaging to the level of non-invasive ion channel assays. PMID:26342528

Estrogen and Thyroid Hormone Receptor Activation by Medicinal Plants from Bahia, Brazil

PubMed Central

da Silva, Magnus Régios Dias; Costa, Silvia Lima; Velozo, Eudes da Silva

2018-01-01

Background: A number of medicinal plants are traditionally used for metabolic disorders in Bahia state, Brazil. The aim of this study was to evaluate the estrogen receptor (ER) and thyroid receptor (TR) activation of crude extracts prepared from 20 plants. Methods: Species were extracted and assayed for receptor activation through both ER and TR gene-reporter assays, using 17β-estradiol and triiodothyronine (T3), respectively, as the positive controls. Results: Cajanus cajan (Fabaceae), Abarema cochliacarpus (Fabaceae), and Borreria verticillata (Rubiaceae) were able to activate ER as much as the positive control (17β-estradiol). These three plant species were also assayed for TR activation. At the concentration of 50 µg/mL, C. cajans exerted the highest positive modulation on TR, causing an activation of 59.9%, while B. verticillata and A. cochliacarpus caused 30.8% and 23.3%, respectively. Conclusions: Our results contribute towards the validation of the traditional use of C. cajans, B. verticillata, and A. cochliacarpus in the treatment of metabolic disorders related to ER and TR functions. The gene-reporter assay was proven effective in screening crude plant extracts for ER/TR activation, endorsing this methodology as an important tool for future bioprospection studies focused on identifying novel starting molecules for the development of estrogen and thyroid agonists. PMID:29342924

Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

PubMed

Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

2015-03-01

Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina

PubMed Central

Irie, Shoichi; Sanuki, Rikako; Muranishi, Yuki; Kato, Kimiko; Chaya, Taro

2015-01-01

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina. PMID:25986607

New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michalska, Karolina; Brown, Roslyn N.; Li, Hui

Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene frommore » Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.« less

COMPARISON OF GeneXpert MTB/RIF ASSAY WITH CONVENTIONAL AFB SMEAR FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS IN NORTHEASTERN THAILAND.

PubMed

Reechaipichitkul, Wipa; Suleesathira, Tanapong; Chaimanee, Prajaub

2017-03-01

Among infectious agents, Mycobacterium tuberculosis remains one of the most significant causes of death worldwide. Rapid and accurate diagnosis of pulmonary tuberculosis (TB) remains a great challenge. GeneXpert MTB/RIF assay is a novel integrated diagnostic system for rapid diagnosis of TB and particularly of rifampicin-resistant strains. A study was conducted between January 2010 and December 2014 to compare the performance of the sputum GeneXpert MTB/RIF assay with the conventional sputum AFB smear for diagnosis of active pulmonary TB in Thailand, a country with a high burden of this disease. Of the 125 patients who had cough and/or prolonged fever together with abnormal chest radiograph, 63 were diagnosed as having pulmonary TB by mycobacterium culture assay, while the remaining subjects were considered of having TB-like conditions, viz non-tuberculous mycobacterium infection (NTM), bacterial pneumonia or bronchogenic carcinoma. Two-thirds of the patients had underlying diseases, eg, diabetes mellitus (19 patients), autoimmune diseases (14), and HIV (6). Among patients with positive diagnosis of M. tuberculosis infection, 30 were AFB smear positive and 53 by sputum GeneXpert MTB/RIF method; among patients negative for M. tuberculosis infection, 4 were AFB smear positive and 5 by GeneXpert MTB/ RIF assay. Sensitivity and specificity of the sputum AFB smear and GeneXpertMTB/ RIF assay test were 48% (95% CI: 35-61) and 84% (95% CI: 73-92), and 94% (95% CI: 84-98) and 92% (95% CI: 82-97), respectively. Diagnostic performance of the GeneXpert MTB/RIF assay among AFB smear positive patients was higher than among AFB smear negative patients (adjusted OR 6.7; 95% CI: 2.3-19.9). Earlier diagnosis of pulmonary TB using GeneXpert MTB/RIF assay will lead to earlier appropriate treatment and provide opportunities to interrupt TB transmission.

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

PubMed

Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

2014-02-01

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis.

PubMed

Del Toro-De León, Gerardo; García-Aguilar, Marcelina; Gillmor, C Stewart

2014-10-30

Zygotic genome activation in metazoans typically occurs several hours to a day after fertilization, and thus maternal RNAs and proteins drive early animal embryo development. In plants, despite several molecular studies of post-fertilization transcriptional activation, the timing of zygotic genome activation remains a matter of debate. For example, two recent reports that used different hybrid ecotype combinations for RNA sequence profiling of early Arabidopsis embryo transcriptomes came to divergent conclusions. One identified paternal contributions that varied by gene, but with overall maternal dominance, while the other found that the maternal and paternal genomes are transcriptionally equivalent. Here we assess paternal gene activation functionally in an isogenic background, by performing a large-scale genetic analysis of 49 EMBRYO DEFECTIVE genes and testing the ability of wild-type paternal alleles to complement phenotypes conditioned by mutant maternal alleles. Our results demonstrate that wild-type paternal alleles for nine of these genes are completely functional 2 days after pollination, with the remaining 40 genes showing partial activity beginning at 2, 3 or 5 days after pollination. Using our functional assay, we also demonstrate that different hybrid combinations exhibit significant variation in paternal allele activation, reconciling the apparently contradictory results of previous transcriptional studies. The variation in timing of gene function that we observe confirms that paternal genome activation does not occur in one early discrete step, provides large-scale functional evidence that maternal and paternal genomes make non-equivalent contributions to early plant embryogenesis, and uncovers an unexpectedly profound effect of hybrid genetic backgrounds on paternal gene activity.

The loss-of-allele assay for ES cell screening and mouse genotyping.

PubMed

Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

2010-01-01

Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction (qPCR) as our method of allele quantification, but any method that can reliably distinguish the difference between one and two copies of the target gene can be used to develop an LOA assay. We have designed qPCR LOA assays for deletions, insertions, point mutations, domain swaps, conditional, and humanized alleles and have used the insert assays to quantify the copy number of random insertion BAC transgenics. Because of its quantitative precision, specificity, and compatibility with high throughput robotic operations, the LOA assay eliminates bottlenecks in ES cell screening and mouse genotyping and facilitates maximal speed and throughput for knockout mouse production. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Je Min, E-mail: jemin@knu.ac.kr; Department of Horticultural Science, Kyungpook National University, Daegu; Lee, Sang-Jik

Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function inmore » fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.« less

Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

PubMed Central

Paget, Vincent; Lechevrel, Mathilde; André, Véronique; Le Goff, Jérémie; Pottier, Didier; Billet, Sylvain; Garçon, Guillaume; Shirali, Pirouz; Sichel, François

2012-01-01

Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B1 exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B1 and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers. PMID:22319594

Corylin increases the sensitivity of hepatocellular carcinoma cells to chemotherapy through long noncoding RNA RAD51-AS1-mediated inhibition of DNA repair.

PubMed

Chen, Chin-Chuan; Chen, Chi-Yuan; Ueng, Shir-Hwa; Hsueh, Chuen; Yeh, Chau-Ting; Ho, Jar-Yi; Chou, Li-Fang; Wang, Tong-Hong

2018-05-10

Corylin, a biologically active agent extracted from Psoralea corylifolia L. (Fabaceae), promotes bone differentiation and inhibits inflammation. Currently, few reports have addressed the biological functions that are regulated by corylin, and to date, no studies have investigated its antitumor activity. In this study, we used cell functional assays to analyze the antitumor activity of corylin in hepatocellular carcinoma (HCC). Furthermore, whole-transcriptome assays were performed to identify the downstream genes that were regulated by corylin, and gain-of-function and loss-of-function experiments were conducted to examine the regulatory roles of the above genes. We found that corylin significantly inhibited the proliferation, migration, and invasion of HCC cells and increased the toxic effects of chemotherapeutic agents against HCC cells. These properties were due to the induction of a long noncoding RNA, RAD51-AS1, which bound to RAD51 mRNA, thereby inhibiting RAD51 protein expression, thus inhibiting the DNA damage repair ability of HCC cells. Animal experiments also showed that a combination treatment with corylin significantly increased the inhibitory effects of the chemotherapeutic agent etoposide (VP16) on tumor growth. These findings indicate that corylin has strong potential as an adjuvant drug in HCC treatment and that corylin can strengthen the therapeutic efficacy of chemotherapy and radiotherapy.

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

PubMed

Theile, Dirk; Spalwisz, Adriana; Weiss, Johanna

2013-06-15

Luminescence-based reporter gene assays are widely used in biochemistry. Signals from reporter genes (e.g., firefly luminescence) are usually normalized to signals from constantly luminescing luciferases such as Renilla luciferase. This normalization step can be performed by modern luminometry devices automatically providing final results. Here we demonstrate paclitaxel to strikingly enhance Renilla luminescence, thereby potentially flawing results from reporter gene assays. In consequence, these data advocate for careful examination of raw data and militate against automatic data processing. Copyright © 2013 Elsevier Inc. All rights reserved.

Interaction between mating-type proteins from the homothallic fungus Sordaria macrospora.

PubMed

Jacobsen, Sabine; Wittig, Michael; Pöggeler, Stefanie

2002-06-01

Mating-type genes control sexual development in ascomycetes. Little is known about their function in homothallic species, which are self-fertile and do not require a mating partner for sexual reproduction. The function of mating-type genes in the homothallic fungus Sordaria macrospora was assayed using a yeast system in order to find properties typical of eukaryotic transcription factors. We were able to demonstrate that the mating-type proteins SMTA-1 and SMTa-1 have domains capable of activating transcription of yeast reporter genes. Two-hybrid analysis for heterodimerization and homodimerization revealed the ability of SMTA-1 to interact with SMTa-1 and vice versa. These two proteins are encoded by different mating types in the related heterothallic species Neurospora crassa. The interaction between SMTA-1 and SMTa-1 was defined by experiments with truncated versions of SMTA-1 and in vitro by means of protein cross-linking. Moreover, we gained evidence for homodimerization of SMTA-1. Possible functions of mating-type proteins in the homothallic ascomycete S. macrospora are discussed.

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

PubMed Central

Cherry, Jonathan J; Osman, Erkan Y; Evans, Matthew C; Choi, Sungwoon; Xing, Xuechao; Cuny, Gregory D; Glicksman, Marcie A; Lorson, Christian L; Androphy, Elliot J

2013-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA. PMID:23740718

Emerging techniques for the discovery and validation of therapeutic targets for skeletal diseases.

PubMed

Cho, Christine H; Nuttall, Mark E

2002-12-01

Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure-activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis. While current treatments exist in the form of hormone replacement therapy, antiresorptive and anabolic agents for osteoporosis, there are no disease-modifying therapies for the treatment of the most common human joint disease, osteoarthritis. A massive market potential for improved options with better safety and efficacy still remains. Therefore, the application of genomics and proteomics for both diseases should provide much needed novel therapeutic approaches to treating these major world health problems.

International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials.

PubMed

Galipeau, Jacques; Krampera, Mauro; Barrett, John; Dazzi, Francesco; Deans, Robert J; DeBruijn, Joost; Dominici, Massimo; Fibbe, Willem E; Gee, Adrian P; Gimble, Jeffery M; Hematti, Peiman; Koh, Mickey B C; LeBlanc, Katarina; Martin, Ivan; McNiece, Ian K; Mendicino, Michael; Oh, Steve; Ortiz, Luis; Phinney, Donald G; Planat, Valerie; Shi, Yufang; Stroncek, David F; Viswanathan, Sowmya; Weiss, Daniel J; Sensebe, Luc

2016-02-01

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

PubMed

Li, Dayong; Zhang, Huijuan; Song, Qiuming; Wang, Lu; Liu, Shixia; Hong, Yongbo; Huang, Lei; Song, Fengming

2015-06-14

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

Wnt/β-catenin signaling stimulates the expression and synaptic clustering of the autism-associated Neuroligin 3 gene.

PubMed

Medina, Matías A; Andrade, Víctor M; Caracci, Mario O; Avila, Miguel E; Verdugo, Daniela A; Vargas, Macarena F; Ugarte, Giorgia D; Reyes, Ariel E; Opazo, Carlos; De Ferrari, Giancarlo V

2018-03-05

Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/β-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/β-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous β-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/β-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.

Structure-function analysis of RBP-J-interacting and tubulin-associated (RITA) reveals regions critical for repression of Notch target genes.

PubMed

Tabaja, Nassif; Yuan, Zhenyu; Oswald, Franz; Kovall, Rhett A

2017-06-23

The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance, and aberrant Notch signaling has been implicated in various cancers, congenital defects, and cardiovascular diseases. Notch signaling activates the expression of target genes, which are regulated by the transcription factor CSL (CBF1/RBP-J, Su(H), Lag-1). CSL interacts with both transcriptional corepressor and coactivator proteins, functioning as both a repressor and activator, respectively. Although Notch activation complexes are relatively well understood at the structural level, less is known about how CSL interacts with corepressors. Recently, a new RBP-J (mammalian CSL ortholog)-interacting protein termed RITA has been identified and shown to export RBP-J out of the nucleus, thereby leading to the down-regulation of Notch target gene expression. However, the molecular details of RBP-J/RITA interactions are unclear. Here, using a combination of biochemical/cellular, structural, and biophysical techniques, we demonstrate that endogenous RBP-J and RITA proteins interact in cells, map the binding regions necessary for RBP-J·RITA complex formation, and determine the X-ray structure of the RBP-J·RITA complex bound to DNA. To validate the structure and glean more insights into function, we tested structure-based RBP-J and RITA mutants with biochemical/cellular assays and isothermal titration calorimetry. Whereas our structural and biophysical studies demonstrate that RITA binds RBP-J similarly to the RAM (RBP-J-associated molecule) domain of Notch, our biochemical and cellular assays suggest that RITA interacts with additional regions in RBP-J. Taken together, these results provide molecular insights into the mechanism of RITA-mediated regulation of Notch signaling, contributing to our understanding of how CSL functions as a transcriptional repressor of Notch target genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Contrasting Effects of Human, Canine, and Hybrid Adenovirus Vectors on the Phenotypical and Functional Maturation of Human Dendritic Cells: Implications for Clinical Efficacy▿

PubMed Central

Perreau, Matthieu; Mennechet, Franck; Serratrice, Nicolas; Glasgow, Joel N.; Curiel, David T.; Wodrich, Harald; Kremer, Eric J.

2007-01-01

Antipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis of Adenoviridae immunogenicity and that each may have specific clinical advantages. PMID:17229706

A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

PubMed Central

Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

2016-01-01

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

Identification of the gene transcription repressor domain of Gli3.

PubMed

Tsanev, Robert; Tiigimägi, Piret; Michelson, Piret; Metsis, Madis; Østerlund, Torben; Kogerman, Priit

2009-01-05

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

Multiplex ligation-dependent probe amplification analysis on capillary electrophoresis instruments for a rapid gene copy number study.

PubMed

Jankowski, Stéphane; Currie-Fraser, Erica; Xu, Licen; Coffa, Jordy

2008-09-01

Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies.

