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1.
J Thromb Haemost ; 14(12): 2478-2492, 2016 12.
Article in English | MEDLINE | ID: mdl-27685947

ABSTRACT

Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. SUMMARY: Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc-/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc-/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein secretion from autologous B cells in vivo in the treatment of hemophilia and other acquired or inherited diseases.


Subject(s)
B-Lymphocytes/cytology , Factor IX/metabolism , Genetic Vectors , Hemophilia A/blood , Lentivirus/genetics , Animals , Antigen-Presenting Cells/cytology , CD4-Positive T-Lymphocytes/cytology , Cell Proliferation , Gene Transfer Techniques , HEK293 Cells , Hemophilia A/therapy , Humans , Immunotherapy/methods , Membrane Glycoproteins/chemistry , Mice , Mice, Inbred NOD , Mice, SCID , Papio , Plasmids , Transduction, Genetic , Transgenes , Treatment Outcome , Viral Envelope Proteins/chemistry
2.
Transgenic Res ; 19(5): 799-808, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20069454

ABSTRACT

Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.


Subject(s)
Animals, Genetically Modified/genetics , Genetic Vectors/genetics , Rabbits/genetics , Simian Immunodeficiency Virus/genetics , Transgenes , Animals , Embryo Transfer , Feasibility Studies , Female , Gene Expression Regulation , Gene Silencing , Genes, Reporter , Green Fluorescent Proteins/genetics , Male , Microinjections , Mosaicism , Organ Specificity , Spermatozoa/chemistry , Zygote
3.
Hum Reprod ; 24(11): 2890-901, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19671624

ABSTRACT

BACKGROUND: Adiponectin is involved in the regulation of energy homeostasis and more recently in the reproductive functions. We have previously shown that adiponectin receptors (AdipoR1 and AdipoR2) are expressed in human granulosa cells. However, it remains to be investigated whether both AdipoR1 and AdipoR2 or only one of these receptors serve as the major receptor(s) for adiponectin in human granulosa cells. METHODS: The RNA interference (RNAi) technology was used to specifically knockdown the expression of either AdipoR1 or AdipoR2. Progesterone and estradiol levels in the conditioned media were measured by radioimmunoassay, and determination of cell proliferation by tritiated thymidine incorporation. The levels of adiponectin receptors and proteins involved in the steroidogenesis and in the signalling pathways were examined by western blot. RESULTS: We generated AdipoR1 (R1) and AdipoR2 (R2) knockdown KGN cell lines. R1 cells were apoptotic and had increased expression levels of cleaved caspase 3 and decreased levels of BAD phosphorylation and PCNA as compared with control or parental KGN cells. R2 cells had similar morphology to control or KGN cells. However, they produced less progesterone and estradiol and expressed lower levels of StAR protein in response to FSH or IGF-1 stimulation compared with control cells. Furthermore, the increase of MAPK ERK1/2 phosphorylation in response to human recombinant adiponectin and FSH was lower in R2 than control cells. CONCLUSIONS: In the human granulosa KGN cell-line, AdipoR1 seems to be involved in the cell survival whereas AdipoR2, through MAPK ERK1/2 activation, may be implicated in the regulation of steroid production.


Subject(s)
Estradiol/biosynthesis , Progesterone/biosynthesis , Receptors, Adiponectin/physiology , Adiponectin/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Cell Survival , Culture Media, Conditioned , Female , Follicle Stimulating Hormone/biosynthesis , Granulosa Cells , Humans , Insulin-Like Growth Factor I/biosynthesis , MAP Kinase Kinase 2/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , RNA Interference , Receptors, Adiponectin/genetics , Receptors, Adiponectin/metabolism , Signal Transduction
4.
Methods Mol Biol ; 434: 99-112, 2008.
Article in English | MEDLINE | ID: mdl-18470641

ABSTRACT

A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34+ cells that reside in the G0 phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. We describe here novel LVs that overcome this restriction by displaying early-acting cytokines on their surface. Display of thrombopoietin, stem cell factor or both cytokines on LV surface allows high transfer into quiescent cord blood CD34+ cells. Moreover, these surface-engineered LVs preferentially transduce and promote survival of resting CD34+ cells rather than cycling cells. These novel LVs allow superior gene transfer in the most immature CD34+ cells compared to conventional LVs, even in the presence of recombinant cytokines. This is demonstrated by their capacity to promote selective transduction in long-term culture initiating cell colonies (LTC-ICs) and of long-term non-obese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs). Here we describe the production of these "early acting cytokine" displaying vectors and the methodology to confirm the capacity of these vectors to promote selective transduction of HSCs.


