Characterization ofndvD, the third gene involved in the synthesis of cyclic β-(13),(16)-D-glucans inBradyrhizobium japonicum

2002 ◽  
Vol 48 (11) ◽  
pp. 1008-1016 ◽  
Author(s):  
Rongji Chen ◽  
Arvind A Bhagwat ◽  
Robert Yaklich ◽  
Donald L Keister
Keyword(s):  
Gene Deletion ◽  
Transmembrane Domain ◽  
Nodule Development ◽  
Symbiotic Interaction ◽  
Reading Frame ◽  
New Gene ◽  
Conjugative Gene Transfer ◽  
Glucan Synthesis

Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic β-(1[Formula: see text]3),(1[Formula: see text]6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for β-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo β-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral β-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for β-glucan synthesis. Our hypothesis for the mechanism of β-(1[Formula: see text]3),(1[Formula: see text]6)-D-glucan synthesis is presented. Key Words: β-glucans,Bradyrhizobium, soybean, nitrogen fixation.

scholarly journals Ectopic Expression of the Rhizobium etli amtB Gene Affects the Symbiosome Differentiation Process and Nodule Development

1999 ◽  
Vol 12 (6) ◽  
pp. 515-525 ◽  
Author(s):  
Rosarita Taté ◽  
Michele Cermola ◽  
Anna Riccio ◽  
Maurizio Iaccarino ◽  
Mike Merrick ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Ectopic Expression ◽  
Host Cells ◽  
Nodule Development ◽  
Differentiation Process ◽  
Symbiotic Interaction ◽  
Methylammonium Transport ◽  
Bacterial Genes

Under conditions of nitrogen limitation, soil bacteria of the genus Rhizobium are able to induce the development of symbiotic nodules on the roots of leguminous plants. During nodule organogenesis, bacteria are released endocytotically inside the invaded plant cells where they differentiate into their endosymbiotic form called bacteroids. Bacteroids surrounded by a plant-derived peribacteroid membrane are nondividing, organelle-like structures, called symbiosomes, that use nitrogenase to reduce N2 to ammonia. Experiments performed in vitro with isolated symbiosomes have previously led to the suggestion that the NH3 produced by the bacteroids is released as NH4+ into the plant cytosol. Furthermore, it was observed that the bacterial amtB (ammonium/methylammonium transport B) gene is switched off very early during symbiosis, just when bacteria are released into the host cells. We report here that the ectopic expression of amtB in bacteroids alters the ability of bacteria to invade the host cells and the symbiosome differentiation process. Both the NtrC protein, which controls the expression of the bacterial genes involved in NH4+ assimilation, and the nitrogenase activity are essential to observe the amtB-mediated effect. Our results support the idea that in vivo bacteroids do not take up NH4+ and demonstrate that the transcriptional down-regulation of the amtB gene is essential for an effective symbiotic interaction.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

scholarly journals Regulated Targeting of BAX to Mitochondria

1998 ◽  
Vol 143 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Ing Swie Goping ◽  
Atan Gross ◽  
Josée N. Lavoie ◽  
Mai Nguyen ◽  
Ronald Jemmerson ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Transmembrane Domain ◽  
Apoptotic Cell ◽  
Membrane Insertion ◽  
Cell Extracts ◽  
Proapoptotic Protein ◽  
Signal Anchor ◽  
Discrete Domains

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.

scholarly journals CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo

2018 ◽  
Author(s):  
Benjamin L. Duchêne ◽  
Khadija Cherif ◽  
Jean-Paul Iyombe-Engembe ◽  
Antoine Guyon ◽  
Joel Rousseau ◽  
...  
Keyword(s):  
Hereditary Disease ◽  
Reading Frame ◽  
Dystrophin Expression ◽  
Dmd Gene ◽  
Dystrophin Protein ◽  
Further Development ◽  
Muscle Biopsies ◽  
Complete Deletion

AbstractDuchenne Muscular Dystrophy (DMD), a severe hereditary disease, affecting 1 boy out of 3500, mainly results from the deletion of one or more exons leading to a reading frame shift of the DMD gene that abrogates dystrophin protein synthesis. We used the Cas9 of Staphylococcus aureus (SaCas9) to edit the human DMD gene. Pairs of sgRNAs were meticulously chosen to induce a genomic deletion to not only restore the reading frame but also produced a dystrophin protein with normally phased spectrin-like repeats. The formation of a dystrophin protein with spectrin-like repeats normally phased is not usually obtained by skipping or by deletion of complete exons. This can however be obtained in rare instances where the exon/intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion are at similar positions in the spectrin-like repeat. We used pairs of sgRNAs, targeting exons 47 and 58 and a normal reading frame was restored in 67 to 86% of the resulting hybrid exons in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. The restoration of the DMD reading frame and restoration of the dystrophin expression was also obtained in vivo in the heart of the del52hDMD/mđx. Our results provide a proof-of-principle that SaCas9 could be used to edit the human DMD gene and could be considered for the further development of a therapy for DMD.

scholarly journals Characterization of a Chlamydia psittaciDNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle

1998 ◽  
Vol 66 (3) ◽  
pp. 1167-1173 ◽  
Author(s):  
Li Zhang ◽  
Annemarie L. Douglas ◽  
Thomas P. Hatch
Keyword(s):  
Promoter Region ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Upstream Open Reading Frame ◽  
Developmental Cycle ◽  
Binding Property ◽  
Logarithmic Growth Phase ◽  
Reading Frame

ABSTRACT The EUO gene (for early upstream open reading frame) ofChlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crpDNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.

scholarly journals A Sinorhizobium meliloti Lipopolysaccharide Mutant Altered in Cell Surface Sulfation

2002 ◽  
Vol 184 (23) ◽  
pp. 6681-6689 ◽  
Author(s):  
David H. Keating ◽  
Michael G. Willits ◽  
Sharon R. Long
Keyword(s):  
Cell Surface ◽  
Sinorhizobium Meliloti ◽  
Glucuronic Acid ◽  
Initial Study ◽  
Nodule Development ◽  
Nod Factor ◽  
Legume Symbiosis ◽  
Surface Polysaccharides

ABSTRACT The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates. Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a β-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end. This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3′-phosphoadenosine-5′-phosphosulfate. It was previously reported that S. meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ. We have further characterized this unique form of bacterial carbohydrate modification. Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation. We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant. The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo. Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.

scholarly journals Applications of CRISPR/Cas9 for the Treatment of Duchenne Muscular Dystrophy

2018 ◽  
Vol 8 (4) ◽  
pp. 38 ◽  
Author(s):  
Kenji Lim ◽  
Chantal Yoon ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Wide Spectrum ◽  
Membrane Integrity ◽  
Dystrophin Gene ◽  
Muscle Membrane ◽  
Reading Frame ◽  
Long Term Treatment

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein that leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.

scholarly journals Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
Author(s):  
Sarah L. Barker ◽  
Linda Lee ◽  
B. Daniel Pierce ◽  
Lymarie Maldonado-Báez ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

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