Breeding and Genetics Symposium: networks and pathways to guide genomic selection.

PubMed

Snelling, W M; Cushman, R A; Keele, J W; Maltecca, C; Thomas, M G; Fortes, M R S; Reverter, A

2013-02-01

Many traits affecting profitability and sustainability of meat, milk, and fiber production are polygenic, with no single gene having an overwhelming influence on observed variation. No knowledge of the specific genes controlling these traits has been needed to make substantial improvement through selection. Significant gains have been made through phenotypic selection enhanced by pedigree relationships and continually improving statistical methodology. Genomic selection, recently enabled by assays for dense SNP located throughout the genome, promises to increase selection accuracy and accelerate genetic improvement by emphasizing the SNP most strongly correlated to phenotype although the genes and sequence variants affecting phenotype remain largely unknown. These genomic predictions theoretically rely on linkage disequilibrium (LD) between genotyped SNP and unknown functional variants, but familial linkage may increase effectiveness when predicting individuals related to those in the training data. Genomic selection with functional SNP genotypes should be less reliant on LD patterns shared by training and target populations, possibly allowing robust prediction across unrelated populations. Although the specific variants causing polygenic variation may never be known with certainty, a number of tools and resources can be used to identify those most likely to affect phenotype. Associations of dense SNP genotypes with phenotype provide a 1-dimensional approach for identifying genes affecting specific traits; in contrast, associations with multiple traits allow defining networks of genes interacting to affect correlated traits. Such networks are especially compelling when corroborated by existing functional annotation and established molecular pathways. The SNP occurring within network genes, obtained from public databases or derived from genome and transcriptome sequences, may be classified according to expected effects on gene products. As illustrated by functionally informed genomic predictions being more accurate than naive whole-genome predictions of beef tenderness, coupling evidence from livestock genotypes, phenotypes, gene expression, and genomic variants with existing knowledge of gene functions and interactions may provide greater insight into the genes and genomic mechanisms affecting polygenic traits and facilitate functional genomic selection for economically important traits.

ECR-MAPK regulation in liver early development.

PubMed

Zhao, Xiu-Ju; Zhuo, Hexian

2014-01-01

Early growth is connected to a key link between embryonic development and aging. In this paper, liver gene expression profiles were assayed at postnatal day 22 and week 16 of age. Meanwhile another independent animal experiment and cell culture were carried out for validation. Significance analysis of microarrays, qPCR verification, drug induction/inhibition assays, and metabonomics indicated that alpha-2u globulin (extracellular region)-socs2 (-SH2-containing signals/receptor tyrosine kinases)-ppp2r2a/pik3c3 (MAPK signaling)-hsd3b5/cav2 (metabolism/organization) plays a vital role in early development. Taken together, early development of male rats is ECR and MAPK-mediated coordination of cancer-like growth and negative regulations. Our data represent the first comprehensive description of early individual development, which could be a valuable basis for understanding the functioning of the gene interaction network of infant development.

Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay.

PubMed

Bowley, Lucas A; Alam, Farhana; Marentette, Julie R; Balshine, Sigal; Wilson, Joanna Y

2010-12-01

A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region. Copyright © 2010 SETAC.

Genetic Analysis of X-Chromosome Dosage Compensation in Caenorhabditis elegans

PubMed Central

Meneely, Philip M.; Wood, William B.

1987-01-01

We have shown that the phenotypes resulting from hypomorphic mutations (causing reduction but not complete loss of function) in two X-linked genes can be used as a genetic assay for X-chromosome dosage compensation in Caenorhabditis elegans between males ( XO) and hermaphrodites (XX). In addition we show that recessive mutations in two autosomal genes, dpy-21 V and dpy-26 IV, suppress the phenotypes resulting from the X-linked hypomorphic mutations, but not the phenotypes resulting from comparable autosomal hypomorphic mutations. This result strongly suggests that the dpy-21 and dpy-26 mutations cause increased X expression, implying that the normal function of these genes may be to lower the expression of X-linked genes. Recessive mutations in two other dpy genes, dpy-22 X and dpy-23 X, increase the severity of phenotypes resulting from some X-linked hypomorphic mutations, although dpy-23 may affect the phenotypes resulting from the autosomal hypomorphs as well. The mutations in all four of the dpy genes show their effects in both XO and XX animals, although to different degrees. Mutations in 18 other dpy genes do not show these effects. PMID:3666440

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences

PubMed Central

Moyle, Richard L.; Carvalhais, Lilia C.; Pretorius, Lara-Simone; Nowak, Ekaterina; Subramaniam, Gayathery; Dalton-Morgan, Jessica; Schenk, Peer M.

2017-01-01

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence. PMID:28979287

Functional Characterization of Promoter Variants of the Adiponectin Gene Complemented by Epidemiological Data

PubMed Central

Laumen, Helmut; Saningong, Akuma D.; Heid, Iris M.; Hess, Jochen; Herder, Christian; Claussnitzer, Melina; Baumert, Jens; Lamina, Claudia; Rathmann, Wolfgang; Sedlmeier, Eva-Maria; Klopp, Norman; Thorand, Barbara; Wichmann, H.-Erich; Illig, Thomas; Hauner, Hans

2009-01-01

OBJECTIVE Adiponectin (APM1, ACDC) is an adipocyte-derived protein with downregulated expression in obesity and insulin-resistant states. Several potentially regulatory single nucleotide polymorphisms (SNPs) within the APM1 gene promoter region have been associated with circulating adiponectin levels. None of them have been functionally characterized in adiponectin-expressing cells. Hence, we investigated three SNPs (rs16861194, rs17300539, and rs266729) for their influence on adiponectin promoter activity and their association with circulating adiponectin levels. RESEARCH DESIGN AND METHODS Basal and rosiglitazone-induced promoter activity of different SNP combinations (haplotypes) was analyzed in 3T3-L1 adipocytes using luciferase reporter gene assays and DNA binding studies comparing all possible APM1 haplotypes. This functional approach was complemented with analysis of epidemiological population-based data of 1,692 participants of the MONICA/KORA S123 cohort and 696 participants from the KORA S4 cohort for SNP and haplotype association with circulating adiponectin levels. RESULTS Major to minor allele replacements of the three SNPs revealed significant effects on promoter activity in luciferase assays. Particularly, a minor variant in rs16861194 resulted in reduced basal and rosiglitazone-induced promoter activity and hypoadiponectinemia in the epidemiological datasets. The haplotype with the minor allele in all three SNPs showed a complete loss of promoter activity, and no subject carried this haplotype in either of the epidemiological samples (combined P value for statistically significant difference from a random sample was 0.006). CONCLUSIONS Our results clearly demonstrate that promoter variants associated with hypoadiponectinemia in humans substantially affect adiponectin promoter activity in adipocytes. Our combination of functional experiments with epidemiological data overcomes the drawback of each approach alone. PMID:19074982

Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

PubMed Central

Hakami, Nora Y.; Ranjan, Amaresh K.; Hardikar, Anandwardhan A.; Dusting, Greg J.; Peshavariya, Hitesh M.

2017-01-01

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization. PMID:28386230

Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development.

PubMed

Xu, Jing; Zhang, Lin; Yang, Dong-Lei; Li, Qun; He, Zuhua

2015-12-01

Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated. Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development. Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 5'-Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 2'-deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms. Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development. © 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist © 2015 New Phytologist Trust.

Discovery of functional non-coding conserved regions in the α-synuclein gene locus

PubMed Central

Sterling, Lori; Walter, Michael; Ting, Dennis; Schüle, Birgitt

2014-01-01

Several single nucleotide polymorphisms (SNPs) and the Rep-1 microsatellite marker of the α-synuclein ( SNCA) gene have consistently been shown to be associated with Parkinson’s disease, but the functional relevance is unclear. Based on these findings we hypothesized that conserved cis-regulatory elements in the SNCA genomic region regulate expression of SNCA, and that SNPs in these regions could be functionally modulating the expression of SNCA, thus contributing to neuronal demise and predisposing to Parkinson’s disease. In a pair-wise comparison of a 206kb genomic region encompassing the SNCA gene, we revealed 34 evolutionary conserved DNA sequences between human and mouse. All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays.  We found that 12 out of 34 elements exhibited either an enhancement or reduction of the expression of the reporter gene. Three elements upstream of the SNCA gene displayed an approximately 1.5 fold (p<0.009) increase in expression. Of the intronic regions, three showed a 1.5 fold increase and two others indicated a 2 and 2.5 fold increase in expression (p<0.002). Three elements downstream of the SNCA gene showed 1.5 fold and 2.5 fold increase (p<0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity. Our results demonstrate that the SNCA gene contains cis-regulatory regions that might regulate the transcription and expression of SNCA. Further studies in disease-relevant tissue types will be important to understand the functional impact of regulatory regions and specific Parkinson’s disease-associated SNPs and its function in the disease process. PMID:25566351

Molecular Cloning, Functional Characterization, and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates

PubMed Central

Kollitz, Erin M.; Zhang, Guozhu; Hawkins, Mary Beth; Whitfield, G. Kerr; Reif, David M.; Kullman, Seth W.

2015-01-01

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators. PMID:25855982

Molecular Characterization of the NLRC4 Expression in Relation to Interleukin-18 Levels

PubMed Central

Zeller, Tanja; Haase, Tina; Müller, Christian; Riess, Helene; Lau, Denise; Zeller, Simon; Krause, Jasmin; Baumert, Jens; Pless, Ole; Dupuis, Josée; Wild, Philipp S.; Eleftheriadis, Medea; Waldenberger, Melanie; Zeilinger, Sonja; Ziegler, Andreas; Peters, Annette; Tiret, Laurence; Proust, Carole; Marzi, Carola; Munzel, Thomas; Strauch, Konstantin; Prokisch, Holger; Lackner, Karl J.; Herder, Christian; Thorand, Barbara; Benjamin, Emilia J.; Blankenberg, Stefan; Koenig, Wolfgang; Schnabel, Renate B.

2015-01-01

Background Interleukin-18 (IL-18) is a pleiotropic cytokine centrally involved in the cytokine cascade with complex immunomodulatory functions in innate and acquired immunity. Circulating IL-18 concentrations are associated with type 2 diabetes, cardiovascular events and diverse inflammatory and autoimmune disorders. Methods and Results To identify causal variants affecting circulating IL-18 concentrations, we applied various omics and molecular biology approaches. By GWAS, we confirmed association of IL-18 levels with a SNP in the untranslated exon 2 of the inflammasome component NLRC4 (NLR family, CARD domain containing 4) gene on chromosome 2 (rs385076, P=2.4×10−45). Subsequent molecular analyses by gene expression analysis and reporter gene assays indicated an effect of rs385076 on NLRC4 expression and differential isoform usage by modulating binding of the transcription factor PU.1. Conclusions Our study provides evidence for the functional causality of SNP rs385076 within the NLRC4 gene in relation to IL-18 activation. PMID:26362438

Effect of Resveratrol, a SIRT1 Activator, on the Interactions of the CLOCK/BMAL1 Complex

PubMed Central

Park, Insung; Lee, Yool; Kim, Hee-Dae

2014-01-01

Background In mammals, the CLOCK/BMAL1 heterodimer is a key transcription factor complex that drives the cyclic expression of clock-controlled genes involved in various physiological functions and behavioral consequences. Recently, a growing number of studies have reported a molecular link between the circadian clock and metabolism. In the present study, we explored the regulatory effects of SIRTUIN1 (SIRT1), an NAD+-dependent deacetylase, on CLOCK/BMAL1-mediated clock gene expression. Methods To investigate the interaction between SIRT1 and CLOCK/BMAL1, we conducted bimolecular fluorescence complementation (BiFC) analyses supplemented with immunocytochemistry assays. BiFC experiments employing deletion-specific mutants of BMAL1 were used to elucidate the specific domains that are necessary for the SIRT1-BMAL1 interaction. Additionally, luciferase reporter assays were used to delineate the effects of SIRT1 on circadian gene expression. Results BiFC analysis revealed that SIRT1 interacted with both CLOCK and BMAL1 in most cell nuclei. As revealed by BiFC assays using various BMAL1 deletion mutants, the PAS-B domain of BMAL1 was essential for interaction with SIRT1. Activation of SIRT1 with resveratrol did not exert any significant change on the interaction with the CLOCK/BMAL1 complex. However, promoter analysis using Per1-Luc and Ebox-Luc reporters showed that SIRT1 significantly downregulated both promoter activities. This inhibitory effect was intensified by treatment with resveratrol, indicating a role for SIRT1 and its activator in CLOCK/BMAL1-mediated transcription of clock genes. Conclusion These results suggest that SIRT1 may form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression, probably via deacetylase activity. PMID:25309798

Chitin utilization by the insect-transmitted bacterium Xylella fastidiosa.

PubMed

Killiny, Nabil; Prado, Simone S; Almeida, Rodrigo P P

2010-09-01

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa.

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

PubMed Central

Kojima, Hiroyuki; Takeda, Yukimasa; Muromoto, Ryuta; Takahashi, Miki; Hirao, Toru; Takeuchi, Shinji; Jetten, Anton M.; Matsuda, Tadashi

2018-01-01

The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10−6 M to 1 × 10−5 M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells. PMID:25583575

Survivin is a novel transcription regulator of KIT and is downregulated by miRNA-494 in gastrointestinal stromal tumors.

PubMed

Yun, SeongJu; Kim, Won Kyu; Kwon, Yujin; Jang, Mi; Bauer, Sebastian; Kim, Hoguen

2018-05-15

Gain-of-function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT-independent targets of KIT-regulating mRNAs. We sought to investigate how miR-494 inhibits GIST proliferation and to identify novel target gene. We used microarray-based gene expression analyses to identify pathways and target genes affected by miR-494. The expressional relationship between survivin and miR-494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR-494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR-494, and its expression showed an inverse correlation with miR-494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = -0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR-494-mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR-494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Survivin is a novel transcription regulator of KIT and is downregulated by miRNA‐494 in gastrointestinal stromal tumors

PubMed Central

Yun, SeongJu; Kim, Won Kyu; Kwon, Yujin; Jang, Mi; Bauer, Sebastian

2018-01-01

Gain‐of‐function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT‐independent targets of KIT‐regulating mRNAs. We sought to investigate how miR‐494 inhibits GIST proliferation and to identify novel target gene. We used microarray‐based gene expression analyses to identify pathways and target genes affected by miR‐494. The expressional relationship between survivin and miR‐494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR‐494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR‐494, and its expression showed an inverse correlation with miR‐494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = −0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR‐494‐mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR‐494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT. PMID:29277888

Chitin Utilization by the Insect-Transmitted Bacterium Xylella fastidiosa▿ †

PubMed Central

Killiny, Nabil; Prado, Simone S.; Almeida, Rodrigo P. P.

