Effect of target gene CpG content on spontaneous mutation in transgenic mice

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10⁻⁵ range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.     

The interaction between saliva and Actinobacillus actinomycetemcomitans influenced by the Zeta potential

The adhesion of Actinobacillus actinomycetemcomitans is a virulence factor in the aetiology of periodontitis and is determined by physico-chemical properties, e.g. surface charge and hydrophobicity, of the bacterial cell surface. Although oral surfaces are constantly coated with saliva, few studies have dealt with the binding of A. actinomycetemcomitans with saliva. In this report, the charge properties of A. actinomycetemcomitans have been studied through measurement of the zeta potential and the saliva-bacteria interaction investigated at different pH-values.At physiological conditions the zeta potential was negative, varying from -11 to -26 mV, for two laboratory and two fresh isolates of A. actinomycetemcomitans. Under these conditions, binding of the low-molecular-weight salivary mucin, lactoferrin, and S-IgA was confirmed using salivary samples and purified salivary fractions in liquid-phase and in ELISA. The iso-electric points of the laboratory and fresh clinical isolates of A. actinomycetemcomitans were determined at pH 4.6 and 3.8, respectively. At pH below the iso-electric point, giving positive values of the zeta potential, additional salivary protein species bound to A. actinomycetemcomitans, including the high-molecular-weight salivary mucin (MG1) and agglutinin. Binding of the low-molecular-weight salivary mucin (MG2), lactoferrin, and S-IgA, was hardly affected by this change in zeta potential. A salivary coating formed on the bacterium at pH 7 reduced the zeta potential of the laboratory strain Y4 greatly and an iso-electric point for the bacterium could not be determined. Overall, the study suggests that upon changes in environmental pH additional salivary attachment sites on the micro-organism are exposed.     

Soil organic matter molecular composition with long-term detrital alterations is controlled by site-specific forest properties

Forest ecosystems are important global soil carbon (C) reservoirs, but their capacity to sequester C is susceptible to climate change factors that alter the quantity and quality of C inputs. To better understand forest soil C responses to altered C inputs, we integrated three molecular composition published data sets of soil organic matter (SOM) and soil microbial communities for mineral soils after 20 years of detrital input and removal treatments in two deciduous forests: Bousson Forest (BF), Harvard Forest (HF), and a coniferous forest: H.J. Andrews Forest (HJA). Soil C turnover times were estimated from radiocarbon measurements and compared with the molecular-level data (based on nuclear magnetic resonance and specific analysis of plant- and microbial-derived compounds) to better understand how ecosystem properties control soil C biogeochemistry and dynamics. Doubled aboveground litter additions did not increase soil C for any of the forests studied likely due to long-term soil priming. The degree of SOM decomposition was higher for bacteria-dominated sites with higher nitrogen (N) availability while lower for the N-poor coniferous forest. Litter exclusions significantly decreased soil C, increased SOM decomposition state, and led to the adaptation of the microbial communities to changes in available substrates. Finally, although aboveground litter determined soil C dynamics and its molecular composition in the coniferous forest (HJA), belowground litter appeared to be more influential in broadleaf deciduous forests (BH and HF). This synthesis demonstrates that inherent ecosystem properties regulate how soil C dynamics change with litter manipulations at the molecular-level. Across the forests studied, 20 years of litter additions did not enhance soil C content, whereas litter reductions negatively impacted soil C concentrations. These results indicate that soil C biogeochemistry at these temperate forests is highly sensitive to changes in litter deposition, which are a product of environmental change drivers.     

Occluding Junctions in MDCK Cells: Modulation of Transepithelial Permeability by the Cytoskeleton

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In addition we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with M_r of ≥ 120K daltons as well as peptides with M_r = 56K, 50K, 36K, and 18K daltons.     

Acid phosphatase interactions with organo-mineral complexes: influence on catalytic activity

The influence of montmorillonite intercalated with representatives of three major classes of biological molecules-lysine (amino acid), –glucose (carbohydrate) and rhamnolipid (lipid) on the catalytic activity of acid phosphatase was investigated. In comparison to pure clay, the presence of the organic intercalates preserves residual activity at extreme pH values of 3 and 11 and temperatures as low as 10°C. Thermodynamic parameters of free energy, enthalpy and entropy, suggest that catalytic activity on the lysine and rhamnolipid intercalated surfaces is more spontaneous and favorable than that of pure clay. Michaelis-constants (K _m values) and maximum reaction velocities (V _max values) were determined and confirmed the enhancement of activity on the organo-mineral surfaces. The catalytic reaction product was measured as a function of time and the data fitted to equations describing the behavior of first and second order rates of reaction. All processes apart from the glucose-intercalated clay (second order) could be described by first order reactions. Catalytic activity was generally less on the glucose-mineral surface compared to the other organo-mineral surfaces and the pure clay. However, when all surfaces were saturated with acid phosphatase the glucose complex exhibited the highest level of catalytic activity.     

Replication of non-repetitive DNA during early, middle and late S phase : The general class and the subset transcribed

Synchronized cultures of Chinese hamster ovary cells were pulse-labelled with 5-bromodeoxyuridine (BrdU) during early (0-2.0 h), middle (2.5-4.0 h) and late (4.5-6.0 h) S phase in two successive cell cycles. In each case, the DNA containing BrdU in both strands was duplicated at the same time in both cycles and was isolated for further characterization by centrifugation in CsCl gradients. These DNAs were then radiolabelled by nick-translation and used in either DNA-DNA or RNA-DNA hybridization experiments. In the DNA-DNA experiments, advantage was taken of the substantial rate increases attainable in high concentrations of dextran sulfate to obtain complete reassociation curves with relatively small amounts of material. Assuming that no unresolved low repetition frequency components exist, renaturation kinetics suggest that early replicating DNA contains a greater proportion of non-repetitive sequences than DNA synthesized at later times, the order being early greater than middle greater than late. However, in terms of complexity the non-repeated DNA duplicated early had only 74% of the diverse sequences present in log-phase cells, whereas that replicated in middle and late S phase had 82 and 79.5%, respectively. It therefore appears that while DNA synthesized at different times in S phase may contain varying proportions of non-repetitive sequences, when their diversity is taken into account very few of these sequences (25% or less) exhibit temporal control of replication. Finally, measurements with total cell RNA indicated that the transcribed fraction of non-repeated DNA showed a slight preference for replication in early S phase.     

Interaction between the yellow fever virus nonstructural protein NS3 and the host protein Alix contributes to the release of infectious particles

The ESCRT (endosomal sorting complex required for transport) machinery normally executes cargo sorting and internalization during multivesicular body biogenesis, but is also utilized by several enveloped viruses to facilitate their budding from cellular membranes. Although the mechanisms of flavivirus infectious particle assembly and release are poorly understood, the nonstructural protein NS3 has been reported to have an essential role via an undescribed mechanism. Here, we shed light on the role of NS3 by connecting it to the host factor Alix, a protein intimately connected with the ESCRT machinery. We demonstrate that NS3 and Alix interact and show that dominant negative versions of Alix inhibit YFV release. Furthermore, we show that NS3 supplied in trans rescues this effect. We propose that the interaction between NS3 and Alix contributes to YFV release.