Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.     

Purification of the herpes simplex virus type 1 65-kilodalton DNA-binding protein: properties of the protein and evidence of its association with the virus-encoded DNA polymerase.

Using a combination of conventional column chromatography and velocity sedimentation, we have purified the 65-kilodalton DNA-binding protein (65KDBP) encoded by herpes simplex virus (HSV) greater than 625-fold. The HSV type 1 (HSV-1)-encoded DNA polymerase (pol) cofractionated with 65KDBP through DEAE-Sephacel, Blue Sepharose, and Mono Q columns and was only separated from 65KDBP by sedimentation through a glycerol gradient. Immunoaffinity columns containing monoclonal antibody (MAb) 6898 immunoglobulin effectively bound most of the HSV-1 pol activity which coeluted with 65KDBP. The pattern of reactivities of HSV-1/HSV-2 recombinants with MAbs specific for HSV-1 65KDBP or the HSV-2-infected cell-specific protein ICSP34,35 strongly suggests that these two species are serotype equivalents of the same protein. Taken together, all these data indicate that 65KDBP is a pol-associated protein and the HSV-1 counterpart of HSV-2 ICSP34,35 previously reported to have similar properties (P. J. Vaughan, D. J. M. Purifoy, and K. L. Powell, J. Virol. 53:501-508, 1985). Purified preparations of 65KDBP were capable of binding to double-stranded DNA, as determined by filter retention and mobility shift assays. The protein-DNA complex formed with 65KDBP was distinct from that produced by pol and could be further shifted by the addition of immunoglobulin specific for 65KDBP. These results demonstrate that 65KDBP has been purified substantially free from pol and indicate that DNA binding is an inherent property of the protein.     

UNCOORDINATED PHYLOGEOGRAPHY OF BORRELIA BURGDORFERI AND ITS TICK VECTOR,IXODES SCAPULARIS

Vector‐borne microbes necessarily co‐occur with their hosts and vectors, but the degree to which they share common evolutionary or biogeographic histories remains unexplored. We examine the congruity of the evolutionary and biogeographic histories of the bacterium and vector of the Lyme disease system, the most prevalent vector‐borne disease in North America. In the eastern and midwestern US, Ixodes scapularis ticks are the primary vectors of Borrelia burgdorferi, the bacterium that causes Lyme disease. Our phylogeographic and demographic analyses of the 16S mitochondrial rDNA suggest that northern I. scapularis populations originated from very few migrants from the southeastern US that expanded rapidly in the Northeast and subsequently in the Midwest after the recession of the Pleistocene ice sheets. Despite this historical gene flow, current tick migration is restricted even between proximal sites within regions. In contrast, B. burgdorferi suffers no barriers to gene flow within the northeastern and midwestern regions but shows clear interregional migration barriers. Despite the intimate association of B. burgdorferi and I. scapularis, the population structure, evolutionary history, and historical biogeography of the pathogen are all contrary to its arthropod vector. In the case of Lyme disease, movements of infected vertebrate hosts may play a larger role in the contemporary expansion and homogenization of the pathogen than the movement of tick vectors whose populations continue to bear the historical signature of climate‐induced range shifts.     

Transcriptomic response to differentiation induction

Background  

Microarrays used for gene expression studies yield large amounts of data. The processing of such data typically leads to lists of differentially-regulated genes. A common terminal data analysis step is to map pathways of potentially interrelated genes.     

Urban amphibian assemblages as metacommunities

1. Urban ecosystems are expanding throughout the world, and urban ecology is attracting increasing research interest. Some authors have questioned the value of existing ecological theories for understanding the processes and consequences of urbanization. 2. In order to assess the applicability of metacommunity theory to urban systems, I evaluated three assumptions that underlie the theory - the effect of patch area, the effect of patch isolation, and species-environment relations - using data on assemblages of pond-breeding amphibians in the Greater Melbourne area of Australia. I also assessed the relative impact of habitat fragmentation, habitat isolation, and changes to habitat quality on these assemblages. 3. Poisson regression modelling provided support for an important increase in species richness with patch area (pond size) and a decrease in species richness with increasing patch isolation, as measured by surrounding road cover. Holding all other variables constant, species richness was predicted to be 2.8-5.5 times higher at the largest pond than at the smallest, while the most isolated pond was predicted to have 12-19% of the species richness of the least isolated pond. Thus, the data were consistent with the first two assumptions of metacommunity theory evaluated. 4. The quality of habitat at a pond was also important, with a predicted 44-56% decrease in the number of species detected at ponds with a surrounding vertical wall compared with those with a gently sloping bank. This demonstrates that environmental differences between habitat patches were also influencing amphibian assemblages, providing support for the species-sorting and/or mass-effect perspectives of metacommunity theory. 5. Without management intervention, urbanization may lead to a reduction in the number of amphibian species persisting in urban ponds, particularly where increasing isolation of ponds by roads and associated infrastructure reduces the probability of re-colonization following local extinction. Journal of Animal Ecology (2006) 75, 757-764 doi: 10.1111/j.1365-2656.2006.01096.x.     

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).     

Extracellular polymeric substances of the marine fouling diatom amphora rostrata Wm.Sm

Amphora rostrata was grown under continuous illumination at 27°C in batch cultures using f/2 medium. Cell biomass (measured as chllorophyll a and cell counts) reached a maximum on day 7. Thereafter, cell biomass as chl a showed a small decrease. Planktonic('free') and biofilm extracellular polymeric substances (EPS) from the adherent cells of A. rostrata were studied. Both types of EPS were produced during the logarithmic phase of growth. However, production was higher during the stationary growth phase. Enhanced EPS production was associated with nutrient deficient conditions. Planktonic and biofilm EPS were purified by gel filtration using Sephadex G‐200 and ion exchange chromatography using DEAE‐cellulose. Both polymers showed the presence of a single peak. Capillary gas Chromatographie analysis of both planktonic and biofilm EPS showed that fucose (36.7%) and galactose (27.6%) were the most abundant monosaccharides, with small quantities of rhamnose, xylose, arabinose, mannose and glucose. Other chemical analysis showed the presence of sulphate, uronic acids, hexoamines, pyruvate and proteins in both the planktonic and bio‐film EPS. Uronic acid, pyruvate and sulphate together were found to contribute ～50 to 60% (W/W) to the EPS of A. rostrata. Such a high content of non‐sugar components indicates their importance to the diatom in metal binding, desiccation prevention and flexibility.     

Structure and fragmentation of growling grass frog metapopulations

Metapopulations occur in fragmented landscapes, and consist of demographically-independent populations connected by dispersal. Nevertheless, anthropogenic habitat fragmentation may be fatal to metapopulations, as it disrupts dispersal and gene flow, and undermines the balance between population extinction and colonization. Understanding the extent to which particular land-use practices disrupt dispersal and gene flow is therefore crucial for conserving metapopulations. We examined the structure and fragmentation of metapopulations of the endangered growling grass frog (Litoria raniformis) in an urbanizing landscape in southern Australia. Population clustering analyses revealed three distinct genetic units, corresponding to the three wetland clusters sampled. Isolation-by-distance was apparent between populations, and genetic distance was significantly correlated with the presence of urban barriers between populations. Our study provides evidence that urbanization fragments metapopulations of L. raniformis. Managers of L. raniformis in urbanizing landscapes should seek to mitigate effects of urbanization on dispersal and gene flow.