2010-01-01

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa. PMID:20656858

Folate and Breast Cancer: Role of Intake, Blood Levels, and Metabolic Gene Polymorphisms

DTIC Science & Technology

2006-06-01

polymorphisms . The specific aims are 1) methodological training in the analysis of gene - gene and gene -environment interactions by studying folate...evaluation of folate intake, plasma folate, and metabolic gene polymorphisms in relation to breast cancer risk: Months 1-19. b. Prepare blood samples...isolated for the folate and gene polymorphism assays among the 184 cases and matched controls. The folate assays are on-going at this time and over

An efficient platform for genetic selection and screening of gene switches in Escherichia coli

PubMed Central

Muranaka, Norihito; Sharma, Vandana; Nomura, Yoko; Yokobayashi, Yohei

2009-01-01

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay. PMID:19190095

Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils.

PubMed

Etebu, E; Osborn, A M

2009-05-01

The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes (PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0-5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3.88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes. The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils.

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

PubMed Central

Holman, Edna C.; Campbell, Leah J.; Hines, John; Crews, Craig M.

2012-01-01

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes. PMID:23028429

Receptor Tyrosine Kinase MET Interactome and Neurodevelopmental Disorder Partners at the Developing Synapse.

PubMed

Xie, Zhihui; Li, Jing; Baker, Jonathan; Eagleson, Kathie L; Coba, Marcelo P; Levitt, Pat

2016-12-15

Atypical synapse development and plasticity are implicated in many neurodevelopmental disorders (NDDs). NDD-associated, high-confidence risk genes have been identified, yet little is known about functional relationships at the level of protein-protein interactions, which are the dominant molecular bases responsible for mediating circuit development. Proteomics in three independent developing neocortical synaptosomal preparations identified putative interacting proteins of the ligand-activated MET receptor tyrosine kinase, an autism risk gene that mediates synapse development. The candidates were translated into interactome networks and analyzed bioinformatically. Additionally, three independent quantitative proximity ligation assays in cultured neurons and four independent immunoprecipitation analyses of synaptosomes validated protein interactions. Approximately 11% (8/72) of MET-interacting proteins, including SHANK3, SYNGAP1, and GRIN2B, are associated with NDDs. Proteins in the MET interactome were translated into a novel MET interactome network based on human protein-protein interaction databases. High-confidence genes from different NDD datasets that encode synaptosomal proteins were analyzed for being enriched in MET interactome proteins. This was found for autism but not schizophrenia, bipolar disorder, major depressive disorder, or attention-deficit/hyperactivity disorder. There is correlated gene expression between MET and its interactive partners in developing human temporal and visual neocortices but not with highly expressed genes that are not in the interactome. Proximity ligation assays and biochemical analyses demonstrate that MET-protein partner interactions are dynamically regulated by receptor activation. The results provide a novel molecular framework for deciphering the functional relations of key regulators of synaptogenesis that contribute to both typical cortical development and to NDDs. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Effect of supercoiling on formation of protein-mediated DNA loops

NASA Astrophysics Data System (ADS)

Purohit, P. K.; Nelson, P. C.

2006-12-01

DNA loop formation is one of several mechanisms used by organisms to regulate genes. The free energy of forming a loop is an important factor in determining whether the associated gene is switched on or off. In this paper we use an elastic rod model of DNA to determine the free energy of forming short (50-100 basepair), protein mediated DNA loops. Superhelical stress in the DNA of living cells is a critical factor determining the energetics of loop formation, and we explicitly account for it in our calculations. The repressor protein itself is regarded as a rigid coupler; its geometry enters the problem through the boundary conditions it applies on the DNA. We show that a theory with these ingredients is sufficient to explain certain features observed in modulation of in vivo gene activity as a function of the distance between operator sites for the lac repressor. We also use our theory to make quantitative predictions for the dependence of looping on superhelical stress, which may be testable both in vivo and in single-molecule experiments such as the tethered particle assay and the magnetic bead assay.

Functional and Promoter Analysis of ChiIV3, a Chitinase of Pepper Plant, in Response to Phytophthora capsici Infection

PubMed Central

Liu, Zhiqin; Shi, Lanping; Yang, Sheng; Lin, Youquan; Weng, Yahong; Li, Xia; Hussain, Ansar; Noman, Ali; He, Shuilin

2017-01-01

Despite the involvement of many members of the chitinase family in plant immunity, the precise functions of the majority of the members remain poorly understood. Herein, the gene ChiIV3 in Capsicum annuum encoding a chitinase protein containing a chitin binding domain and targeting to the plasma membrane was found to be induced by Phytophthora capsici inoculation (PCI) and applied chitin treatment. Besides its direct inhibitory effect on growth of Phytophthora capsici (P. capsici), ChiIV3 was also found by virus-induced gene silencing (VIGS) and transient overexpression (TOE) in pepper plants to act as a positive regulator of plant cell death and in triggering defense signaling and upregulation of PR (pathogenesis related) genes against PCI. A 5′ deletion assay revealed that pChiIV3−712 to −459 bp was found to be sufficient for ChiIV3’ response to PCI. Furthermore, a mutation assay indicated that W-box−466 to −461 bp in pChiIV3−712 to −459 bp was noted to be the PCI-responsible element. These results collectively suggest that ChiIV3 acts as a likely antifungal protein and as a receptor for unidentified chitin in planta to trigger cell death and defense signaling against PCI. PMID:28763001

Sequence variations of the partially dominant DELLA gene Rht-B1c in wheat and their functional impacts

PubMed Central

Ma, Zhengqiang

2013-01-01

Rht-B1c, allelic to the DELLA protein-encoding gene Rht-B1a, is a natural mutation documented in common wheat (Triticum aestivum). It confers variation to a number of traits related to cell and plant morphology, seed dormancy, and photosynthesis. The present study was conducted to examine the sequence variations of Rht-B1c and their functional impacts. The results showed that Rht-B1c was partially dominant or co-dominant for plant height, and exhibited an increased dwarfing effect. At the sequence level, Rht-B1c differed from Rht-B1a by one 2kb Veju retrotransposon insertion, three coding region single nucleotide polymorphisms (SNPs), one 197bp insertion, and four SNPs in the 1kb upstream sequence. Haplotype investigations, association analyses, transient expression assays, and expression profiling showed that the Veju insertion was primarily responsible for the extreme dwarfing effect. It was found that the Veju insertion changed processing of the Rht-B1c transcripts and resulted in DELLA motif primary structure disruption. Expression assays showed that Rht-B1c caused reduction of total Rht-1 transcript levels, and up-regulation of GATA-like transcription factors and genes positively regulated by these factors, suggesting that one way in which Rht-1 proteins affect plant growth and development is through GATA-like transcription factor regulation. PMID:23918966

Novel transcriptional networks regulated by CLOCK in human neurons.

PubMed

Fontenot, Miles R; Berto, Stefano; Liu, Yuxiang; Werthmann, Gordon; Douglas, Connor; Usui, Noriyoshi; Gleason, Kelly; Tamminga, Carol A; Takahashi, Joseph S; Konopka, Genevieve

2017-11-01

The molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to human cognition and disease. In this study, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin immunoprecipitation sequencing for endogenous CLOCK in adult neocortices and RNA sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates the expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts coexpressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks is driven by hub genes with human-specific patterns of expression. These data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function. © 2017 Fontenot et al.; Published by Cold Spring Harbor Laboratory Press.

Comparative genomic analysis of SET domain family reveals the origin, expansion, and putative function of the arthropod-specific SmydA genes as histone modifiers in insects.

PubMed

Jiang, Feng; Liu, Qing; Wang, Yanli; Zhang, Jie; Wang, Huimin; Song, Tianqi; Yang, Meiling; Wang, Xianhui; Kang, Le

2017-06-01

The SET domain is an evolutionarily conserved motif present in histone lysine methyltransferases, which are important in the regulation of chromatin and gene expression in animals. In this study, we searched for SET domain-containing genes (SET genes) in all of the 147 arthropod genomes sequenced at the time of carrying out this experiment to understand the evolutionary history by which SET domains have evolved in insects. Phylogenetic and ancestral state reconstruction analysis revealed an arthropod-specific SET gene family, named SmydA, that is ancestral to arthropod animals and specifically diversified during insect evolution. Considering that pseudogenization is the most probable fate of the new emerging gene copies, we provided experimental and evolutionary evidence to demonstrate their essential functions. Fluorescence in situ hybridization analysis and in vitro methyltransferase activity assays showed that the SmydA-2 gene was transcriptionally active and retained the original histone methylation activity. Expression knockdown by RNA interference significantly increased mortality, implying that the SmydA genes may be essential for insect survival. We further showed predominantly strong purifying selection on the SmydA gene family and a potential association between the regulation of gene expression and insect phenotypic plasticity by transcriptome analysis. Overall, these data suggest that the SmydA gene family retains essential functions that may possibly define novel regulatory pathways in insects. This work provides insights into the roles of lineage-specific domain duplication in insect evolution. © The Authors 2017. Published by Oxford University Press.

Comparative genomic analysis of SET domain family reveals the origin, expansion, and putative function of the arthropod-specific SmydA genes as histone modifiers in insects

PubMed Central

Jiang, Feng; Liu, Qing; Wang, Yanli; Zhang, Jie; Wang, Huimin; Song, Tianqi; Yang, Meiling

2017-01-01

Abstract The SET domain is an evolutionarily conserved motif present in histone lysine methyltransferases, which are important in the regulation of chromatin and gene expression in animals. In this study, we searched for SET domain–containing genes (SET genes) in all of the 147 arthropod genomes sequenced at the time of carrying out this experiment to understand the evolutionary history by which SET domains have evolved in insects. Phylogenetic and ancestral state reconstruction analysis revealed an arthropod-specific SET gene family, named SmydA, that is ancestral to arthropod animals and specifically diversified during insect evolution. Considering that pseudogenization is the most probable fate of the new emerging gene copies, we provided experimental and evolutionary evidence to demonstrate their essential functions. Fluorescence in situ hybridization analysis and in vitro methyltransferase activity assays showed that the SmydA-2 gene was transcriptionally active and retained the original histone methylation activity. Expression knockdown by RNA interference significantly increased mortality, implying that the SmydA genes may be essential for insect survival. We further showed predominantly strong purifying selection on the SmydA gene family and a potential association between the regulation of gene expression and insect phenotypic plasticity by transcriptome analysis. Overall, these data suggest that the SmydA gene family retains essential functions that may possibly define novel regulatory pathways in insects. This work provides insights into the roles of lineage-specific domain duplication in insect evolution. PMID:28444351

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

PubMed

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.

Force dependent internalization of magnetic nanoparticles results in highly loaded endothelial cells for use as potential therapy delivery vectors.

PubMed

MacDonald, Cristin; Barbee, Kenneth; Polyak, Boris

2012-05-01

To investigate the kinetics, mechanism and extent of MNP loading into endothelial cells and the effect of this loading on cell function. MNP uptake was examined under field on/off conditions, utilizing varying magnetite concentration MNPs. MNP-loaded cell viability and functional integrity was assessed using metabolic respiration, cell proliferation and migration assays. MNP uptake in endothelial cells significantly increased under the influence of a magnetic field versus non-magnetic conditions. Larger magnetite density of the MNPs led to a higher MNP internalization by cells under application of a magnetic field without compromising cellular respiration activity. Two-dimensional migration assays at no field showed that higher magnetite loading resulted in greater cell migration rates. In a three-dimensional migration assay under magnetic field, the migration rate of MNP-loaded cells was more than twice that of unloaded cells and was comparable to migration stimulated by a serum gradient. Our results suggest that endothelial cell uptake of MNPs is a force dependent process. The in vitro assays determined that cell health is not adversely affected by high MNP loadings, allowing these highly magnetically responsive cells to be potentially beneficial therapy (gene, drug or cell) delivery systems.

Tuning Riboswitch Regulation through Conformational Selection

PubMed Central

Wilson, Ross C.; Smith, Angela M.; Fuchs, Ryan T.; Kleckner, Ian R.; Henkin, Tina M.; Foster, Mark P.

2010-01-01

SUMMARY The SMK box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the SMK box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound and ligand-free states. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element. PMID:21075119

Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

PubMed Central

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-01-01

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

PubMed

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-08-27

The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

A quantitative PCR assay for aerobic, vinyl chloride- and ethene-assimilating microorganisms in groundwater.

PubMed

Jin, Yang Oh; Mattes, Timothy E

2010-12-01

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plumes that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 × 10(3) - 1.0 × 10(5) copies/L groundwater while etnE abundance ranged from 4.3 × 10(3) - 6.3 × 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting microbial ecology studies involving etheneotrophs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengthsmore » of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.« less

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

PubMed

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-03-12

ZmbZIP25 ( Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter

PubMed Central

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-01-01

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from −2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from −2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5′-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5′-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5′-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5′-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from −1117 to −957 that were responsible for the specificity of the ZmbZIP25 5′-flanking sequence. PMID:29534529

Avoiding false positives and optimizing identification of true ...

EPA Pesticide Factsheets

The potential for chemicals to affect endocrine signaling is commonly evaluated via in vitro receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented from screening 94 chemicals from 54 chemical groups for estrogen receptor (ER) activation in a competitive rainbow trout ER (rtER) binding assay and a trout liver slice vitellogenin mRNA expression assay. Results from true competitive agonists and antagonists, and inactive chemicals with little or no indication of ER binding or gene activation were easily interpreted. However, results for numerous industrial chemicals were more challenging to interpret, including chemicals with: (1) apparent competitive binding curves but no gene activation, (2) apparent binding and gene inhibition with evidence of either cytotoxicity or changes in assay media pH, (3) apparent binding but non-competitive gene inhibition of unknown cause, or (4) no rtER binding and gene inhibition not due to competitive ER interaction but due to toxicity, pH change, or some unknown cause. The use of endpoints such as toxicity, pH, precipitate formation, and determination of inhibitor dissociation constants (Ki) for interpreting the results of antagonism and binding assays for diverse chemicals is presented. Of the 94 chemicals tested for antagonism only two, tamoxifen and ICI-182,780, were found to be true competitive

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents.

PubMed

Fukushima, Toshikazu; Hara-Yamamura, Hiroe; Nakashima, Koji; Tan, Lea Chua; Okabe, Satoshi

2017-12-01

Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

PubMed

Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

2013-11-27

The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter.

PubMed

Biamonti, G; Bassi, M T; Cartegni, L; Mechta, F; Buvoli, M; Cobianchi, F; Riva, S

1993-03-05

hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

Development and evaluation of a simple and effective real time PCR assay for mitochondrial quantification in racing camels.

PubMed

Soman, Soja Saghar; Tinson, Alex

2016-10-01

Camel racing is a popular sport in the Middle East region, where the demand is high for racing camels with higher stamina and endurance. Devising a technique to measure oxidative capacity and endurance in camels should be useful. Mitochondria are highly specialized organelles involved in metabolism in all higher organisms for sustaining life and providing energy for physical functions. The ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is often used as an estimate for the metabolic status of the tissue. A greater quantity of mitochondria per unit of tissue translates into greater oxidative capacity and endurance. In this report, we describe a simple, sensitive and efficient real-time PCR assay for the quantification of blood mitochondria in racing camels. The primer sequences selected for the SYBR green-based PCR assay included mitochondrial D-loop region, mitochondrial ATP6ase gene and the nuclear β-actin gene. The assay was validated using two groups of camels comprising racing and dairy camels. The racing camels demonstrated a higher mtDNA/nDNA ratio compared with dairy camels based on the ΔΔCt values, with a higher variability among racing camels. The mean ΔΔCt values of adult and young racing camels did not vary considerably. The findings show that the present assay can be used as an evaluative tool for racing camels. Copyright © 2016 Elsevier Ltd. All rights reserved.

