Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

Purification of synthetic peptides. Immobilized metal ion affinity chromatography (IMAC).

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N-terminal metal-binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side-products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30-40 amino acid residues) were efficiently purified by this approach.     

Microbial colonization and succession on the faucial mucosa in newborn infants rooming in with their mothers in a maternity hospital]

The formation of microflora on the laryngeal mucosa in newborn infants during the first 5 days of their life was studied in one of the maternity hospitals of Moscow. In this work modern methods of the isolation and identification of aerobic and anaerobic microorganisms were used, and the results thus obtained were computer-processed. In the maternity hospital of the "mother-child" type the microbial colonization of the laryngeal mucosa by normal and opportunistic microorganisms was noted in newborn infants. A wave-like course of the formation of laryngeal microflora, indicative of microbial succession occurring in the child, was revealed. The attempt to establish the cases of microbial interference between the species colonizing the laryngeal mucosa revealed that it was very rarely observed in 5-day-old newborns. This feature was seemingly the cause of low resistance of the larynx to colonization in newborn infants, which determined frequent colonization of their laryngeal mucosa with Staphylococcus aureus and Klebsiella.     

One-step purification procedure for UDP-N-acetylmuramyl-peptide murein precursors from Bacillus cereus

A method is described for the rapid isolation of the activated murein precursors UDP-N-acetyl-muramyl-pentapeptide (UDP-MurNAc-pentapeptide) and UDP-MurNAc-tripeptide from Bacillus cereus. After accumulation of the precursors by inhibition of murein synthesis either in the presence of vancomycin (for the pentapeptide precursor) or D-cycloserine (for the tripeptide precursor) the cells were extracted with boiling water. Prior to high pressure liquid chromatography the material was freed from acid precipitable material. UDP-MurNAc-penta- and tripeptide were separated from other components by reversed-phase HPLC on Hypersil ODS using isocratic elution conditions with sodium phosphate buffer. The precursors were obtained with at least 98% purity and a yield of about 50 mumol from a 10-l culture of B. cereus.     

Bacteriophage receptors of Lactococcus lactis subsp. 'diacetylactis' F7/2 and Lactococcus lactis subsp. cremoris Wg2-1

Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L. lactis subsp. 'diacetylactis' F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L. lactis subsp. cremoris Wg2-1. Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions. However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments. This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors of strains F7/2 and Wg2-1.     

The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression

Complementary DNAs coding for four Desmodus rotundus salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPA alpha 1 and -alpha 2) contain a signal peptide, a finger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPA beta and -gamma lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAs.     

Polymorphic restriction sites of type II collagen gene: their location and frequencies in the Finnish population

Restriction fragment length polymorphism (RFLP) of the cartilage-specific type II collagen gene has been studied in the Finnish population. Two high-frequency alleles, also reported in other populations, were detected. The HindIII allele had a frequency of 0.33, and that detected with PvuII a frequency of 0.46. Both of these frequencies resembled the ones reported for other populations. Also one BamHI allele, not earlier reported, was found at a low frequency. Two other previously reported polymorphisms for BamHI and EcoRI were not detected in the Finnish population. The RFLPs showed a fair agreement with the Hardy-Weinberg equilibrium. A linkage disequilibrium was found between PvuII and HindIII markers. The alpha 1(II) collagen gene seems to be more conserved in populations of various origins than the alpha 2(I) collagen gene. These polymorphic collagen markers would be useful in linkage studies of various inherited cartilage disorders.     

Polymorphism of alpha-1-antitrypsin (Pi) in the Swiss population determined by isoelectric focusing with an immobilized pH gradient

The distribution of the phenotypes of alpha-1-antitrypsin (Pi) was investigated in a Swiss population sample of 1,148 unrelated individuals using isoelectric focusing with a immobilized pH gradient. A short focusing period of only 2 h using high-voltage is an additional asset of this modified method. All common as well as the rarer phenotypes were reliably detected. However, detection of Pi M4 required a narrower pH range as chosen for routine work. The allele frequencies found were: PiM1:0.7121; PiM2:0.1381; PiM3:0.0976; PiS:0.0383; PiZ:0.0113; PiVar(I, N, V.Vdon):0.0026.