ABSTRACT
We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far. We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl. Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation. All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells. For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were up-regulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was up-regulated in benzoate-grown cells. The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C(1) metabolic pathway genes was observed. The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest. The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.
Subject(s)
Bacterial Proteins/metabolism , Benzoates/metabolism , Burkholderia/metabolism , Gene Expression Regulation, Bacterial , Genome, Bacterial , Oligonucleotide Array Sequence Analysis/methods , Polychlorinated Biphenyls/metabolism , Bacterial Proteins/genetics , Burkholderia/genetics , Burkholderia/growth & development , Culture Media , Gene Expression Profiling , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the antigen-binding domain of immunoglobulins and T-cell receptors, by introducing site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte development. These breaks are generated in two steps, nicking of one strand (hydrolysis), followed by hairpin formation (transesterification). The nature and location of the RAG active site(s) have remained unknown. Because acidic amino acids have a critical role in catalyzing DNA cleavage by nucleases and recombinases that require divalent metal ions as cofactors, we hypothesized that acidic active site residues are likewise essential for RAG-mediated DNA cleavage. We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed mutagenesis, and examined >100 mutants using a combination of in vivo and in vitro analyses. No conserved acidic amino acids in RAG2 were critical for catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically inactive for both nicking and hairpin formation. These data argue that one active site in RAG1 performs both steps of the cleavage reaction. Amino acid substitution experiments that changed the metal ion specificity suggest that at least one of these three residues contacts the metal ion(s) directly. These data suggest that RAG-mediated DNA cleavage involves coordination of divalent metal ion(s) by RAG1.
Subject(s)
DNA Nucleotidyltransferases/metabolism , Gene Rearrangement/physiology , Homeodomain Proteins/physiology , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , CHO Cells , Catalysis , Catalytic Domain , Cricetinae , Cricetulus , Cysteine/chemistry , DNA/genetics , DNA/metabolism , DNA-Binding Proteins , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Humans , Lymphocytes/metabolism , Manganese/physiology , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins , Recombinant Fusion Proteins/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Transfection , VDJ Recombinases , Vertebrates/geneticsABSTRACT
The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).
Subject(s)
Antibodies, Monoclonal/metabolism , Antigen-Antibody Complex , Muramidase/metabolism , Animals , Arginine/metabolism , Aspartic Acid/metabolism , Chickens , Colinus , Crystallography, X-Ray , Glutamine/metabolism , Immunoglobulin Fab Fragments/metabolism , Models, Chemical , Muramidase/immunology , Protein Binding , Protein Conformation , Protein Denaturation , Protein Folding , Structure-Activity Relationship , TemperatureABSTRACT
A scaleable method for the liquid-phase separation of plasmid DNA from RNA.
Subject(s)
DNA/isolation & purification , Plasmids/genetics , RNA/isolation & purification , Chemical Precipitation , Chromatography, Ion Exchange , Electrophoresis, Agar Gel , Indicators and ReagentsABSTRACT
Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced. The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding. Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains. The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG. Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system.
Subject(s)
Antibodies/genetics , Immunoglobulin Fab Fragments/genetics , Muramidase/immunology , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibodies/metabolism , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation , Hybridomas/immunology , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Molecular Sequence Data , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.