Clonal hematopoiesis as determined by the HUMARA assay is a marker for acquired mutations in epigenetic regulators in older women.

PubMed

Wiedmeier, Julia Erin; Kato, Catherine; Zhang, Zhenzhen; Lee, Hyunjung; Dunlap, Jennifer; Nutt, Eric; Rattray, Rogan; McKay, Sarah; Eide, Christopher; Press, Richard; Mori, Motomi; Druker, Brian; Dao, Kim-Hien

2016-09-01

Recent large cohort studies revealed that healthy older individuals harbor somatic mutations that increase their risk for hematologic malignancy and all-cause cardiovascular deaths. The majority of these mutations are in chromatin and epigenetic regulatory genes (CERGs). CERGs play a key role in regulation of DNA methylation (DNMT3A and TET2) and histone function (ASXL1) and in clonal proliferation of hematopoietic stem cells. We hypothesize that older women manifesting clonal hematopoiesis, defined here as a functional phenomenon in which a hematopoietic stem cell has acquired a survival and proliferative advantage, harbor a higher frequency of somatic mutations in CERGs. The human androgen receptor gene (HUMARA) assay was used in our study to detect the presence of nonrandom X inactivation in women, a marker for clonal hematopoiesis. In our pilot study, we tested 127 blood samples from women ≥65 years old without a history of invasive cancer or hematologic malignancies. Applying stringent qualitative criteria, we found that 26% displayed clonal hematopoiesis; 52.8% displayed polyclonal hematopoiesis; and 21.3% had indeterminate patterns (too close to call by qualitative assessment). Using Illumina MiSeq next-generation sequencing, we identified somatic mutations in CERGs in 15.2% of subjects displaying clonal hematopoiesis (three ASXL1 and two DNMT3A mutations with an average variant allele frequency of 15.7%, range: 6.3%-23.3%). In a more limited sequencing analysis, we evaluated the frequency of ASXL1 mutations by Sanger sequencing and found mutations in 9.7% of the clonal samples and 0% of the polyclonal samples. By comparing several recent studies (with some caveats as described), we determined the fold enrichment of detecting CERG mutations by using the HUMARA assay as a functional screen for clonal hematopoiesis. We conclude that a functional assay of clonal hematopoiesis is enriching for older women with somatic mutations in CERGs, particularly for ASXL1 and TET2 mutations and less so for DNMT3A mutations. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

miR-324-3p promotes gastric cancer development by activating Smad4-mediated Wnt/beta-catenin signaling pathway.

PubMed

Sun, Guang-Li; Li, Zheng; Wang, Wei-Zhi; Chen, Zheng; Zhang, Lei; Li, Qing; Wei, Song; Li, Bo-Wen; Xu, Jiang-Hao; Chen, Liang; He, Zhong-Yuan; Ying, Kai; Zhang, Xuan; Xu, Hao; Zhang, Dian-Cai; Xu, Ze-Kuan

2018-06-01

Emerging evidence suggested that miRNAs can function as oncogenes or tumor suppressors by regulating downstream target genes. miR-324-3p has been reported to function in several carcinomas, but its role in gastric cancer (GC) is still unknown. This study aims to explore the effects of miR-324-3p on the development of GC. Expression of miR-324-3p was examined in GC cells and tissues by qRT-PCR. Effects of miR-324-3p on GC cells were evaluated by cell vitality assay, colony formation assay, cell migration assay, and flow cytometric assay. The dual luciferase assay was used to verify whether miR-324-3p could interact with the potential target genes. Western blot was used to assess the expression level of Smad4 and beta-catenin. Intracellular ATP level was also examined. The tumor xenografts were established using nude mice. A gastric organoid model was made from fresh stomach tissue. miR-324-3p was expressed at higher levels in the tumor tissues compared with adjacent normal tissues. Overexpression of miR-324-3p promoted cell growth, migration, and decreased apoptosis. miR-324-3p repressed the expression of Smad4, and loss of Smad4 activated the Wnt/beta-catenin signaling pathway. Overexpression of Smad4 rescued the effects of miR-324-3p on GC cells. The intracellular ATP level was upregulated with overexpression of miR-324-3p. miR-324-3p facilitated tumor cell colonization and growth in vivo and contributed to the growth of gastric organoids. The results suggested that miR-324-3p promoted GC through activating the Smad4-mediated Wnt/beta-catenin signaling pathway. The miR-324-3p/Smad4/Wnt signaling axis may be a potential therapeutic target to prevent GC progression.

Mannosyltransferase is required for cell wall biosynthesis, morphology and control of asexual development in Neurospora crassa.

PubMed

Bowman, Shaun M; Piwowar, Amy; Ciocca, Maria; Free, Stephen J

2005-01-01

Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.

Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements.

PubMed

Papantonis, Argyris; Sourmeli, Sissy; Lecanidou, Rena

2008-05-09

From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway.

PubMed

Wang, Yingjun; Zhang, Mingzhi; Xu, Huanan; Wang, Yifei; Li, Zhaoming; Chang, Yu; Wang, Xinhuan; Fu, Xiaorui; Zhou, Zhiyuan; Yang, Siyuan; Wang, Bei; Shang, Yufeng

2017-09-22

Diffuse large B-cell lymphoma (DLBCL) is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel prognostic markers and therapeutic targets of DLBCL. Recent studies have shown that long non-coding RNAs (lncRNAs) play an important role in the development of cancer. By using the next generation HiSeq sequencing assay, we determined lncRNAs exhibiting differential expression between DLBCL patients and healthy controls. Then, RT-qPCR was performed for identification in clinical samples and cell materials, and lncRNA PANDA was verified to be down-regulated in DLBCL patients and have considerable diagnostic potential. In addition, decreased serum PANDA level was correlated to poorer clinical outcome and lower overall survival in DLBCL patients. Subsequently, we determined the experimental role of lncRNA PANDA in DLBCL progression. Luciferase reporter assay and chromatin immunoprecipitation assay suggested that lncRNA PANDA was induced by p53 and p53 interacts with the promoter region of PANDA. Cell functional assay further indicated that PANDA functioned as a tumor suppressor gene through the suppression of cell growth by a G0/G1 cell cycle arrest in DLBCL. More importantly, Cignal Signal Transduction Reporter Array and western blot assay showed that lncRNA PANDA inactivated the MAPK/ERK signaling pathway. In conclusion, our integrated approach demonstrates that PANDA in DLBCL confers a tumor suppressive function through inhibiting cell proliferation and silencing MAPK/ERK signaling pathway. Thus, PANDA may be a promising therapeutic target for patients with DLBCL.

Identification of Heat Shock Transcription Factor Genes Involved in Thermotolerance of Octoploid Cultivated Strawberry

PubMed Central

Liao, Wan-Yu; Lin, Lee-Fong; Jheng, Jing-Lian; Wang, Chun-Chung; Yang, Jui-Hung; Chou, Ming-Lun

2016-01-01

Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a, which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress. PMID:27999304

Identification of Heat Shock Transcription Factor Genes Involved in Thermotolerance of Octoploid Cultivated Strawberry.

PubMed

Liao, Wan-Yu; Lin, Lee-Fong; Jheng, Jing-Lian; Wang, Chun-Chung; Yang, Jui-Hung; Chou, Ming-Lun

2016-12-17

Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a , which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress.

VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA.

PubMed

Dai, Yuanyuan; Chang, Wenjiao; Zhao, Changcheng; Peng, Jing; Xu, Liangfei; Lu, Huaiwei; Zhou, Shusheng; Ma, Xiaoling

2017-05-01

Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene ( vraR ) and decreased expression of virulence genes ( hla , hlb , and coa ) and virulence-regulated genes (RNAIII, agrA , and saeR ). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla , hlb , and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence. Copyright © 2017 American Society for Microbiology.

Cross-talk among HMGA1 and FoxO1 in control of nuclear insulin signaling.

PubMed

Chiefari, Eusebio; Arcidiacono, Biagio; Palmieri, Camillo; Corigliano, Domenica Maria; Morittu, Valeria Maria; Britti, Domenico; Armoni, Michal; Foti, Daniela Patrizia; Brunetti, Antonio

2018-06-04

As a mediator of insulin-regulated gene expression, the FoxO1 transcription factor represents a master regulator of liver glucose metabolism. We previously reported that the high-mobility group AT-hook 1 (HMGA1) protein, a molecular switch for the insulin receptor gene, functions also as a downstream target of the insulin receptor signaling pathway, representing a critical nuclear mediator of insulin function. Here, we investigated whether a functional relationship existed between FoxO1 and HMGA1, which might help explain insulin-mediated gene transcription in the liver. To this end, as a model study, we investigated the canonical FoxO1-HMGA1-responsive IGFBP1 gene, whose hepatic expression is regulated by insulin. By using a conventional GST-pull down assay combined with co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) analyses, we provide evidence of a physical interaction between FoxO1 and HMGA1. Further investigation with chromatin immunoprecipitation, confocal microscopy, and Fluorescence Recovery After Photobleaching (FRAP) technology indicated a functional significance of this interaction, in both basal and insulin-stimulated states, providing evidence that, by modulating FoxO1 transactivation, HMGA1 is essential for FoxO1-induced IGFBP1 gene expression, and thereby a critical modulator of insulin-mediated FoxO1 regulation in the liver. Collectively, our findings highlight a novel FoxO1/HMGA1-mediated mechanism by which insulin may regulate gene expression and metabolism.

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

PubMed

Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

2016-01-01

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor.

PubMed

Faucheux, M; Roignant, J-Y; Netter, S; Charollais, J; Antoniewski, C; Théodore, L

2003-02-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

batman Interacts with Polycomb and trithorax Group Genes and Encodes a BTB/POZ Protein That Is Included in a Complex Containing GAGA Factor

PubMed Central

Faucheux, M.; Roignant, J.-Y.; Netter, S.; Charollais, J.; Antoniewski, C.; Théodore, L.

2003-01-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes. PMID:12556479

The analytical validation of the Oncotype DX Recurrence Score assay

PubMed Central

Baehner, Frederick L

2016-01-01

In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score® result (scale: 0–100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time. PMID:27729940

The analytical validation of the Oncotype DX Recurrence Score assay.

PubMed

Baehner, Frederick L

2016-01-01

In vitro diagnostic multivariate index assays are highly complex molecular assays that can provide clinically actionable information regarding the underlying tumour biology and facilitate personalised treatment. These assays are only useful in clinical practice if all of the following are established: analytical validation (i.e., how accurately/reliably the assay measures the molecular characteristics), clinical validation (i.e., how consistently/accurately the test detects/predicts the outcomes of interest), and clinical utility (i.e., how likely the test is to significantly improve patient outcomes). In considering the use of these assays, clinicians often focus primarily on the clinical validity/utility; however, the analytical validity of an assay (e.g., its accuracy, reproducibility, and standardisation) should also be evaluated and carefully considered. This review focuses on the rigorous analytical validation and performance of the Oncotype DX ® Breast Cancer Assay, which is performed at the Central Clinical Reference Laboratory of Genomic Health, Inc. The assay process includes tumour tissue enrichment (if needed), RNA extraction, gene expression quantitation (using a gene panel consisting of 16 cancer genes plus 5 reference genes and quantitative real-time RT-PCR), and an automated computer algorithm to produce a Recurrence Score ® result (scale: 0-100). This review presents evidence showing that the Recurrence Score result reported for each patient falls within a tight clinically relevant confidence interval. Specifically, the review discusses how the development of the assay was designed to optimise assay performance, presents data supporting its analytical validity, and describes the quality control and assurance programmes that ensure optimal test performance over time.

Coordinated Gene Regulation in the Initial Phase of Salt Stress Adaptation*

PubMed Central

Vanacloig-Pedros, Elena; Bets-Plasencia, Carolina; Pascual-Ahuir, Amparo; Proft, Markus

2015-01-01

Stress triggers complex transcriptional responses, which include both gene activation and repression. We used time-resolved reporter assays in living yeast cells to gain insights into the coordination of positive and negative control of gene expression upon salt stress. We found that the repression of “housekeeping” genes coincides with the transient activation of defense genes and that the timing of this expression pattern depends on the severity of the stress. Moreover, we identified mutants that caused an alteration in the kinetics of this transcriptional control. Loss of function of the vacuolar H+-ATPase (vma1) or a defect in the biosynthesis of the osmolyte glycerol (gpd1) caused a prolonged repression of housekeeping genes and a delay in gene activation at inducible loci. Both mutants have a defect in the relocation of RNA polymerase II complexes at stress defense genes. Accordingly salt-activated transcription is delayed and less efficient upon partially respiratory growth conditions in which glycerol production is significantly reduced. Furthermore, the loss of Hog1 MAP kinase function aggravates the loss of RNA polymerase II from housekeeping loci, which apparently do not accumulate at inducible genes. Additionally the Def1 RNA polymerase II degradation factor, but not a high pool of nuclear polymerase II complexes, is needed for efficient stress-induced gene activation. The data presented here indicate that the finely tuned transcriptional control upon salt stress is dependent on physiological functions of the cell, such as the intracellular ion balance, the protective accumulation of osmolyte molecules, and the RNA polymerase II turnover. PMID:25745106

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3

PubMed Central

Loya, Travis J.; O’Rourke, Thomas W.; Reines, Daniel

2012-01-01

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 ‘tail’ forms an α-helical multimerization domain that helps assemble it onto an RNA substrate. PMID:22564898

Lessons from the canine Oxtr gene: populations, variants and functional aspects.

PubMed

Bence, M; Marx, P; Szantai, E; Kubinyi, E; Ronai, Z; Banlaki, Z

2017-04-01

Oxytocin receptor (OXTR) acts as a key behavioral modulator of the central nervous system, affecting social behavior, stress, affiliation and cognitive functions. Variants of the Oxtr gene are known to influence behavior both in animals and humans; however, canine Oxtr polymorphisms are less characterized in terms of possible relevance to function, selection criteria in breeding and domestication. In this report, we provide a detailed characterization of common variants of the canine Oxtr gene. In particular (1) novel polymorphisms were identified by direct sequencing of wolf and dog samples, (2) allelic distributions and pairwise linkage disequilibrium patterns of several canine populations were compared, (3) neighbor joining (NJ) tree based on common single nucleotide polymorphisms (SNPs) was constructed, (4) mRNA expression features were assessed, (5) a novel splice variant was detected and (6) in vitro functional assays were performed. Results indicate marked differences regarding Oxtr variations between purebred dogs of different breeds, free-ranging dog populations, wolf subspecies and golden jackals. This, together with existence of explicitly dog-specific alleles and data obtained from the NJ tree implies that Oxtr could indeed have been a target gene during domestication and selection for human preferred aspects of temperament and social behavior. This assumption is further supported by the present observations on gene expression patterns within the brain and luciferase reporter experiments, providing a molecular level link between certain canine Oxtr polymorphisms and differences in nervous system function and behavior. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9

PubMed Central

Capitanio, Juliana S; Montpetit, Ben; Wozniak, Richard W

2017-01-01

Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases. DOI: http://dx.doi.org/10.7554/eLife.18825.001 PMID:28221134

Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

PubMed Central

Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

2015-01-01

The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the “lipolysome.” Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

Characterization of gossypol biosynthetic pathway

PubMed Central

Tian, Xiu; Ruan, Ju-Xin; Huang, Jin-Quan; Fang, Xin; Chen, Zhi-Wen; Hong, Hui; Wang, Ling-Jian; Mao, Ying-Bo; Lu, Shan; Zhang, Tian-Zhen; Chen, Xiao-Ya

2018-01-01

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion. PMID:29784821

Gene quantification by the NanoGene assay is resistant to inhibition by humic acids.

