A new method for DNA surface-property display and its application to E. coli promoter sequences

A personal computer program to visualize and compare the property of double-stranded DNA surface has been developed. Comparison of the surface property between Watson-Crick base-pairs in B-form DNA has elucidated that the base-pair replacement between a "degenerated base-pairs" conserves the pattern of potential hydrogen-bonding sites in both major and minor grooves. The idea of the "degenerated base-pairs" was applied for the problem of the base-sequence variation from the consensus sequence in the -35 region of E. coli promoter. The sequence variation is found to have tendency to occur among the degenerated base-pairs.     

A new technique for selection of sensitive and auxotrophic mutants of E. coli: isolation of a strain sensitive to an excess of one-carbon metabolites.

Summary An E. coli strain deleted in the region malasd is used for the selection of conditional or auxotrophic mutants. Thermosensitive and auxotrophic strains have thus been isolated on plates. After selection in liquid medium, a strain has been isolated which is sensitive to excess one-carbon metabolites. It carries two mutations, smg A (near metA and argH), probably identical to rel C, and smg B (between asn and ilv), probably part of the E. coli membrane ATPase.Abbreviations dap 1 meso diaminopimelic acid - smg serine+methionine+glycine - 1:1 1 per weight     

DNA cleavage by phenylhydroquinone: the major metabolite of a fungicide o-phenylphenol

The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.     

Development of an E. coli strain for cell-free ADC manufacturing

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody–drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.     

Two new E colicins, E8 and E9, produced by a strain of Escherichia coli

We have isolated a strain of Escherichia coli from chicken caeca which produces two E colicins and colicin M. This strain has seven plasmids, five of which have been transferred to E. coli K12. Two E. coli K12 derivatives which produce the two E colicins separately have been tested against seven standard E colicin producing strains which define seven different immunity groups. Our results indicate that these new E colicins define two further immunity groups, E8 and E9.     

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes,prepared without passaging through E. coli

Background  

Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.     

Construction and characterization of an Escherichia coli strain with a uncI mutation.

A strain of Escherichia coli with a mutation in the promoter proximal gene ( uncI ) of the unc operon has been constructed by using a new gene replacement method. The mutation is a deletion of a defined sequence of 196 base pairs. It was constructed by homologous integration and segregation of a ColE1-derived recombinant plasmid containing the mutation, in a temperature-sensitive polA strain. The mutant strain is phenotypically unc+ but has a reduced growth yield compared to a normal sibling strain.     

Stereoselective analysis of metoprolol and its metabolites in rat plasma with application to oxidative metabolism

We investigated the stereoselective kinetic disposition and metabolism of metoprolol (MET) in rats. The racemic MET (15 mg/kg) was given by oral gavage and blood samples were collected from 0 to 10h (n=6 at each time point). The enantiomeric concentrations of MET and its metabolites alpha-hydroxymetoprolol (alpha-OHM) and O-demethylmetoprolol (ODM) were determined by HPLC using a Chiralpak AD chiral column and fluorescence detection. The pharmacokinetic parameters of unchanged MET and the formation of ODM did not show to be stereoselective. In contrast, the AUC (ng h/mL) of alpha-hydroxymetoprolol isomers were higher to I'R [638.2(525.2-706.2) for 1'R2R and 659.6(580.4-698.1) for 1'R,2S, mean, (95%CI)] than to I'S products [58.3(47.4-66.1) for 1'S,2R and 57.1(44.7-67.9) for 1'S,2S, mean, (95%CI)]. We conclude that the kinetic disposition of unchanged MET and the formation of ODM are not enantioselective in rats but the metabolism of alpha-OHM yields predominantly the 1'R-product.     

N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli

274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.     

Complement-mediated bactericidal activity of human milk to a serum-susceptible strain of E. coli 0111

There has been a lot of controversy concerning the physiological significance of the complement system in human breast-milk. This is mainly due to the observation that human milk contains predominantly non-inflammatory and many anti-inflammatory factors, while simultaneously protecting the infant against a wide range of infectious and other diseases. The present study was carried out to assess the contribution of the complement system to the bactericidal activity of the human colostrum and early lactational milk. Using a serum-sensitive strain of Escherichia coli, different fractions of human breast-milk were assessed for their ability to kill the bacteria, with and without inactivation of their complement components, in comparison to another strain of the bacteria species. Deposition of activated C3 fragments on the killed bacteria, using an established ELISA technique, was demonstrated, further proving that the human milk complement could be activated in vitro. The bactericidal activities of human milk were almost completely abolished by complement heat inactivation at 56 degrees C or by the addition of EDTA.