scholarly journals Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome

2008 ◽  
Vol 74 (21) ◽  
pp. 6756-6765 ◽  
Author(s):  
Jodi L. Humann ◽  
Brenda K. Schroeder ◽  
Michael W. Mortimer ◽  
Brent L. House ◽  
Svetlana N. Yurgel ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Open Reading Frames ◽  
Vector System ◽  
Destination Vector ◽  
Entry Vector ◽  
Gateway Entry Vector ◽  
Sugar Kinase

ABSTRACT The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

scholarly journals Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

2017 ◽  
Author(s):  
Vincent A Bielinski ◽  
Tayah M Bolt ◽  
Christopher L Dupont ◽  
Philip D Weyman
Keyword(s):  
Phaeodactylum Tricornutum ◽  
Expression Vectors ◽  
Dna Assembly ◽  
Destination Vector ◽  
Genetic Tools ◽  
Entry Vector ◽  
Polymerase Iii ◽  
Recombination System ◽  
Low Efficiency ◽  
Gateway Entry Vector

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals Targeted Nuclear Import of Open Reading Frame 1 Protein Is Required for In Vivo Retrotransposition of a Telomere-Specific Non-Long Terminal Repeat Retrotransposon, SART1

2004 ◽  
Vol 24 (1) ◽  
pp. 105-122 ◽  
Author(s):  
Takumi Matsumoto ◽  
Hidekazu Takahashi ◽  
Haruhiko Fujiwara
Keyword(s):  
Long Terminal Repeat ◽  
Nuclear Import ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Terminal Repeat ◽  
Ltr Retrotransposons ◽  
Reading Frame ◽  
Localization Pattern ◽  
Nuclear Localization Signals

ABSTRACT Non-long terminal repeat (non-LTR) retrotransposons, most of which carry two open reading frames (ORFs), are abundant mobile elements that are distributed widely among eukaryotes. ORF2 encodes enzymatic domains, such as reverse transcriptase, that are conserved in all retroelements, but the functional roles of ORF1 in vivo are little understood. We show with green fluorescent protein-ORF1 fusion proteins that the ORF1 proteins of SART1, a telomeric repeat-specific non-LTR retrotransposon in Bombyx mori, are transported into the nucleus to produce a dotted localization pattern. Nuclear localization signals N1 (RRKR) and N2 (PSKRGRG) at the N terminus and a highly basic region in the center of SART1 ORF1 are involved in nuclear import and the dotted localization pattern in the nucleus, respectively. An in vivo retrotransposition assay clarified that at least three ORF1 domains, N1/N2, the central basic domain, and CCHC zinc fingers are required for SART1 retrotransposition. The nuclear import activity of SART1 ORF1 makes it clear that the ORF1 proteins of non-LTR retrotransposons work mainly in the nucleus, in contrast to the cytoplasmic action of Gag proteins of LTR elements. The functional domains found here in SART1 ORF1 will be useful for developing a more efficient and target-specific LINE-based gene delivery vector.

scholarly journals Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

2017 ◽  
Author(s):  
Vincent A Bielinski ◽  
Tayah M Bolt ◽  
Christopher L Dupont ◽  
Philip D Weyman
Keyword(s):  
Phaeodactylum Tricornutum ◽  
Expression Vectors ◽  
Dna Assembly ◽  
Destination Vector ◽  
Genetic Tools ◽  
Entry Vector ◽  
Polymerase Iii ◽  
Recombination System ◽  
Low Efficiency ◽  
Gateway Entry Vector

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

scholarly journals In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

Blood ◽  
2016 ◽  
Vol 128 (18) ◽  
pp. 2206-2217 ◽  
Author(s):  
Maximilian Richter ◽  
Kamola Saydaminova ◽  
Roma Yumul ◽  
Rohini Krishnan ◽  
Jing Liu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Genetically Modify ◽  
Blood Stream ◽  
Vector System ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice

Abstract Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin−Sca1+Kit− cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy.

scholarly journals Gene Therapy: A Lipofection Approach for Gene Transfer into Primary Endothelial Cells

2002 ◽  
Vol 11 (6) ◽  
pp. 573-582 ◽  
Author(s):  
A. T. L. Young ◽  
J. R. T. Lakey ◽  
A. G. Murray ◽  
R. B. Moore
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Umbilical Vein ◽  
Vector System ◽  
Microscopic Evaluation