PubMed

Kim, Gha-Young; Wang, Xiaofang; Ahn, Hosang; Son, Ahjeong

2011-10-15

NanoGene assay is a magnetic bead and quantum dot nanoparticles based gene quantification assay. It relies on a set of probe and signaling probe DNAs to capture the target DNA via hybridization. We have demonstrated the inhibition resistance of the NanoGene assay using humic acids laden genomic DNA (gDNA). At 1 μg of humic acid per mL, quantitiative PCR (qPCR) was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 60% of its quantification capability. To further increase the inhibition resistance of NanoGene assay at high concentration of humic acids, we have identified the specific mechanisms that are responsible for the inhibition. We examined five potential mechanisms with which the humic acids can partially inhibit our NanoGene assay. The mechanisms examined were (1) adsorption of humic acids on the particle surface; (2) particle aggregation induced by humic acids; (3) fluorescence quenching of quantum dots by humic acids during hybridization; (4) humic acids mimicking of target DNA; and (5) nonspecific binding between humic acids and target gDNA. The investigation showed that no adsorption of humic acids onto the particles' surface was observed for the humic acids' concentration. Particle aggregation and fluorescence quenching were also negligible. Humic acids also did not mimic the target gDNA except 1000 μg of humic acids per mL and hence should not contribute to the partial inhibition. Four of the above mechanisms were not related to the inhibition effect of humic acids particularly at the environmentally relevant concentrations (<100 μg/mL). However, a substantial amount of nonspecific binding was observed between the humic acids and target gDNA. This possibly results in lesser amount of target gDNA being captured by the probe and signaling DNA.

DETECTION OF ESCHERICHIA COLI IN WATER USING A COLORIMETRIC GENE PROBE ASSAY

EPA Science Inventory

A commercially available DNA hydribization assay (Gene-trak , Framingham, MA. USA) was compared with the EC-MUG procedure for the detection of Escherichia coli in water. The gene probe gave positive responses for pure cultures of E. coli 0157:H7, E. fergusonii, Shigella sonnei, S...

Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

PubMed Central

Tan, Yaohong; Hu, Yunhui; Zhou, Yuan; Liu, Juanni; Xu, Yuanfu; Xie, Yinliang; Wang, Caiyun; Gao, Yingdai; Wang, Jianxiang; Cheng, Tao; Yang, Chunzheng; Xiong, Dongsheng; Miao, Hua

2012-01-01

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux. PMID:22403708

The roles of HOXB7 in promoting migration, invasion, and anti-apoptosis in gastric cancer.

PubMed

Joo, Moon Kyung; Park, Jong-Jae; Yoo, Hyo Soon; Lee, Beom Jae; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

2016-10-01

The aim of this study was to compare HOXB7 expression level between gastric cancer and non-cancerous gastric tissues. Additionally, the functional effects of HOXB7, including its pro-migration or invasion and anti-apoptosis roles, were evaluated in gastric cancer cells. Both gene and protein expression levels of HOXB7 were examined in gastric cancer cell lines, and HOXB7 expression was compared between primary or metastatic gastric cancer tissues and chronic gastritis or intestinal metaplasia tissues. Functional studies included a wound healing assay, a Matrigel invasion assay, and an Annexin-V assay were performed, and Akt/PTEN activity was measured by western blotting. Both gene and protein expression levels of HOXB7 could be clearly detected in various gastric cancer cell lines except MKN-28 cell. HOXB7 expression was significantly higher in primary or metastatic gastric cancer tissues than in chronic gastritis or intestinal metaplasia tissues. HOXB7 knockdown led to inhibition of cell invasion and migration, had an apoptotic effect, downregulated phosphor-Akt, and upregulated PTEN in AGS and SNU-638 cells. Reinforced expression of HOXB7 caused the opposite effects in MKN-28 and MKN-45 cells. Our study suggests that HOXB7 has an oncogenic role in gastric cancer, which might be related to the modulation of Akt/PTEN activity to induce cell migration/invasion and anti-apoptotic effects. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene.

PubMed

Dobrovolsky, Vasily N; Revollo, Javier; Petibone, Dayton M; Heflich, Robert H

2017-01-01

The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

PubMed

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Regulatory single nucleotide polymorphisms (rSNPs) at the promoters 1A and 1B of the human APC gene.

PubMed

Matveeva, Marina Yu; Kashina, Elena V; Reshetnikov, Vasily V; Bryzgalov, Leonid O; Antontseva, Elena V; Bondar, Natalia P; Merkulova, Tatiana I

2016-12-22

Germline mutations in the coding sequence of the tumour suppressor APC gene give rise to familial adenomatous polyposis (which leads to colorectal cancer) and are associated with many other oncopathologies. The loss of APC function because of deletion of putative promoter 1A or 1B also results in the development of colorectal cancer. Since the regions of promoters 1A and 1B contain many single nucleotide polymorphisms (SNPs), the aim of this study was to perform functional analysis of some of these SNPs by means of an electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay. First, it was shown that both putative promoters of APC (1A and 1B) drive transcription in an in vitro reporter experiment. From eleven randomly selected SNPs of promoter 1A and four SNPs of promoter 1B, nine and two respectively showed differential patterns of binding of nuclear proteins to oligonucleotide probes corresponding to alternative alleles. The luciferase reporter assay showed that among the six SNPs tested, the rs75612255 C allele and rs113017087 C allele in promoter 1A as well as the rs138386816 T allele and rs115658307 T allele in promoter 1B significantly increased luciferase activity in the human erythromyeloblastoid leukaemia cell line K562. In human colorectal cancer HCT-116 cells, none of the substitutions under study had any effect, with the exception of minor allele G of rs79896135 in promoter 1B. This allele significantly decreased the luciferase reporter's activity CONCLUSION: Our results indicate that many SNPs in APC promoters 1A and 1B are functionally relevant and that allele G of rs79896135 may be associated with the predisposition to colorectal cancer.

Study of goldfish (Carassius auratus) growth hormone structure-function relationship by domain swapping.

PubMed

Chan, Y H; Cheng, C H K; Chan, K M

2007-03-01

Using goldfish as a model, the structure-function relationship of goldfish growth hormone was studied using the strategy of homologous domain swapping. Chimeric mutants were constructed by exchanging homologous regions between goldfish growth hormone (gfGH II) and goldfish prolactin (gfPRL) with their cloned complementary DNAs. Six mutants, with their domain-swapped, were generated to have different combinations of three target regions, including the helix a, helix d and the large section in between these helices (possess the helices b, c and other random coiled regions). After expression in E. coli and refolding, these mutants were characterized by using competitive receptor binding assay (RRA) and growth hormone responding promoter activation assay. The different activity profiles of mutants in Spi 2.1 gene promoter assays from that in RRA shows that, for gfGH, receptor binding dose not confer receptor signal activations. When either helices a or d of gfGH was maintained with other helices replaced by their gfPRL counterparts, both receptor binding and hence gene activation activities are reduced. In mutants with helices b and c in gfGH maintained, containing the gfGH middle section, and helices a and d swapped with gfPRL, the had reduced RRA activities but the promoter activation activities retained. In conclusion, as in the case of human GH, the gfGH molecule possesses two functional sites: one of them is composed of discontinuous epitopes located on the target regions of this study and is for receptor binding; another site is located on the middle section of the molecule that helices a and d are not involved, and it is for activation of GH receptor and intracellular signals.

A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions

PubMed Central

Lee, Daniel Y.; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W.; Deng, Zhaoqun; Yang, Burton B.

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy. PMID:19223980

Differentially Expressed Plasma MicroRNAs and the Potential Regulatory Function of Let-7b in Chronic Thromboembolic Pulmonary Hypertension

PubMed Central

Guo, Lijuan; Yang, Yuanhua; Liu, Jie; Wang, Lei; Li, Jifeng; Wang, Ying; Liu, Yan; Gu, Song; Gan, Huili; Cai, Jun; Yuan, Jason X.-J.; Wang, Jun; Wang, Chen

2014-01-01

Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive disease characterized by misguided thrombolysis and remodeling of pulmonary arteries. MicroRNAs are small non-coding RNAs involved in multiple cell processes and functions. During CTEPH, circulating microRNA profile endued with characteristics of diseased cells could be identified as a biomarker, and might help in recognition of pathogenesis. Thus, in this study, we compared the differentially expressed microRNAs in plasma of CTEPH patients and healthy controls and investigated their potential functions. Microarray was used to identify microRNA expression profile and qRT-PCR for validation. The targets of differentially expressed microRNAs were identified in silico, and the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway database were used for functional investigation of target gene profile. Targets of let-7b were validated by fluorescence reporter assay. Protein expression of target genes was determined by ELISA or western blotting. Cell migration was evaluated by wound healing assay. The results showed that 1) thirty five microRNAs were differentially expressed in CTEPH patients, among which, a signature of 17 microRNAs, which was shown to be related to the disease pathogenesis by in silico analysis, gave diagnostic efficacy of both sensitivity and specificity >0.9. 2) Let-7b, one of the down-regulated anti-oncogenic microRNAs in the signature, was validated to decrease to about 0.25 fold in CTEPH patients. 3) ET-1 and TGFBR1 were direct targets of let-7b. Altering let-7b level influenced ET-1 and TGFBR1 expression in pulmonary arterial endothelial cells (PAECs) as well as the migration of PAECs and pulmonary arterial smooth muscle cells (PASMCs). These results suggested that CTEPH patients had aberrant microRNA signature which might provide some clue for pathogenesis study and biomarker screening. Reduced let-7b might be involved in the pathogenesis of CTEPH by affecting ET-1 expression and the function of PAECs and PASMCs. PMID:24978044

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

PubMed Central

Hiller, Ekkehard; Istel, Fabian; Tscherner, Michael; Brunke, Sascha; Ames, Lauren; Firon, Arnaud; Green, Brian; Cabral, Vitor; Marcet-Houben, Marina; Jacobsen, Ilse D.; Quintin, Jessica; Seider, Katja; Frohner, Ingrid; Glaser, Walter; Jungwirth, Helmut; Bachellier-Bassi, Sophie; Chauvel, Murielle; Zeidler, Ute; Ferrandon, Dominique; Gabaldón, Toni; Hube, Bernhard; d'Enfert, Christophe; Rupp, Steffen; Cormack, Brendan; Haynes, Ken; Kuchler, Karl

2014-01-01

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes. PMID:24945925

The rec A operon: a novel stress response gene cluster in Bacteroides fragilis

PubMed Central

Nicholson, Samantha A; Smalley, Darren; Smith, C. Jeffrey; Abratt, Valerie R

2014-01-01

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections. PMID:24703997

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells

PubMed Central

Peters, Derek T.; Henderson, Christopher A.; Warren, Curtis R.; Friesen, Max; Xia, Fang; Becker, Caroline E.; Musunuru, Kiran; Cowan, Chad A.

2016-01-01

ABSTRACT Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. PMID:27143754

Identification and Functional Analysis of Healing Regulators in Drosophila

PubMed Central

Álvarez-Fernández, Carmen; Tamirisa, Srividya; Prada, Federico; Chernomoretz, Ariel; Podhajcer, Osvaldo; Blanco, Enrique; Martín-Blanco, Enrique

2015-01-01

Wound healing is an essential homeostatic mechanism that maintains the epithelial barrier integrity after tissue damage. Although we know the overall steps in wound healing, many of the underlying molecular mechanisms remain unclear. Genetically amenable systems, such as wound healing in Drosophila imaginal discs, do not model all aspects of the repair process. However, they do allow the less understood aspects of the healing response to be explored, e.g., which signal(s) are responsible for initiating tissue remodeling? How is sealing of the epithelia achieved? Or, what inhibitory cues cancel the healing machinery upon completion? Answering these and other questions first requires the identification and functional analysis of wound specific genes. A variety of different microarray analyses of murine and humans have identified characteristic profiles of gene expression at the wound site, however, very few functional studies in healing regulation have been carried out. We developed an experimentally controlled method that is healing-permissive and that allows live imaging and biochemical analysis of cultured imaginal discs. We performed comparative genome-wide profiling between Drosophila imaginal cells actively involved in healing versus their non-engaged siblings. Sets of potential wound-specific genes were subsequently identified. Importantly, besides identifying and categorizing new genes, we functionally tested many of their gene products by genetic interference and overexpression in healing assays. This non-saturated analysis defines a relevant set of genes whose changes in expression level are functionally significant for proper tissue repair. Amongst these we identified the TCP1 chaperonin complex as a key regulator of the actin cytoskeleton essential for the wound healing response. There is promise that our newly identified wound-healing genes will guide future work in the more complex mammalian wound healing response. PMID:25647511

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

PubMed

Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

2008-01-01

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.

Transrepressive Function of TLX Requires the Histone Demethylase LSD1 ▿ †

PubMed Central

Yokoyama, Atsushi; Takezawa, Shinichiro; Schüle, Roland; Kitagawa, Hirochika; Kato, Shigeaki

2008-01-01

TLX is an orphan nuclear receptor (also called NR2E1) that regulates the expression of target genes by functioning as a constitutive transrepressor. The physiological significance of TLX in the cytodifferentiation of neural cells in the brain is known. However, the corepressors supporting the transrepressive function of TLX have yet to be identified. In this report, Y79 retinoblastoma cells were subjected to biochemical techniques to purify proteins that interact with TLX, and we identified LSD1 (also called KDM1), which appears to form a complex with CoREST and histone deacetylase 1. LSD1 interacted with TLX directly through its SWIRM and amine oxidase domains. LSD1 potentiated the transrepressive function of TLX through its histone demethylase activity as determined by a luciferase assay using a genomically integrated reporter gene. LSD1 and TLX were recruited to a TLX-binding site in the PTEN gene promoter, accompanied by the demethylation of H3K4me2 and deacetylation of H3. Knockdown of either TLX or LSD1 derepressed expression of the endogenous PTEN gene and inhibited cell proliferation of Y79 cells. Thus, the present study suggests that LSD1 is a prime corepressor for TLX. PMID:18391013

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

PubMed Central

Jones, Morgan; Osawa, Gail; Regal, Joshua A.; Weinberg, Daniel N.; Taggart, James; Kocak, Hande; Friedman, Ann; Ferguson, David O.; Keegan, Catherine E.; Maillard, Ivan

2013-01-01

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors. PMID:24316971

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

PubMed

Wang, Nan; Zhang, Yeting; Gedvilaite, Erika; Loh, Jui Wan; Lin, Timothy; Liu, Xiuping; Liu, Chang-Gong; Kumar, Dibyendu; Donnelly, Robert; Raymond, Kimiyo; Schuchman, Edward H; Sleat, David E; Lobel, Peter; Xing, Jinchuan

2017-11-01

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect. © 2017 Wiley Periodicals, Inc.

