scholarly journals Violaxanthin conversion by recombinant diatom and plant de-epoxidases, expressed in Escherichia coli – comparative analysis

2019 ◽  
Author(s):  
Monika Olchawa-Pajor ◽  
Monika Bojko ◽  
Wojciech Strzałka ◽  
Kazimierz Strzałka ◽  
Dariusz Latowski
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Metal Affinity ◽  
Comparable Activity ◽  
First Time ◽  
Kinetics Of ◽  
Reading Frames

The purpose of this research was to obtain recombinant violaxanthin de-epoxidases (VDEs) from two species. The first one was VDE of Arabidopsis thaliana (L.) Heynh. (WT Columbia strain) (AtVDE) which in vivo catalyzes conversion of violaxanthin (Vx) to zeaxanthin (Zx) via anteraxanthin (Ax). The second one was VDE of Phaeodactylum tricornutum Bohlin, 1897 (CCAP 1055/1 strain) (PtVDE) which is responsible for de-epoxidation of diadinoxanthin (Ddx) to diatoxanthin (Dtx). As the first step of our experiments, open reading frames coding for studied enzymes were amplified and subsequently cloned into pET-15b plasmid. For recombinant proteins production Escherichia coli Origami b strain was used. The molecular weight of the produced enzymes were estimated approximately at 45kDa and 50kDa for AtVDE and PtVDE, respectively. Both enzymes, purified under native conditions by immobilized metal affinity chromatography, displayed comparable activity in assay mixture and converted up to 90% Vx in 10 min in two steps enzymatic de-epoxidation, irrespective of enzyme origin. No statistically significant differences were observed when kinetics of the reactions catalyzed by these enzymes were compared. Putative role of selected amino-acid residues of AtVDE and PtVDE was also considered. The significance of the first time obtained recombinant PtVDE as a useful tool in various comparative investigations of de-epoxidation reactions in main types of xanthophyll cycles existing in nature are also indicated.

scholarly journals Chromatin Remodeling around Nucleosome-Free Regions Leads to Repression of Noncoding RNA Transcription

2010 ◽  
Vol 30 (21) ◽  
pp. 5110-5122 ◽  
Author(s):  
Adam N. Yadon ◽  
Daniel Van de Mark ◽  
Ryan Basom ◽  
Jeffrey Delrow ◽  
Iestyn Whitehouse ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Noncoding Rna ◽  
Open Reading Frames ◽  
Yeast Genome ◽  
Transcription Start Sites ◽  
Transcriptional Regulatory ◽  
First Time ◽  
Reading Frames

ABSTRACT Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5′ and 3′ ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA.

scholarly journals Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome

mSystems ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Kaneyoshi Yamamoto ◽  
Yuki Yamanaka ◽  
Tomohiro Shimada ◽  
Paramita Sarkar ◽  
Myu Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Small Subunit ◽  
Open Reading Frames ◽  
Pcr Assay ◽  
E Coli ◽  
K 12 ◽  
Chip Chip ◽  
Reading Frames

The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (β′, of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.

scholarly journals Gravity Affects the Closure of the Traps inDionaea muscipula

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Camilla Pandolfi ◽  
Elisa Masi ◽  
Boris Voigt ◽  
Sergio Mugnai ◽  
Dieter Volkmann ◽  
...  
Keyword(s):  
Parabolic Flight ◽  
Large Diameter ◽  
Carnivorous Plant ◽  
Venus Flytrap ◽  
Dionaea Muscipula ◽  
Fast Movement ◽  
First Time ◽  
Kinetics Of

Venus flytrap (Dionaea muscipulaEllis) is a carnivorous plant known for its ability to capture insects thanks to the fast snapping of its traps. This fast movement has been long studied and it is triggered by the mechanical stimulation of hairs, located in the middle of the leaves. Here we present detailed experiments on the effect of microgravity on trap closure recorded for the first time during a parabolic flight campaign. Our results suggest that gravity has an impact on trap responsiveness and on the kinetics of trap closure. The possible role of the alterations of membrane permeability induced by microgravity on trap movement is discussed. Finally we show how the Venus flytrap could be an easy and effective model plant to perform studies on ion channels and aquaporin activities, as well as on electrical activityin vivoon board of parabolic flights and large diameter centrifuges.