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37°C, 24°C, and 6°C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 μg; Fugene 6, 0.5 μg; DOTAP, 2.5 μg) and lipids (Effectene, 10 μl; Fugene 6, 6 μl; DOTAP, 15 μl) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 ± 1.3% (mean ± SEM), 33 ± 1.4%, and 18 ± 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37°C. Transfection results were lower at 24°C with mean efficiencies of 26 ± 2.4% for Effectene, 14 ± 2.9% for Fugene 6, and 15 ± 3.2% for DOTAP. Furthermore, mean efficiencies at 6°C were 6 ± 0.5%, 8 ± 1.5%, and 6 ± 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37°C vs. 24°C: p < 0.0001; 37°C vs. 6°C: p < 0.0001; 24°C vs. 6°C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

scholarly journals Short Tetracysteine Tags to β-Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging

2004 ◽  
Vol 15 (12) ◽  
pp. 5616-5622 ◽  
Author(s):  
Martin Andresen ◽  
Rita Schmitz-Salue ◽  
Stefan Jakobs
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Open Reading Frames ◽  
Host Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent ◽  
Β Tubulin

Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein (GFP) were fused by integrative epitope tagging to the C terminus of β-tubulin (Tub2) in the budding yeast Saccharomyces cerevisiae. The increasing tag size correlated with functional interference to the host protein. Tub2 tagged with either 1×TetCys (10 amino acids [aa]) or 2×TetCys (20 aa) was able to substitute Tub2 in haploid cells. In contrast, C-terminal tagging of Tub2 with 3×TetCys (29 aa) or with GFP (244 aa) resulted in nonviable haploid cells. Cells expressing Tub2-1×TetCys or Tub2-2×TetCys were stained with FlAsH, which selectively binds to the TetCys-tag. The stained cells displayed dynamic FlAsH-labeled microtubules and low cellular background fluorescence. The presented approach to tag open reading frames (ORFs) at their native loci with very small TetCys-tags and the subsequent visualization of the tagged proteins in vivo can be extended in principle to any ORF in S. cerevisiae.

scholarly journals Development of a Functional Genomics Platform for Sinorhizobium meliloti: Construction of an ORFeome

2005 ◽  
Vol 71 (10) ◽  
pp. 5858-5864 ◽  
Author(s):  
Brenda K. Schroeder ◽  
Brent L. House ◽  
Michael W. Mortimer ◽  
Svetlana N. Yurgel ◽  
Scott C. Maloney ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Symbiotic Bacterium ◽  
Bioinformatic Analysis ◽  
Open Reading Frames ◽  
Successful Implementation ◽  
Plant Microbe Interaction ◽  
Genes Encoding ◽  
Genomic Scale

ABSTRACT The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

scholarly journals Global Transcriptional Programs Reveal a Carbon Source Foraging Strategy byEscherichia coli

2005 ◽  
Vol 280 (16) ◽  
pp. 15921-15927 ◽  
Author(s):  
Mingzhu Liu ◽  
Tim Durfee ◽  
Julio E. Cabrera ◽  
Kai Zhao ◽  
Ding J. Jin ◽  
...  
Keyword(s):  
Carbon Source ◽  
Fluorescent Protein ◽  
Gene Activation ◽  
Carbon Sources ◽  
Growth Potential ◽  
Rrna Synthesis ◽  
Carbon Substrate ◽  
Protein Fusion ◽  
Substrate Quality

By exploring global gene expression ofEscherichia coligrowing on six different carbon sources, we discovered a striking genome transcription pattern: as carbon substrate quality declines, cells systematically increase the number of genes expressed. Gene induction occurs in a hierarchical manner and includes many factors for uptake and metabolism of better but currently unavailable carbon sources. Concomitantly, cells also increase their motility. Thus, as the growth potential of the environment decreases, cells appear to devote progressively more energy on the mere possibility of improving conditions. This adaptation is not what would be predicated by classic regulatory models alone. We also observe an inverse correlation between gene activation and rRNA synthesis suggesting that reapportioning RNA polymerase (RNAP) contributes to the expanded genome activation. Significant differences in RNAP distributionin vivo, monitored using an RNAP-green fluorescent protein fusion, from energy-rich and energy-poor carbon source cultures support this hypothesis. Together, these findings represent the integration of both substrate-specific and global regulatory systems, and may be a bacterial approximation to metazoan risk-prone foraging behavior.

Sign in / Sign up

Export Citation Format

Share Document