Development of Multilocus Sequence Typing (MLST) for Mycoplasma synoviae.

PubMed

El-Gazzar, Mohamed; Ghanem, Mostafa; McDonald, Kristina; Ferguson-Noel, Naola; Raviv, Ziv; Slemons, Richard D

2017-03-01

Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.

Translating tumor biology into personalized treatment planning: analytical performance characteristics of the Oncotype DX® Colon Cancer Assay

PubMed Central

2010-01-01

Background The Oncotype DX® Colon Cancer Assay is a new diagnostic test for determining the likelihood of recurrence in stage II colon cancer patients after surgical resection using fixed paraffin embedded (FPE) primary colon tumor tissue. Like the Oncotype DX Breast Cancer Assay, this is a high complexity, multi-analyte, reverse transcription (RT) polymerase chain reaction (PCR) assay that measures the expression levels of specific cancer-related genes. By capturing the biology underlying each patient's tumor, the Oncotype DX Colon Cancer Assay provides a Recurrence Score (RS) that reflects an individualized risk of disease recurrence. Here we describe its analytical performance using pre-determined performance criteria, which is a critical component of molecular diagnostic test validation. Results All analytical measurements met pre-specified performance criteria. PCR amplification efficiency for all 12 assays was high, ranging from 96% to 107%, while linearity was demonstrated over an 11 log2 concentration range for all assays. Based on estimated components of variance for FPE RNA pools, analytical reproducibility and precision demonstrated low SDs for individual genes (0.16 to 0.32 CTs), gene groups (≤0.05 normalized/aggregate CTs) and RS (≤1.38 RS units). Conclusions Analytical performance characteristics shown here for both individual genes and gene groups in the Oncotype DX Colon Cancer Assay demonstrate consistent translation of specific biology of individual tumors into clinically useful diagnostic information. The results of these studies illustrate how the analytical capability of the Oncotype DX Colon Cancer Assay has enabled clinical validation of a test to determine individualized recurrence risk after colon cancer surgery. PMID:21176237

A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf-Hirschhorn syndrome.

PubMed

McQuibban, Angus G; Joza, Nicholas; Megighian, Aram; Scorzeto, Michele; Zanini, Damiano; Reipert, Siegfried; Richter, Constance; Schweyen, Rudolf J; Nowikovsky, Karin

2010-03-15

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.

Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces cerevisiae

PubMed Central

McNabb, David S.; Reed, Robin; Marciniak, Robert A.

2005-01-01

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements. PMID:16151247

Human Cells Display Reduced Apoptotic Function Relative to Chimpanzee Cells

PubMed Central

McDonald, John F.

2012-01-01

Previously published gene expression analyses suggested that apoptotic function may be reduced in humans relative to chimpanzees and led to the hypothesis that this difference may contribute to the relatively larger size of the human brain and the increased propensity of humans to develop cancer. In this study, we sought to further test the hypothesis that humans maintain a reduced apoptotic function relative to chimpanzees by conducting a series of apoptotic function assays on human, chimpanzee and macaque primary fibroblastic cells. Human cells consistently displayed significantly reduced apoptotic function relative to the chimpanzee and macaque cells. These results are consistent with earlier findings indicating that apoptotic function is reduced in humans relative to chimpanzees. PMID:23029431

Biomarkers of adult and developmental neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slikker, William; Bowyer, John F.

2005-08-07

Neurotoxicity may be defined as any adverse effect on the structure or function of the central and/or peripheral nervous system by a biological, chemical, or physical agent. A multidisciplinary approach is necessary to assess adult and developmental neurotoxicity due to the complex and diverse functions of the nervous system. The overall strategy for understanding developmental neurotoxicity is based on two assumptions: (1) significant differences in the adult versus the developing nervous system susceptibility to neurotoxicity exist and they are often developmental stage dependent; (2) a multidisciplinary approach using neurobiological, including gene expression assays, neurophysiological, neuropathological, and behavioral function is necessarymore » for a precise assessment of neurotoxicity. Application of genomic approaches to developmental studies must use the same criteria for evaluating microarray studies as those in adults including consideration of reproducibility, statistical analysis, homogenous cell populations, and confirmation with non-array methods. A study using amphetamine to induce neurotoxicity supports the following: (1) gene expression data can help define neurotoxic mechanism(s) (2) gene expression changes can be useful biomarkers of effect, and (3) the site-selective nature of gene expression in the nervous system may mandate assessment of selective cell populations.« less

OncoBinder facilitates interpretation of proteomic interaction data by capturing coactivation pairs in cancer.

PubMed

Van Coillie, Samya; Liang, Lunxi; Zhang, Yao; Wang, Huanbin; Fang, Jing-Yuan; Xu, Jie

2016-04-05

High-throughput methods such as co-immunoprecipitationmass spectrometry (coIP-MS) and yeast 2 hybridization (Y2H) have suggested a broad range of unannotated protein-protein interactions (PPIs), and interpretation of these PPIs remains a challenging task. The advancements in cancer genomic researches allow for the inference of "coactivation pairs" in cancer, which may facilitate the identification of PPIs involved in cancer. Here we present OncoBinder as a tool for the assessment of proteomic interaction data based on the functional synergy of oncoproteins in cancer. This decision tree-based method combines gene mutation, copy number and mRNA expression information to infer the functional status of protein-coding genes. We applied OncoBinder to evaluate the potential binders of EGFR and ERK2 proteins based on the gastric cancer dataset of The Cancer Genome Atlas (TCGA). As a result, OncoBinder identified high confidence interactions (annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) or validated by low-throughput assays) more efficiently than co-expression based method. Taken together, our results suggest that evaluation of gene functional synergy in cancer may facilitate the interpretation of proteomic interaction data. The OncoBinder toolbox for Matlab is freely accessible online.

Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene.

PubMed

Tu, N; Chen, H; Winnikes, U; Reinert, I; Marmann, G; Pirke, K M; Lentes, K U

1999-11-19

As a member of the uncoupling protein family, UCP2 is ubiquitously expressed in rodents and humans, implicating a major role in thermogenesis. To analyze promoter function and regulatory motifs involved in the transcriptional regulation of UCP2 gene expression, 3.3 kb of 5'-flanking region of the human UCP2 (hUCP2) gene have been cloned. Sequence analysis showed that the promoter region of hUCP2 lacks a classical TATA or CAAT box, however, appeared GC-rich resulting in the presence of several Sp-1 motifs and Ap-1/-2 binding sites near the transcription initiation site. Functional characterization of human UCP2 promoter-CAT fusion constructs in transient expression assays showed that minimal promoter activity was observed within 65 bp upstream of the transcriptional start site (+1). 75 bp further upstream (from nt -141 to -66) a strong cis-acting regulatory element (or enhancer) was identified, which significantly enhanced basal promoter activity. The regulation of human UCP2 gene expression involves complex interactions among positive and negative regulatory elements distributed over a minimum of 3.3 kb of the promoter region. Copyright 1999 Academic Press.

Development of a Recombinant Multifunctional Biomacromolecule for Targeted Gene Transfer to Prostate Cancer Cells.

PubMed

Hatefi, Arash; Karjoo, Zahra; Nomani, Alireza

2017-09-11

The objective of this study was to genetically engineer a fully functional single chain fusion peptide composed of motifs from diverse biological and synthetic origins that can perform multiple tasks including DNA condensation, cell targeting, cell transfection, particle shielding from immune system and effective gene transfer to prostate tumors. To achieve the objective, a single chain biomacromolecule (vector) consisted of four repeatative units of histone H2A peptide, fusogenic peptide GALA, short elastin-like peptide, and PC-3 cell targeting peptide was designed. To examine the functionality of each motif in the vector sequence, it was characterized in terms of size and zeta potential by Zetasizer, PC-3 cell targeting and transfection by flowcytometry, IgG induction by immunogenicity assay, and PC-3 tumor transfection by quantitative live animal imaging. Overall, the results of this study showed the possibility of using genetic engineering techniques to program various functionalities into one single chain vector and create a multifunctional nonimmunogenic biomacromolecule for targeted gene transfer to prostate cancer cells. This proof-of-concept study is a significant step forward toward creating a library of vectors for targeted gene transfer to any cancer cell type at both in vitro and in vivo levels.

Machine Learning Analysis Identifies Drosophila Grunge/Atrophin as an Important Learning and Memory Gene Required for Memory Retention and Social Learning.

PubMed

Kacsoh, Balint Z; Greene, Casey S; Bosco, Giovanni

2017-11-06

High-throughput experiments are becoming increasingly common, and scientists must balance hypothesis-driven experiments with genome-wide data acquisition. We sought to predict novel genes involved in Drosophila learning and long-term memory from existing public high-throughput data. We performed an analysis using PILGRM, which analyzes public gene expression compendia using machine learning. We evaluated the top prediction alongside genes involved in learning and memory in IMP, an interface for functional relationship networks. We identified Grunge/Atrophin ( Gug/Atro ), a transcriptional repressor, histone deacetylase, as our top candidate. We find, through multiple, distinct assays, that Gug has an active role as a modulator of memory retention in the fly and its function is required in the adult mushroom body. Depletion of Gug specifically in neurons of the adult mushroom body, after cell division and neuronal development is complete, suggests that Gug function is important for memory retention through regulation of neuronal activity, and not by altering neurodevelopment. Our study provides a previously uncharacterized role for Gug as a possible regulator of neuronal plasticity at the interface of memory retention and memory extinction. Copyright © 2017 Kacsoh et al.

Functional autonomy of distant-acting human enhancers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Visel, Axel; Akiyama, Jennifer A.; Shoukry, Malak

2009-02-19

Many human genes are associated with dispersed arrays of transcriptional enhancers that regulate their expression in time and space. Studies in invertebrate model systems have suggested that these elements function as discrete and independent regulatory units, but the in vivo combinatorial properties of vertebrate enhancers remain poorly understood. To explore the modularity and regulatory autonomy of human developmental enhancers, we experimentally concatenated up to four enhancers from different genes and used a transgenic mouse assay to compare the in vivo activity of these compound elements with that of the single modules. In all of the six different combinations of elementsmore » tested, the reporter gene activity patterns were additive without signs of interference between the individual modules, indicating that regulatory specificity was maintained despite the presence of closely-positioned heterologous enhancers. Even in cases where two elements drove expression in close anatomical proximity, such as within neighboring subregions of the developing limb bud, the compound patterns did not show signs of cross-inhibition between individual elements or novel expression sites. These data indicate that human developmental enhancers are highly modular and functionally autonomous and suggest that genomic enhancer shuffling may have contributed to the evolution of complex gene expression patterns in vertebrates« less

Multiplex Ligation-Dependent Probe Amplification Analysis on Capillary Electrophoresis Instruments for a Rapid Gene Copy Number Study

PubMed Central

Jankowski, Stéphane; Currie-Fraser, Erica; Xu, Licen; Coffa, Jordy

2008-01-01

Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies. PMID:19137113

Quality Control Test for Sequence-Phenotype Assignments

PubMed Central

Ortiz, Maria Teresa Lara; Rosario, Pablo Benjamín Leon; Luna-Nevarez, Pablo; Gamez, Alba Savin; Martínez-del Campo, Ana; Del Rio, Gabriel

2015-01-01

Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas. PMID:25700273

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

PubMed

Gonzalez, S M; Ferland, L H; Robert, B; Abdelhay, E

1998-06-01

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

PubMed Central

Dodds, Peter N.; Lawrence, Gregory J.; Catanzariti, Ann-Maree; Teh, Trazel; Wang, Ching-I. A.; Ayliffe, Michael A.; Kobe, Bostjan; Ellis, Jeffrey G.

2006-01-01

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes. PMID:16731621

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer

PubMed Central

Liu, Yali; Liu, Li; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Chen, Mingwei; Li, Jianfa; Cai, Zhiming; Huang, Weiren; Zhang, Yong

2016-01-01

CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer–binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits. PMID:27231846

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer.

PubMed

Xu, Wen; Liu, Yuchen; Liu, Yali; Liu, Li; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Chen, Mingwei; Li, Jianfa; Cai, Zhiming; Huang, Weiren; Zhang, Yong

2016-08-23

CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer-binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits.

Performance of a commercial assay for the diagnosis of influenza A (H1N1) infection in comparison to the Centers for Disease Control and Prevention protocol of real time RT-PCR.

PubMed

Barbás, María G; Gallego, Sandra V; Castro, Gonzalo M; Baumeister, Elsa; Kademian, Silvia; De Leon, Juan; Cudolá, Analía

2012-01-01

At the time of influenza A (H1N1) emergency, the WHO responded with remarkable speed by releasing guidelines and a protocol for a real-time RT-PCR assay (rRT-PCR). The aim of the present study was to evalúate the performance of the "Real Time Ready Influenza A/H1N1 Detection Set" (June 2009)-Roche kit in comparison to the CDC reference rRT-PCR protocol. The overall sensitivity of the Roche assay for detection of the Inf A gene in the presence or absence of the H1 gene was 74.5 %. The sensitivity for detecting samples that were only positive for the Inf A gene (absence of the H1 gene) was 53.3 % whereas the sensitivity for H1N1-positive samples (presence of the Inf A gene and any other swine gene) was 76.4 %. The specificity of the assay was 97.1 %. A new version of the kit (November 2009) is now available, and a recent evaluation of its performance showed good sensitivity to detect pandemic H1N1 compared to other molecular assays.

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina.