Subject(s)
Gene Transfer Techniques , Genetic Vectors , Hematopoietic Stem Cells/physiology , Lentivirus/physiology , Stem Cell Factor/metabolism , Thrombopoietin/metabolism , Animals , Antigens, CD34/metabolism , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Transduction, Genetic
5.
J Endocrinol ; 194(3): 557-68, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17761895

ABSTRACT

IGF-I regulates pituitary and gonadal functions, and is pivotal for sexual development and fertility in mammalian species. To better understand the function of autocrine IGF-I in Sertoli cell physiology, we established a system for Cre-mediated conditional inactivation of the IGF-I receptor (IGF-IR) in cultured Sertoli cells. We show here that loss of IGF-IR decreased the number of viable Sertoli cells as a consequence of diminished Sertoli cell proliferation and increased Sertoli cell death. Furthermore, the lack of IGF-IR altered the morphology of cultured Sertoli cells and decreased lactate and transferrin secretions. Collectively, our data indicate that autocrine IGF-I contributes significantly to Sertoli cell homeostasis. The described in vitro system for loss-of-function analysis of the IGF-IR can be readily transposed to study the role of other intratesticular growth factors involved in spermatogenesis.


Subject(s)
Autocrine Communication/physiology , Insulin-Like Growth Factor I/metabolism , Receptor, IGF Type 1/genetics , Sertoli Cells/metabolism , Spermatogenesis/physiology , Animals , Cell Proliferation , Cell Survival , Genetic Engineering , Immunoblotting , Immunohistochemistry , Male , Mice , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction
7.
Gene Ther ; 7(19): 1613-23, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11083469

ABSTRACT

We describe the generation and the characterization of new lentiviral vectors derived from SIVmac251, a simian immunodeficiency virus (SIV). A methodical approach was used to engineer both efficient and safe packaging constructs allowing the production of SIV viral core proteins. SIV-vectors encoding GFP (green fluorescent protein) were generated as VSV-G-pseudotyped particles upon transient expression of the vector construct and helper functions in 293 cells. The SIV vectors were able to transduce efficiently various target cell types at low multiplicity of infection, including monocyte-differentiated human dendritic cells (DCs) which retained their capacity to differentiate into mature DCs after gene transfer. Transduction of the DCs by the SIV vectors was prevented when infections were performed in the presence of AZT, a reverse-transcriptase inhibitor. After gene transfer, expression of the GFP in the target cells remained constant after several weeks, indicating that the vectors had been stably integrated into the genome of the host cells. Preparations of SIV vectors were systematically checked for the absence of replication-competent and recombinant retroviruses but remained negative, suggesting the innocuousness of these novel gene delivery vectors. Side-to-side comparisons with vectors derived from HIV-1 (human immunodeficiency virus) indicated that the SIV vectors were equally potent in transducing proliferating target cells. Finally, we have determined the infectivity of SIV vectors pseudotyped with surface glycoproteins of several membrane-enveloped viruses.


Subject(s)
Dendritic Cells/metabolism , Genetic Vectors , Simian Immunodeficiency Virus/genetics , Transfection/methods , Animals , Cell Line , Gene Expression , Genetic Engineering , Green Fluorescent Proteins , HIV-1/genetics , Humans , Luminescent Proteins/genetics , Virosomes
8.
J Virol ; 74(18): 8307-15, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10954529

ABSTRACT

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5' cytomegalovirus early promoter, the 5' viral sequences up to the 5' end of gag required for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3' long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 10(7) transducing units/ml on HeLa cells and to deliver a gene without transfer of helper functions to target cells. The central polypurine tract can be included in the minimal vector, resulting in a two- to threefold increase in the transduction titers on dividing or growth-arrested cells. Based on this minimal SIV vector, a sin vector was designed by deleting 151 nucleotides in the 3' LTR U3 region, and this SIV sin vector retained high transduction titers. Furthermore, the minimal SIV vector was efficient at transducing terminally differentiated human CD34(+) cell-derived or monocyte-derived dendritic cells (DCs). Results show that up to 40% of human primary DCs can be transduced by the SIV vectors. This opens a new perspective in the field of immunotherapy.


Subject(s)
Dendritic Cells/metabolism , Gene Transfer Techniques , Simian Immunodeficiency Virus/genetics , Cell Line , Cytomegalovirus/genetics , Genetic Vectors , HeLa Cells , Humans , Polymerase Chain Reaction , Promoter Regions, Genetic , Sequence Deletion , Terminal Repeat Sequences
9.
Biochim Biophys Acta ; 1447(2-3): 278-83, 1999 Oct 28.
Article in English | MEDLINE | ID: mdl-10542327

ABSTRACT

With the aim of identifying genes involved in cartilage differentiation, we have used a subtractive hybridization strategy with cDNAs from a chondrocytic cell line (MC615) and mRNAs from a mesenchymal precursor cell line (10T1/2). We have isolated a cDNA clone representing a novel mouse gene. The predicted 368-amino acid protein, designated ZF-12, contains four C(2)H(2)-type zinc finger motifs and one region homologous to the LeR domain, a finger-associated structural domain. ZF-12 mRNAs are expressed during embryonic development and in different organs in adult, including rib cartilage. These data suggest that ZF-12 might play an important role not only in cartilage differentiation, but also in basic cellular processes.