scholarly journals Hydroxycinnamate (hca) Catabolic Genes from Acinetobacter sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters

2003 ◽  
Vol 69 (9) ◽  
pp. 5398-5409 ◽  
Author(s):  
Donna Parke ◽  
L. Nicholas Ornston
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Genetic Background ◽  
Open Reading Frames ◽  
Aldehyde Dehydrogenases ◽  
E Coli ◽  
Catabolic Genes ◽  
Coa Thioesters ◽  
Reading Frames

ABSTRACT Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Kmr genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.

scholarly journals Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3349-3359 ◽  
Author(s):  
S. Madhusudan ◽  
Andreas Paukner ◽  
Yvonne Klingen ◽  
Karin Schnetz
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Transposon Mutagenesis ◽  
Open Reading Frames ◽  
Wild Type ◽  
Coding Region ◽  
First Time ◽  
Insertion Mutations ◽  
Reading Frames

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

scholarly journals A tyrosine-based trafficking signal in the simian immunodeficiency virus envelope cytoplasmic domain is strongly selected for in pathogenic SIV infection

2021 ◽  
Author(s):  
Scott Lawrence ◽  
Samra E Elser ◽  
Workineh Torben ◽  
Robert V Blair ◽  
Bapi Pahar ◽  
...  
Keyword(s):  
Rhesus Macaques ◽  
Cytoplasmic Domain ◽  
Open Reading Frames ◽  
Virus Envelope ◽  
Viral Loads ◽  
Immunodeficiency Virus ◽  
Siv Infection ◽  
First Time

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion that encodes amino acids 734-736 (ΔQTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naive PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

scholarly journals Escherichia coli Insertion Sequence IS150: Transposition via Circular and Linear Intermediates

2002 ◽  
Vol 184 (21) ◽  
pp. 5833-5841 ◽  
Author(s):  
Markus Haas ◽  
Bodo Rak
Keyword(s):  
Open Reading Frames ◽  
Primary Role ◽  
Translational Frameshifting ◽  
Programmed Translational Frameshifting ◽  
Family Analysis ◽  
Linear Molecules ◽  
Derivatives Of ◽  
Reading Frames

ABSTRACT IS150, a member of the widespread IS3 family, contains two consecutive out-of-phase open reading frames, orfA and orfB, that partially overlap. These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting. We demonstrate that the InsAB protein represents the IS150 element's transposase. In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules. Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family. Our finding supports the assumption that these products represent true transposition intermediates of members of this family. Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules. Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events.

RhoGDI-3, a promising system to investigate the regulatory function of rhoGDIs: uncoupling of inhibitory and shuttling functions of rhoGDIs

2005 ◽  
Vol 33 (4) ◽  
pp. 623-626 ◽  
Author(s):  
E. Dransart ◽  
A. Morin ◽  
J. Cherfils ◽  
B. Olofsson
Keyword(s):  
Membrane Extraction ◽  
Regulatory Function ◽  
Rho Proteins ◽  
Steady State Kinetics ◽  
Rho Protein ◽  
Rho Family ◽  
First Time ◽  
Kinetics Of

rhoGDIs (Rho GDP dissociation inhibitors) are postulated to regulate the activity and the localization of small G-proteins of the Rho family by a shuttling process involving extraction of Rho from donor membranes, formation of inhibitory cytosolic rhoGDI/Rho complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. We investigated here the in vivo functions of two mammalian rhoGDIs: the specific rhoGDI-3 and the well-studied rhoGDI-1 (rhoGDI) after structure-based mutagenesis. We identified two sites in rhoGDIs, forming conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs in vivo. Remarkably, these rhoGDI mutants were detected at Rho-induced membrane ruffles or protrusions, where they co-localized with RhoG or Cdc42, probably identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhang ◽  
Guoyu Yin ◽  
Heping Zhao ◽  
Hanzhi Ling ◽  
Zhen Xie ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Hyaluronic Acid ◽  
Dependent Manner ◽  
Collagen Induced Arthritis ◽  
Intracellular Degradation ◽  
Main Pathway ◽  
First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

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