PubMed

Irie, Shoichi; Sanuki, Rikako; Muranishi, Yuki; Kato, Kimiko; Chaya, Taro; Furukawa, Takahisa

2015-08-01

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Protooncogene TCL1b functions as an Akt kinase co-activator that exhibits oncogenic potency in vivo

PubMed Central

Hashimoto, M; Suizu, F; Tokuyama, W; Noguchi, H; Hirata, N; Matsuda-Lennikov, M; Edamura, T; Masuzawa, M; Gotoh, N; Tanaka, S; Noguchi, M

2013-01-01

Protooncogene T-cell leukemia 1 (TCL1), which is implicated in human T-cell prolymphocytic leukemia (T-PLL), interacts with Akt and enhances its kinase activity, functioning as an Akt kinase co-activator. Two major isoforms of TCL1 Protooncogenes (TCL1 and TCL1b) are present adjacent to each other on human chromosome 14q.32. In human T-PLL, both TCL1 and TCL1b are activated by chromosomal translocation. Moreover, TCL1b-transgenic mice have never been created. Therefore, it remains unclear whether TCL1b itself, independent of TCL1, exhibits oncogenicity. In co-immunoprecipitation assays, both ectopic and endogenous TCL1b interacted with Akt. In in vitro Akt kinase assays, TCL1b enhanced Akt kinase activity in dose- and time-dependent manners. Bioinformatics approaches utilizing multiregression analysis, cluster analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping, Venn diagrams and Gene Ontology (GO) demonstrated that TCL1b showed highly homologous gene-induction signatures similar to Myr-Akt or TCL1. TCL1b exhibited oncogenicity in in vitro colony-transformation assay. Further, two independent lines of β-actin promoter-driven TCL1b-transgenic mice developed angiosarcoma on the intestinal tract. Angiosarcoma is a rare form of cancer in humans with poor prognosis. Using immunohistochemistry, 11 out of 13 human angiosarcoma samples were positively stained with both anti-TCL1b and anti-phospho-Akt antibodies. Consistently, in various cancer tissues, 69 out of 146 samples were positively stained with anti-TCL1b, out of which 46 were positively stained with anti-phospho-Akt antibodies. Moreover, TCL1b structure-based inhibitor ‘TCL1b-Akt-in' inhibited Akt kinase activity in in vitro kinase assays and PDGF (platelet-derived growth factor)-induced Akt kinase activities—in turn, ‘TCL1b-Akt-in' inhibited cellular proliferation of sarcoma. The current study disclosed TCL1b bears oncogenicity and hence serves as a novel therapeutic target for human neoplastic diseases. PMID:24042734

Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping

PubMed Central

Liu, Yanhong; Yan, Xianghe; DebRoy, Chitrita; Fratamico, Pina M.; Needleman, David S.; Li, Robert W.; Wang, Wei; Losada, Liliana; Brinkac, Lauren; Radune, Diana; Toro, Magaly; Hegde, Narasimha; Meng, Jianghong

2015-01-01

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources. PMID:25664526

Technological advances in diagnostic testing for von Willebrand disease: new approaches and challenges.

PubMed

Hayward, C P M; Moffat, K A; Graf, L

2014-06-01

Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays. © 2014 John Wiley & Sons Ltd.

Identification of novel biomass-degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation.

PubMed

Piao, Hailan; Froula, Jeff; Du, Changbin; Kim, Tae-Wan; Hawley, Erik R; Bauer, Stefan; Wang, Zhong; Ivanova, Nathalia; Clark, Douglas S; Klenk, Hans-Peter; Hess, Matthias

2014-08-01

Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this "genomic dark matter." Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as "genomic dark matter" and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications. © 2014 Wiley Periodicals, Inc.

Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications.

PubMed

Hughes, Stephen R; Butt, Tauseef R; Bartolett, Scott; Riedmuller, Steven B; Farrelly, Philip

2011-08-01

The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries. Published by Elsevier Inc.

Fragile X mental retardation protein has a unique, evolutionarily conserved neuronal function not shared with FXR1P or FXR2P

PubMed Central

Coffee, R. Lane; Tessier, Charles R.; Woodruff, Elvin A.; Broadie, Kendal

2010-01-01

SUMMARY Fragile X syndrome (FXS), resulting solely from the loss of function of the human fragile X mental retardation 1 (hFMR1) gene, is the most common heritable cause of mental retardation and autism disorders, with syndromic defects also in non-neuronal tissues. In addition, the human genome encodes two closely related hFMR1 paralogs: hFXR1 and hFXR2. The Drosophila genome, by contrast, encodes a single dFMR1 gene with close sequence homology to all three human genes. Drosophila that lack the dFMR1 gene (dfmr1 null mutants) recapitulate FXS-associated molecular, cellular and behavioral phenotypes, suggesting that FMR1 function has been conserved, albeit with specific functions possibly sub-served by the expanded human gene family. To test evolutionary conservation, we used tissue-targeted transgenic expression of all three human genes in the Drosophila disease model to investigate function at (1) molecular, (2) neuronal and (3) non-neuronal levels. In neurons, dfmr1 null mutants exhibit elevated protein levels that alter the central brain and neuromuscular junction (NMJ) synaptic architecture, including an increase in synapse area, branching and bouton numbers. Importantly, hFMR1 can, comparably to dFMR1, fully rescue both the molecular and cellular defects in neurons, whereas hFXR1 and hFXR2 provide absolutely no rescue. For non-neuronal requirements, we assayed male fecundity and testes function. dfmr1 null mutants are effectively sterile owing to disruption of the 9+2 microtubule organization in the sperm tail. Importantly, all three human genes fully and equally rescue mutant fecundity and spermatogenesis defects. These results indicate that FMR1 gene function is evolutionarily conserved in neural mechanisms and cannot be compensated by either FXR1 or FXR2, but that all three proteins can substitute for each other in non-neuronal requirements. We conclude that FMR1 has a neural-specific function that is distinct from its paralogs, and that the unique FMR1 function is responsible for regulating neuronal protein expression and synaptic connectivity. PMID:20442204

Analysis of the APETALA3- and PISTILLATA-like genes in Hedyosmum orientale (Chloranthaceae) provides insight into the evolution of the floral homeotic B-function in angiosperms

PubMed Central

Liu, Shujun; Sun, Yonghua; Du, Xiaoqiu; Xu, Qijiang; Wu, Feng; Meng, Zheng

2013-01-01

Background and Aims According to the floral ABC model, B-function genes appear to play a key role in the origin and diversification of the perianth during the evolution of angiosperms. The basal angiosperm Hedyosmum orientale (Chloranthaceae) has unisexual inflorescences associated with a seemingly primitive reproductive morphology and a reduced perianth structure in female flowers. The aim of this study was to investigate the nature of the perianth and the evolutionary state of the B-function programme in this species. Methods A series of experiments were conducted to characterize B-gene homologues isolated from H. orientale, including scanning electron microscopy to observe the development of floral organs, phylogenetic analysis to reconstruct gene evolutionary history, reverse transcription–PCR, quantitative real-time PCR and in situ hybridization to identify gene expression patterns, the yeast two-hybrid assay to explore protein dimerization affinities, and transgenic analyses in Arabidopsis thaliana to determine activities of the encoded proteins. Key Results The expression of HoAP3 genes was restricted to stamens, whereas HoPI genes were broadly expressed in all floral organs. HoAP3 was able to partially restore the stamen but not petal identity in Arabidopsis ap3-3 mutants. In contrast, HoPI could rescue aspects of both stamen and petal development in Arabidopsis pi-1 mutants. When the complete C-terminal sequence of HoPI was deleted, however, no or weak transgenic phenotypes were observed and homodimerization capability was completely abolished. Conclusions The results suggest that Hedyosmum AP3-like genes have an ancestral function in specifying male reproductive organs, and that the activity of the encoded PI-like proteins is highly conserved between Hedyosmum and Arabidopsis. Moreover, there is evidence that the C-terminal region is important for the function of HoPI. Our findings indicate that the development of the proposed perianth in Hedyosmum does not rely on the B homeotic function. PMID:23956161

Falsely undetectable TSH in a cohort of South Asian euthyroid patients.

PubMed

Drees, Julia C; Stone, Judith A; Reamer, C Randy; Arboleda, Victoria E; Huang, Karl; Hrynkow, Jane; Greene, Dina N; Petrie, Matthew S; Hoke, Carolyn; Lorey, Thomas S; Dlott, Richard S

2014-04-01

An index case of a clinically euthyroid woman of South Asian descent was identified with discordant TSH results: undetectable TSH on our routine assay and normal TSH on an alternate assay. Low TSH concentrations due to functionally compromising TSH mutations have been reported. Here we describe a new phenomenon of functional TSH that is undetectable by 4 widely used US Food and Drug Administration (FDA)-approved TSH immunoassays marketed by a single vendor. The purpose of this study was to identify additional cases and investigate the cause of the falsely undetectable TSH. All samples with TSH results of <0.01 μIU/mL were retested with a second TSH assay. Discordant samples were evaluated on up to 8 FDA-approved TSH immunoassays and the TSHβ gene was sequenced. Retrospectively, thyroid function tests, diagnoses, and medications from 1.6 million individuals were analyzed. Out of approximately 2 million individuals, we have identified a cohort of 20 hypothyroid and euthyroid patients of shared ethnicity with falsely undetectable TSH (<0.01 μIU/mL) in 4 of 8 commercially available TSH assays. Half of these individuals were initially treated based on repeated falsely undetectable TSH values (7 euthyroid patients were treated with methimazole and 2 hypothyroid patients had doses of levothyroxine decreased). In all cases, a retrospective chart review revealed that clinical assessments and free T4 and total T3 results were inconsistent with the undetectable TSH results. Specific antibodies failing to detect TSH in these cases were identified in the 4 affected assays. A novel TSHβ point mutation was identified. Our data suggest that these individuals have a previously unrecognized, functionally normal, TSH variant to which some monoclonal antibodies fail to bind. To assure appropriate patient management, clinicians and laboratorians need to be aware that certain TSH variants may be undetectable in some hyperselective TSH assays.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

PubMed

Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

2016-07-01

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea. © 2016 The Author(s).

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

USGS Publications Warehouse

Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

2016-01-01

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.E.; Beck, T.W.; Brennscheidt, U.

1994-03-01

The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. The authors have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776more » nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors. 65 refs., 7 figs., 1 tab.« less

Multiple Neuropeptide-Coding Genes Involved in Planarian Pharynx Extension.

PubMed

Shimoyama, Seira; Inoue, Takeshi; Kashima, Makoto; Agata, Kiyokazu

2016-06-01

Planarian feeding behavior involves three steps: moving toward food, extending the pharynx from their planarian's ventral side after arriving at the food, and ingesting the food through the pharynx. Although pharynx extension is a remarkable behavior, it remains unknown what neuronal cell types are involved in its regulation. To identify neurons involved in regulating pharynx extension, we quantitatively analyzed pharynx extension and sought to identify these neurons by RNA interference (RNAi) and in situ hybridization. This assay, when performed using planarians with amputation of various body parts, clearly showed that the head portion is indispensable for inducing pharynx extension. We thus tested the effects of knockdown of brain neurons such as serotonergic, GABAergic, and dopaminergic neurons by RNAi, but did not observe any effects on pharynx extension behavior. However, animals with RNAi of the Prohormone Convertase 2 (PC2, a neuropeptide processing enzyme) gene did not perform the pharynx extension behavior, suggesting the possible involvement of neuropeptide(s in the regulation of pharynx extension. We screened 24 neuropeptide-coding genes, analyzed their functions by RNAi using the pharynx extension assay system, and identified at least five neuropeptide genes involved in pharynx extension. These was expressed in different cells or neurons, and some of them were expressed in the brain, suggesting complex regulation of planarian feeding behavior by the nervous system.

Gene transcription patterns in response to low level petroleum contaminants in Mytilus trossulus from field sites and harbors in southcentral Alaska

NASA Astrophysics Data System (ADS)

Bowen, Lizabeth; Miles, A. Keith; Ballachey, Brenda; Waters, Shannon; Bodkin, James; Lindeberg, Mandy; Esler, Daniel

2018-01-01

The 1989 Exxon Valdez oil spill damaged a wide range of natural resources, including intertidal communities, and post-spill studies demonstrated acute and chronic exposure and injury to an array of species. Standard toxicological methods to evaluate petroleum contaminants have assessed tissue burdens, with fewer assays providing indicators of health or physiology, particularly when contaminant levels are low and chronic. Marine mussels are a ubiquitous and crucial component of the nearshore environment, and new genomic technologies exist to quantify molecular responses of individual mussels to stimuli, including exposure to polycyclic aromatic hydrocarbons (PAHs). We used gene-based assays of exposure and physiological function to assess chronic oil contamination using the Pacific blue mussel, Mytilus trossulus. We developed a diagnostic gene transcription panel to investigate exposure to PAHs and other contaminants and its effects on mussel physiology and health. During 2012-2015, we analyzed mussels from five field sites in western Prince William Sound, Alaska, with varying oil histories from the 1989 Exxon Valdez oil spill, and from three boat harbors in the area. Gene transcription patterns of mussels from harbors were consistent with elevated exposure to PAHs or other contaminants, whereas transcription patterns of mussels sampled from shorelines in areas affected by the oil spill indicated no PAH exposure.

Functional Analysis of the Trichoderma harzianum nox1 Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum▿†

PubMed Central

Montero-Barrientos, M.; Hermosa, R.; Cardoza, R. E.; Gutiérrez, S.; Monte, E.

2011-01-01

The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791

Buffer-dependent regulation of aquaporin-1 expression and function in human peritoneal mesothelial cells.

PubMed

Zhai, Yihui; Bloch, Jacek; Hömme, Meike; Schaefer, Julia; Hackert, Thilo; Philippin, Bärbel; Schwenger, Vedat; Schaefer, Franz; Schmitt, Claus P

2012-07-01

Biocompatible peritoneal dialysis fluids (PDF) are buffered with lactate and/or bicarbonate. We hypothesized that the reduced toxicity of the biocompatible solutions might unmask specific effects of the buffer type on mesothelial cell functions. Human peritoneal mesothelial cells (HPMC) were incubated with bicarbonate (B-)PDF or lactate-buffered (L-)PDF followed by messenger RNA (mRNA) and protein analysis. Gene silencing was achieved using small interfering RNA (siRNA), functional studies using Transwell culture systems, and monolayer wound-healing assays. Incubation with B-PDF increased HPMC migration in the Transwell and monolayer wound-healing assay to 245 ± 99 and 137 ± 11% compared with L-PDF. Gene silencing showed this effect to be entirely dependent on the expression of aquaporin-1 (AQP-1) and independent of AQP-3. Exposure of HPMC to B-PDF increased AQP-1 mRNA and protein abundance to 209  ± 80 and 197  ±  60% of medium control; the effect was pH dependent. L-PDF reduced AQP-1 mRNA. Addition of bicarbonate to L-PDF increased AQP-1 abundance by threefold; mRNA half-life remained unchanged. Immunocytochemistry confirmed opposite changes of AQP-1 cell-membrane abundance with B-PDF and L-PDF. Peritoneal mesothelial AQP-1 abundance and migration capacity is regulated by pH and buffer agents used in PD solutions. In vivo studies are required to delineate the impact with respect to long-term peritoneal membrane integrity and function.

Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure.

PubMed

Wang, Yujia; Wu, Zenghui; Thorin, Eric; Tremblay, Johanne; Lavoie, Julie L; Luo, Hongyu; Peng, Junzheng; Qi, Shijie; Wu, Tao; Chen, Fei; Shen, Jianzhong; Hu, Shenjiang; Wu, Jiangping

2016-04-01

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca(2+)flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals. Copyright © 2016 the American Physiological Society.

Inhibitory effect of cyanide on wastewater nitrification ...