Subject(s)
Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Gene Expression , Mice , Molecular Sequence Data
10.
J Bacteriol ; 181(3): 893-8, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9922253

ABSTRACT

The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.


Subject(s)
Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Isocitrate Dehydrogenase/genetics , Promoter Regions, Genetic , Repressor Proteins/metabolism , Transcription, Genetic , Transcriptional Activation , Amino Acid Substitution , Base Sequence , Deoxyribonuclease I , Isocitrate Dehydrogenase/biosynthesis , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA, Messenger/genetics , Recombinant Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Restriction Mapping
11.
J Mol Biol ; 276(2): 355-65, 1998 Feb 20.
Article in English | MEDLINE | ID: mdl-9512708

ABSTRACT

The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts. The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method. Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene. In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation. Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit. The alpha[L262A], alpha[R265A] and alpha[N268A] substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Phosphotransferases (Paired Acceptors)/genetics , Phosphotransferases (Paired Acceptors)/metabolism , Repressor Proteins/metabolism , Transcriptional Activation , Alanine , Bacterial Proteins/genetics , Base Sequence , Binding Sites , DNA/chemistry , DNA/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA, Messenger , Regulatory Sequences, Nucleic Acid , Repressor Proteins/genetics
12.
Eur J Biochem ; 258(2): 579-85, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9874226

ABSTRACT

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.


Subject(s)
Adenosine/analogs & derivatives , Escherichia coli/enzymology , Phosphoprotein Phosphatases/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Adenosine/metabolism , Adenosine/pharmacology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/analogs & derivatives , Affinity Labels/pharmacology , Disulfides/chemistry , Enzyme Inhibitors/pharmacology , Kinetics , Mutagenesis, Site-Directed/genetics , Peptide Fragments/chemistry , Phosphoprotein Phosphatases/genetics , Protein Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolism
13.
J Mol Biol ; 270(3): 496-510, 1997 Jul 18.
Article in English | MEDLINE | ID: mdl-9237914

ABSTRACT

FruR is an Escherichia coli transcriptional regulator that belongs to the LacI DNA-binding protein family. By using 1H and 15N NMR spectroscopy, we have determined the three-dimensional solution structure of the FruR N-terminal DNA-binding domain consisting of 57 amino acid residues. A total of 809 NMR-derived distances and 54 dihedral angle constraints have been used for molecular modelling with the X-PLOR program. The resulting set of calculated structures presents an average root-mean-square deviation of 0.37 A at the main-chain level for the first 47 residues. This highly defined N-terminal part of the structure reveals a similar topology for the three alpha-helices when compared to the 3D structures of LacI and PurR counterparts. The most striking difference lies in the connection between helix II and helix III, in which three additional residues are present in FruR. This connecting segment is well structured and contains a type III turn. Apart from hydrophobic interactions of non-polar residues with the core of the domain, this connecting segment is stabilised by several hydrogen bonds and by the aromatic ring stacking between Tyr19 of helix II and Tyr28 of the turn. The region containing the putative "hinge helix" (helix IV), that has been described in PurR-DNA complex to make specific base contacts in the minor groove of DNA, is unfolded. Examination of hydrogen bonds highlights the importance of homologous residues that seem to be conserved for their ability to fulfill helix N and C-capping roles in the LacI repressor family.


Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Escherichia coli Proteins , Escherichia coli/chemistry , Magnetic Resonance Spectroscopy/methods , Protein Structure, Secondary , Repressor Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Lac Repressors , Models, Molecular , Molecular Sequence Data , Recombinant Fusion Proteins , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Sequence Alignment
14.
Nucleic Acids Res ; 25(4): 713-8, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9016619

ABSTRACT

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.


Subject(s)
Acetates/metabolism , DNA, Bacterial/metabolism , Escherichia coli/genetics , Promoter Regions, Genetic , Transcription, Genetic , Transcriptional Activation , Base Sequence , Binding Sites/genetics , DNA-Directed RNA Polymerases/metabolism , Genes, Bacterial , Molecular Sequence Data , rRNA Operon
15.
Mol Microbiol ; 21(2): 257-66, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8858581

ABSTRACT

The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein. FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3'. In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family. On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator. However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR.


