Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

Vegetation development on pumice at Mount St. Helens, USA

This study explores early vegetation development on pumice at Mount St.Helens. We monitored species annually in a grid of 200 contiguous100-m² plots between 1989 and 1999. Of interestwere how vegetation changed and if it became more homogeneous over time.Speciesrichness and cover increased annually. Diversity(H) stabilized by 1996 and began to decline aslong-livedstress-tolerant species such as Agrostis pallens, Carexmertensii and Penstemon cardwellii began todominate. Protected sites had more species and higher cover than did exposedones. Plots next to relict vegetation had more species and cover than diddistant plots. The vegetation initially was dominated by species with gooddispersal, but subsequently those with poor dispersal became dominant. Wecompared species expansion patterns to a model based on random colonization.Theresults implied that populations with poor dispersal derived from a fewcolonists that then produced seeds for local expansion. Detrendedcorrespondenceanalysis showed a pronounced shift in species composition. This analysis alsoshowed that species composition was becoming more homogeneous over time.However, significant heterogeneity remained and some plots are diverging fromothers. As yet, this vegetation is not developing towards a regional vegetationtype. Rather, an unusual community with Agrostis spp.,Carex spp., Penstemon cardwellii, Lupinuslepidus, Anaphalis margaritacea and Salixcommutata has developed. The accumulation phase of primarysuccessionis nearly complete. The next phase, in which vertical structure develops asSalix and conifers mature, has scarcely begun. It shouldbemarked by the invasion of forest understory species and loss of subalpinemeadowspecies. Assembly rules based on biotic interactions may then become evident.     

Ephrins in reverse,park and drive

Eph receptors and their membrane-anchored ephrin ligands are thought to orchestrate cell movements by transducing bidirectional tyrosine-kinase-mediated signals into both cells expressing the receptors and cells expressing the ligands. Whether the resulting event is repulsion of an axonal growth cone, directing the orderly segmentation of hindbrain rhombomere cells or controlling angiogenic remodelling, such elaborate and diverse cell movements require intricate changes in the actin cytoskeleton, as well as precise regulation of cellular adhesion. Recent work by several groups has begun to link ephrin reverse signals to intracellular pathways that regulate actin dynamics and might help to explain how these ligands function as receptors to direct cell movement, adhesion and de-adhesion events.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Noncontact dipole effects on channel permeation. VI. 5F- and 6F-Trp gramicidin channel currents

Fluorination of peptide side chains has been shown to perturb gramicidin channel conductance without significantly changing the average side chain structure, which, it is hoped, will allow detailed analysis of electrostatic modulation of current flow. Here we report a 1312-point potassium current-voltage-concentration data set for homodimeric channels formed from gramicidin A (gA) or any of eight fluorinated Trp analogs in both lecithin and monoglyceride bilayers. We fit the data with a three-barrier, two-site, two-ion (3B2S) kinetic model. The fluorination-induced changes in the rate constants were constrained by the same factor in both lipids. The rate constant changes were converted to transition-state free-energy differences for comparison with previous electrostatic potential energy differences based on an ab initio force field. The model allowed a reasonably good fit (chi = 8.29 with 1271 degrees of freedom). The measured changes were subtle. Nevertheless, the fitted energy perturbations agree well with electrostatic predictions for five of the eight peptides. For the other three analogs, the fitted changes suggested a reduced translocation barrier rather than the reduced exit barrier as predicted by electrostatics.     

Dynamics and responses to mortality rates of competing predators undergoing predator-prey cycles

Two or more competing predators can coexist using a single homogeneous prey species if the system containing all three undergoes internally generated fluctuations in density. However, the dynamics of species that coexist via this mechanism have not been extensively explored. Here, we examine both the nature of the dynamics and the responses of the mean densities of each predator to mortality imposed upon it or its competitor. The analysis of dynamics uncovers several previously undescribed behaviors for this model, including chaotic fluctuations, and long-term transients that differ significantly from the ultimate patterns of fluctuations. The limiting dynamics of the system can be loosely classified as synchronous cycles, asynchronous cycles, and chaotic dynamics. Synchronous cycles are simple limit cycles with highly positively correlated densities of the two predator species. Asynchronous cycles are limit cycles, frequently of complex form, including a significant period during which prey density is nearly constant while one predator gradually, monotonically replaces the other. Chaotic dynamics are aperiodic and generally have intermediate correlations between predator densities. Continuous changes in density-independent mortality rates often lead to abrupt transitions in mean population sizes, and increases in the mortality rate of one predator may decrease the population size of the competing predator. Similarly, increases in the immigration rate of one predator may decrease its own density and increase the density of the other predator. Proportional changes in one predator's birth and death rate functions can have significant effects on the dynamics and mean densities of both predator species. All of these responses to environmental change differ from those observed when competitors coexist stably as the result of resource (prey) partitioning. The patterns described here occur in many other competition models in which there are cycles and differences in the linearity of the responses of consumers to their resources.