Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination.
Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages. (+info)
Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.
Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available. (+info)
General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: application to kinetics of open complex formation between Esigma70 RNA polymerase and lambdaP(R) promoter DNA.
A novel analytical method based on the exact solution of equations of kinetics of unbranched first- and pseudofirst-order mechanisms is developed for application to the process of Esigma70 RNA polymerase (R)-lambdaPR promoter (P) open complex formation, which is described by the minimal three-step mechanism with two kinetically significant intermediates (I1, I2), [equation: see text], where the final product is an open complex RPo. The kinetics of reversible and irreversible association (pseudofirst order, [R] >> [P]) to form long-lived complexes (RPo and I2) and the kinetics of dissociation of long-lived complexes both exhibit single exponential behavior. In this situation, the analytical method provides explicit expressions relating observed rate constants to the microscopic rate constants of mechanism steps without use of rapid equilibrium or steady-state approximations, and thereby provides a basis for interpreting the composite rate constants of association (ka), isomerization (ki), and dissociation (kd) obtained from experiment for this or any other sequential mechanism of any number of steps. In subsequent papers, we apply this formalism to analyze kinetic data obtained in the reversible and irreversible binding regimes of Esigma70 RNA polymerase (R)-lambdaP(R) promoter (P) open complex formation. (+info)
Single-polymer dynamics in steady shear flow.
The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased. (+info)
Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli.
Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from Mycoplasma pulmonis. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated mnuA (for "membrane nuclease"). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an M. pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with E. coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studies showed that mnuA sequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma. (+info)
Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes.
The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32. (+info)
X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture.
Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif. The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases. The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four-way DNA junction is presented. (+info)
A RAPID algorithm for sequence database comparisons: application to the identification of vector contamination in the EMBL databases.
MOTIVATION: Word-matching algorithms such as BLAST are routinely used for sequence comparison. These algorithms typically use areas of matching words to seed alignments which are then used to assess the degree of sequence similarity. In this paper, we show that by formally separating the word-matching and sequence-alignment process, and using information about word frequencies to generate alignments and similarity scores, we can create a new sequence-comparison algorithm which is both fast and sensitive. The formal split between word searching and alignment allows users to select an appropriate alignment method without affecting the underlying similarity search. The algorithm has been used to develop software for identifying entries in DNA sequence databases which are contaminated with vector sequence. RESULTS: We present three algorithms, RAPID, PHAT and SPLAT, which together allow vector contaminations to be found and assessed extremely rapidly. RAPID is a word search algorithm which uses probabilities to modify the significance attached to different words; PHAT and SPLAT are alignment algorithms. An initial implementation has been shown to be approximately an order of magnitude faster than BLAST. The formal split between word searching and alignment not only offers considerable gains in performance, but also allows alignment generation to be viewed as a user interface problem, allowing the most useful output method to be selected without affecting the underlying similarity search. Receiver Operator Characteristic (ROC) analysis of an artificial test set allows the optimal score threshold for identifying vector contamination to be determined. ROC curves were also used to determine the optimum word size (nine) for finding vector contamination. An analysis of the entire expressed sequence tag (EST) subset of EMBL found a contamination rate of 0.27%. A more detailed analysis of the 50 000 ESTs in est10.dat (an EST subset of EMBL) finds an error rate of 0.86%, principally due to two large-scale projects. AVAILABILITY: A Web page for the software exists at http://bioinf.man.ac.uk/rapid, or it can be downloaded from ftp://ftp.bioinf.man.ac.uk/RAPID CONTACT: [email protected] (+info)