EPA Pesticide Factsheets

The effect of CN- (CN-) on nitrification was examined with samples from nitrifying wastewater enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT-qPCR to quantify the transcripts of functional genes involved in nitrification. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to cyanide for a period of 12 h. The concentrations of CN- used in the batch assays were 0.03, 0.06, 0.1 and 1.0 mg/L. There was considerable decrease in SOUR with increasing dosages of CN-. A decrease of more than 50% in nitrification activity was observed at 0.1 mg/L CN-. Based on the RT-qPCR data, there was notable reduction in the transcript levels of amoA and hao for increasing CN- dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. The inhibitory effect of cyanide may be attributed to the affinity of cyanide to bind ferric heme proteins, which disrupt protein structure and function. The correspondence between the relative expression of functional genes and SOUR shown in this study demonstrates the efficacy of RNA based function-specific assays for better understanding of the effect of toxic compounds on nitrification activity in wastewater. Nitrification is the first step of nitrogen removal is wastewater, and it is susceptible to inhibition by many industrial chemical. We looked at

Molecular cloning and functional analysis of a UV-B photoreceptor gene, MdUVR8 (UV Resistance Locus 8), from apple.

PubMed

Zhao, Cheng; Mao, Ke; You, Chun-Xiang; Zhao, Xian-Yan; Wang, Shu-Hui; Li, Yuan-Yuan; Hao, Yu-Jin

2016-06-01

UVR8 (UV Resistance Locus 8) is an ultraviolet-B (UV-B; 280-315nm) light receptor that is involved in regulating many aspects of plant growth and development. UV-B irradiation can increase the development of flower and fruit coloration in many fruit trees, such as grape, pear and apple. Previous investigations of the structure and functions of UVR8 in plants have largely focused on Arabidopsis. Here, we isolated the UVR8 gene from apple (Malus domestica) and analyzed its function in transgenic Arabidopsis. Genomic and protein sequence analysis showed that MdUVR8 shares high similarity with the AtUVR8 protein from Arabidopsis, including the conserved seven-bladed β-propeller, the C27 region, the 3 "GWRHT" motifs and crucial amino-acid residues (14 Trps, 2 Args). A point mutation prediction and three-dimensional structural analysis of MdUVR8 indicated that it has a similar structure to AtUVR8 and that the crucial residues are also important in MdUVR8. In terms of transcript levels, MdUVR8 expression was up-regulated by UV-B light, which suggests that its expression follows a 24-h circadian rhythm. Using heterologous expression of MdUVR8 in both uvr8-1 mutant and wild-type (WT) Arabidopsis, we found that MdUVR8 regulates hypocotyl elongation and gene expression under UV-B light. These data provide functional evidence for a role of MdUVR8 in controlling photomorphogenesis under UV-B light and indicate that the function of UVR8 is conserved between Arabidopsis and apple. Furthermore, we examined the interaction between MdUVR8 and MdCOP1 (constitutive photomorphogenic1) using a yeast two-hybrid assay and a co-immunoprecipitation assay. This interaction provides a direction for investigating the regulatory mechanisms of the UV-B-light pathway in apple. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

MicroRNA-137 Negatively Regulates H2O2-Induced Cardiomyocyte Apoptosis Through CDC42

PubMed Central

Wang, Junnan; Xu, Rihao; Wu, Junduo; Li, Zhibo

2015-01-01

Background Oxidative stress, inducing cardiomyocyte apoptosis or myocardial ischemia, is the major denominator of many cardiac diseases. In this study, we intended to explore the regulatory function of microRNA-137 (miR-137) in oxidative stress-induced cardiomyocyte apoptosis. Material/Methods Cardiomyocytes were extracted from newborn C57BL/6 mice and cultured in vitro. Apoptosis was induced by H2O2, and evaluated by TUNEL assay. The effect of cardiomyocyte apoptosis on gene expression of miR-137 was evaluated by qRT-PCR. Lentivirus was used to stably down-regulate miR-137, and the subsequent effects of miR-137 down-regulation on cardiomyocyte apoptosis, its targeted gene CDC42, and caspase pathway were evaluated by TUNEL assay, dual-luciferase reporter assay, and Western blot assay, respectively. Finally, CDC42 was down-regulated by siRNA and its effect on miR-137-mediated cardiomyocyte apoptosis protection was examined. Results H2O2 induced significant apoptosis and up-regulated miR-137 in cardiomyocytes, whereas lentivirus-mediated miR-137 down-regulation protected against apoptosis. CDC42 was the direct target gene of miR-137 and proteins of CDC42, caspase-3, and caspase-9 were all regulated by miR-137 down-regulation in cardiomyocyte apoptosis. SiRNA-mediated CDC42 down-regulation reversed the protection of miR-137 down-regulation against cardiomyocyte apoptosis. Conclusions Our work demonstrated miR-137 and CDC42 are critical regulators in cardiomyocyte apoptosis. It may help to identify the molecular targets to prevent myocardial injury in human patients. PMID:26566162

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

PubMed

He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

2015-03-01

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

Thyroid hormone and COUP-TF1 regulate kallikrein-binding protein (KBP) gene expression.

PubMed

Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen; Brent, Gregory A

2011-03-01

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T(3) and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5' flanking region (-53 to -29) and nTRE2, located in the first intron (104-132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T(3). COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T(3) repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T(3). Nuclear corepressor knockdown resulted in loss of T(3) repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T(3) induction of positive thyroid hormone response elements, reverses T(3) repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T(3) and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T(3).

Thyroid Hormone and COUP-TF1 Regulate Kallikrein-Binding Protein (KBP) Gene Expression

PubMed Central

Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen

2011-01-01

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T3 and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5′ flanking region (−53 to −29) and nTRE2, located in the first intron (104–132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T3. COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T3 repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T3. Nuclear corepressor knockdown resulted in loss of T3 repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T3 induction of positive thyroid hormone response elements, reverses T3 repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T3 and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T3. PMID:21266512

Genome-wide identification of the MADS-box transcription factor family in pear (Pyrus bretschneideri) reveals evolution and functional divergence.

PubMed

Wang, Runze; Ming, Meiling; Li, Jiaming; Shi, Dongqing; Qiao, Xin; Li, Leiting; Zhang, Shaoling; Wu, Jun

2017-01-01

MADS-box transcription factors play significant roles in plant developmental processes such as floral organ conformation, flowering time, and fruit development. Pear ( Pyrus ), as the third-most crucial temperate fruit crop, has been fully sequenced. However, there is limited information about the MADS family and its functional divergence in pear. In this study, a total of 95 MADS-box genes were identified in the pear genome, and classified into two types by phylogenetic analysis. Type I MADS-box genes were divided into three subfamilies and type II genes into 14 subfamilies. Synteny analysis suggested that whole-genome duplications have played key roles in the expansion of the MADS family, followed by rearrangement events. Purifying selection was the primary force driving MADS-box gene evolution in pear, and one gene pairs presented three codon sites under positive selection. Full-scale expression information for PbrMADS genes in vegetative and reproductive organs was provided and proved by transcriptional and reverse transcription PCR analysis. Furthermore, the PbrMADS11(12) gene, together with partners PbMYB10 and PbbHLH3 was confirmed to activate the promoters of the structural genes in anthocyanin pathway of red pear through dual luciferase assay. In addition, the PbrMADS11 and PbrMADS12 were deduced involving in the regulation of anthocyanin synthesis response to light and temperature changes. These results provide a solid foundation for future functional analysis of PbrMADS genes in different biological processes, especially of pigmentation in pear.

Genome-wide identification of the MADS-box transcription factor family in pear (Pyrus bretschneideri) reveals evolution and functional divergence

PubMed Central

Li, Jiaming; Shi, Dongqing; Qiao, Xin; Li, Leiting; Zhang, Shaoling

2017-01-01

MADS-box transcription factors play significant roles in plant developmental processes such as floral organ conformation, flowering time, and fruit development. Pear (Pyrus), as the third-most crucial temperate fruit crop, has been fully sequenced. However, there is limited information about the MADS family and its functional divergence in pear. In this study, a total of 95 MADS-box genes were identified in the pear genome, and classified into two types by phylogenetic analysis. Type I MADS-box genes were divided into three subfamilies and type II genes into 14 subfamilies. Synteny analysis suggested that whole-genome duplications have played key roles in the expansion of the MADS family, followed by rearrangement events. Purifying selection was the primary force driving MADS-box gene evolution in pear, and one gene pairs presented three codon sites under positive selection. Full-scale expression information for PbrMADS genes in vegetative and reproductive organs was provided and proved by transcriptional and reverse transcription PCR analysis. Furthermore, the PbrMADS11(12) gene, together with partners PbMYB10 and PbbHLH3 was confirmed to activate the promoters of the structural genes in anthocyanin pathway of red pear through dual luciferase assay. In addition, the PbrMADS11 and PbrMADS12 were deduced involving in the regulation of anthocyanin synthesis response to light and temperature changes. These results provide a solid foundation for future functional analysis of PbrMADS genes in different biological processes, especially of pigmentation in pear. PMID:28924499

Tumourigenicity and radiation resistance of mesenchymal stem cells.

PubMed

D'Andrea, Filippo P; Horsman, Michael R; Kassem, Moustapha; Overgaard, Jens; Safwat, Akmal

2012-05-01

Cancer stem cells are believed to be more radiation resistant than differentiated tumour cells of the same origin. It is not known, however, whether normal nontransformed adult stem cells share the same radioresistance as their cancerous counterpart. Nontumourigenic (TERT4) and tumourigenic (TRET20) cell lines, from an immortalised mesenchymal stem cell line, were grown in culture prior to irradiation and gene expression analysis. Radiation resistance was measured using a clonogenic assay. Differences in gene expression between the two cell lines, both under nontreated and irradiated conditions, were assessed with microarrays (Affymetrix Human Exon 1.0 ST array). The cellular functions affected by the altered gene expressions were assessed through gene pathway mapping (Ingenuity Pathway Analysis). Based on the clonogenic assay the nontumourigenic cell line was found to be more sensitive to radiation than the tumourigenic cell line. Using the exon chips, 297 genes were found altered between untreated samples of the cell lines whereas only 16 genes responded to radiation treatment. Among the genes with altered expression between the untreated samples were PLAU, PLAUR, TIMP3, MMP1 and LOX. The pathway analysis based on the alteration between the untreated samples indicated cancer and connective tissue disorders. This study has shown possible common genetic events linking tumourigenicity and radiation response. The PLAU and PLAUR genes are involved in apoptosis evasion while the genes TIMP3, MMP1 and LOX are involved in regulation of the surrounding matrix. The first group may contribute to the difference in radiation resistance observed and the latter could be a major contributor to the tumourigenic capabilities by degrading the intercellular matrix. These results also indicate that cancer stem cells are more radiation resistant than stem cells of the same origin.

Metagenomic and functional analyses of the consequences of reduction of bacterial diversity on soil functions and bioremediation in diesel-contaminated microcosms.

PubMed

Jung, Jaejoon; Philippot, Laurent; Park, Woojun

2016-03-14

The relationship between microbial biodiversity and soil function is an important issue in ecology, yet most studies have been performed in pristine ecosystems. Here, we assess the role of microbial diversity in ecological function and remediation strategies in diesel-contaminated soils. Soil microbial diversity was manipulated using a removal by dilution approach and microbial functions were determined using both metagenomic analyses and enzymatic assays. A shift from Proteobacteria- to Actinobacteria-dominant communities was observed when species diversity was reduced. Metagenomic analysis showed that a large proportion of functional gene categories were significantly altered by the reduction in biodiversity. The abundance of genes related to the nitrogen cycle was significantly reduced in the low-diversity community, impairing denitrification. In contrast, the efficiency of diesel biodegradation was increased in the low-diversity community and was further enhanced by addition of red clay as a stimulating agent. Our results suggest that the relationship between microbial diversity and ecological function involves trade-offs among ecological processes, and should not be generalized as a positive, neutral, or negative relationship.

Metagenomic and functional analyses of the consequences of reduction of bacterial diversity on soil functions and bioremediation in diesel-contaminated microcosms

PubMed Central

Jung, Jaejoon; Philippot, Laurent; Park, Woojun

2016-01-01

The relationship between microbial biodiversity and soil function is an important issue in ecology, yet most studies have been performed in pristine ecosystems. Here, we assess the role of microbial diversity in ecological function and remediation strategies in diesel-contaminated soils. Soil microbial diversity was manipulated using a removal by dilution approach and microbial functions were determined using both metagenomic analyses and enzymatic assays. A shift from Proteobacteria- to Actinobacteria-dominant communities was observed when species diversity was reduced. Metagenomic analysis showed that a large proportion of functional gene categories were significantly altered by the reduction in biodiversity. The abundance of genes related to the nitrogen cycle was significantly reduced in the low-diversity community, impairing denitrification. In contrast, the efficiency of diesel biodegradation was increased in the low-diversity community and was further enhanced by addition of red clay as a stimulating agent. Our results suggest that the relationship between microbial diversity and ecological function involves trade-offs among ecological processes, and should not be generalized as a positive, neutral, or negative relationship. PMID:26972977

A new highly sensitive and specific real-time PCR assay targeting the malate dehydrogenase gene of Kingella kingae and application to 201 pediatric clinical specimens.

PubMed

Houmami, Nawal El; Durand, Guillaume André; Bzdrenga, Janek; Darmon, Anne; Minodier, Philippe; Seligmann, Hervé; Raoult, Didier; Fournier, Pierre-Edouard

2018-06-06

Kingella kingae is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium by culture and broad-range 16S rRNA gene polymerase chain reaction (PCR) assays from clinical specimens have proven unsatisfactory and were gradually let out for the benefit of specific real-time PCR tests targeting the groEL gene and RTX locus of K. kingae by the late 2000s. However, recent studies showed that real-time PCR (RT-PCR) assays targeting the Kingella sp. RTX locus that are currently available for the diagnosis of K. kingae infection lack of specificity because they could not distinguish between K. kingae and the recently described K. negevensis species. Furthermore, in silico analysis of the groEL gene from a large collection of 45 K. kingae strains showed that primers and probes from K. kingae groEL -based RT-PCR assays display a few mismatches with K. kingae groEL variations that may result in a decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to groEL - and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a K. kingae -specific RT-PCR assay targeting the malate dehydrogenase ( mdh ) gene was developed for predicting no mismatch against 18 variants of the K. kingae mdh gene from 20 distinct sequences types of K. kingae This novel K. kingae -specific RT-PCR assay demonstrated a high specificity and sensitivity and was successfully used to diagnose K. kingae infections and carriage in 104 clinical specimens from children aged between 7 months and 7 years old. Copyright © 2018 American Society for Microbiology.

Molecular and functional characterization of cry1Ac transgenic pea lines.

PubMed

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-10-01

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

EMSA Analysis of DNA Binding By Rgg Proteins

PubMed Central

LaSarre, Breah; Federle, Michael J.

2016-01-01

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004

EMSA Analysis of DNA Binding By Rgg Proteins.

PubMed

LaSarre, Breah; Federle, Michael J

2013-08-20

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

Molecular and functional characterization of cry1Ac transgenic pea lines

PubMed Central

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-01-01

ABSTRACT Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses. PMID:27764552

Engineered TAL Effector modulators for the large-scale gain-of-function screening

PubMed Central

Zhang, Hanshuo; Li, Juan; Hou, Sha; Wang, Gancheng; Jiang, Mingjun; Sun, Changhong; Hu, Xiongbing; Zhuang, Fengfeng; Dai, Zhifei; Dai, Junbiao; Xi, Jianzhong Jeff

2014-01-01

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE–DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE–TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation. PMID:24939900