Subject(s)
Bacterial Proteins/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Binding Sites/genetics , Consensus Sequence , DNA Footprinting , DNA Methylation , DNA, Bacterial/chemistry , Deoxyribonuclease I , Genes, Bacterial , Molecular Sequence Data , Operon , Phosphofructokinase-1/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Repressor Proteins/genetics , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
16.
Eur J Biochem ; 237(1): 247-54, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8620880

ABSTRACT

The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C. Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP.


Subject(s)
Escherichia coli/enzymology , Phosphoprotein Phosphatases/genetics , Protein Serine-Threonine Kinases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Fluorescence , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Hydrolysis , Kinetics , Molecular Sequence Data , Phosphoprotein Phosphatases/metabolism , Protein Serine-Threonine Kinases/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
17.
Gene ; 153(1): 9-15, 1995 Feb 03.
Article in English | MEDLINE | ID: mdl-7883193

ABSTRACT

A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase. The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin. After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis. Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.


Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli Proteins , Escherichia coli/genetics , Peptide Fragments/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Repressor Proteins/biosynthesis , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Chromatography, Affinity , Cloning, Molecular , DNA, Bacterial/genetics , Glutathione/metabolism , Glutathione Transferase/genetics , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Protein Folding , Protein Structure, Tertiary , Protozoan Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Schistosoma japonicum/genetics , Trypsin/metabolism
18.
J Biol Chem ; 269(21): 14885-91, 1994 May 27.
Article in English | MEDLINE | ID: mdl-8195118

ABSTRACT

The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues. By using a one-step chromatographic procedure, FruR was purified to near-homogeneity. Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons. The positions of interference between FruR and the operator of the acetate operon were examined. The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting. It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon.


Subject(s)
Bacterial Proteins/metabolism , Enzymes/biosynthesis , Escherichia coli Proteins , Glyoxylates/metabolism , Operator Regions, Genetic , Repressor Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Binding Sites , Chromatography, Gel , DNA, Bacterial/metabolism , Electrophoresis, Polyacrylamide Gel , Enzymes/genetics , Escherichia coli/genetics , Hydroxylation , Methylation , Molecular Sequence Data , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Uracil/metabolism
19.
J Mol Biol ; 234(1): 28-44, 1993 Nov 05.
Article in English | MEDLINE | ID: mdl-8230205

ABSTRACT

Evidence has been presented suggesting that the fructose repressor, FruR, is a pleiotropic transcriptional regulatory protein controlling the expression of numerous operons concerned with carbon metabolism in Escherichia coli and Salmonella typhimurium. We have conducted in vitro DNA binding studies to ascertain the nature of the DNA sequences to which FruR binds. Employing both DNA band migration retardation and DNase I footprint analyses, FruR was found to bind to two operators within the regulatory region preceding the structural genes of the fructose operon, fruB(MH)KA. These two operators, O1 and O2, comprise nearly identical palindromes of 12 bp with a half-site of TGAAAC. The binding of FruR to these inverted repeats was found to be reversed by inclusion of micromolar concentrations of fructose-1-phosphate. The two operators are located between the single putative promoter of the fructose operon and the translational initiation site of the fruB gene. Other regulated operons were shown to bind FruR to a single site upstream of the first structural gene as follows: (1) ppsA (positive regulation); (2) icd (positive regulation); (3) aceB (positive regulation); and (4) pts (negative regulation). In all cases, low concentrations of fructose-1-phosphate displaced the protein from the DNA. The binding sites were determined, and a FruR consensus sequence was established. Computer searches revealed the presence of this sequence in numerous functionally diverse operons, implying that FruR is a global transcriptional regulatory protein in enteric bacteria.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Operon , Repressor Proteins/metabolism , Salmonella typhimurium/genetics , Allosteric Regulation , Base Sequence , Consensus Sequence , DNA, Bacterial/metabolism , Fructose , Fructosephosphates/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Operator Regions, Genetic , Protein Binding , Transcription, Genetic
20.
J Mol Biol ; 228(1): 23-9, 1992 Nov 05.
Article in English | MEDLINE | ID: mdl-1447784

ABSTRACT

The positions of interference points between the IclR repressor of the acetate operon of Escherichia coli and its specific operator were examined. The number and nature of nucleotides essential to repressor binding were determined by scanning populations of DNA previously methylated at guanine residues by dimethyl sulfate, or depurinated by treatment with formic acid, or depyrimidated by treatment with hydrazine. A total of 46 nucleotides, distributed almost equally between the two strands of the operator region, were found to be functionally important, although to a varying extent. These are clustered in two successive domains which expand from nucleotide -54 to nucleotide -27 and can organize in a palindrome-like structure containing a large proportion of A and T residues.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Operator Regions, Genetic , Operon , Repressor Proteins/metabolism , Transcription Factors , Amino Acid Sequence , Base Sequence , Binding Sites , DNA, Bacterial , Methylation , Molecular Sequence Data , Oligonucleotides , Polymerase Chain Reaction
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