Joachim Greipel

Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome

In 545 Pseudomonas aeruginosa strains, mainly collected from patients with cystic fibrosis, Spel‐Dral macrorestriction fragment lenght diversity was scanned for using probes of known map position on th P.earuginosa PAO chromosome. Southern analysis of the 60 unrelated clones uncovered a gradient of macrorestriction fragment lenght polymorphisms (RFLPs) from the origin of replication towards the auxotroh‐poor region of the P. aeruginosa population in the region encompassed by the rrn operons. The oriC‐reactive Spel fragment was conserved in nearly all isolates examined. Few fragment lenght classes were seen for the alga60‐, algR‐ and toxA‐encoding Spel fragments. Fragment siz varied within one class by up to 20 kb. Two probes from the auxotroph‐poor region detected a broad size range for the Spel fragment, suggestiong extensive genomic deversity in these reions. Subclonalvariation of fragment size was detected at all investigated loci in at least one of the analysed clones, but within one particlular clone, Spel‐RFLPs were found at only few loci.

Gas chromatographic–tandem mass spectrometric determination of acetylsalicylic acid in human plasma after oral administration of low-dose aspirin and guaimesal

A fully validated gas chromatographic-tandem mass spectrometric (GC-MS-MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[2H3]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-microl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC-MS-MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0-1 microg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0-100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.

Complexes of the single-stranded DNA-binding protein from Escherichia coli (Eco SSB) with poly(dT): an investigation of their structure and internal dynamics by means of electron microscopy and NMR

Based on electron microscopy and NMR spectroscopy it is deduced that Eco SSB binds with moderate cooperativity to polynucleotides. Evidence is provided that the protein binds in its tetrameric form to the nucleic acid forming a nucleosome-like structure. NMR-spectroscopic analysis of the complexes shows that the carboxy-terminal region of the Eco SSB maintains a high flexibility even when the protein is immobilized in large protein-protein clusters.

The single-stranded DNA binding protein of Escherichia coli: physicochemical properties and biological functions

Single-stranded DNA binding proteins fulfil important functions in DNA metabolism. They have been shown to be essential for replication, recombination and repair in bacteria and bacteriophages. The best-studied single-stranded DNA binding proteins are the gene32 protein from T4-phage (gp32) (for reviews, cf. Kowalczykowski et al., 1981; Chase and Williams, 1986; Chase, 1984), the gene5 protein from filamentous phages (gp5) (for reviews, cf. Kowalczykowski et al., 1981), and the E. coli single-stranded DNA binding protein.