Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to >95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.     

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Culex quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in Escherichia coli we have now demonstrated time- and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10× expression compared to the equivalent region (lacking CuRE1) from the susceptible strain. Close correspondence with the gene expression fold-change implicates the upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1 bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic. When applied to C. quinquefasciatus larvae from Ghana we have demonstrated a significant association with permethrin resistance in multiple field sites (mean Odds Ratio?=?3.86) suggesting this marker has relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs contribute regulatory motifs involved in gene expression may be necessary.     

Wood-inhabiting,polyporoid fungi in aspen-dominated forests managed for biomass in the U.S. Lake States

To better understand the potential long-term effects of biomass harvesting on biodiversity, the polyporoid fungi community was characterized from 120 plots in four aspen-dominated forests in Minnesota. Four deadwood variables (substratum species, substratum type, decay class and diameter class) were recorded for each polyporoid species occurrence. A total of 2 358 polyporoid occurrences, representing 86 species, were recorded on 16 tree species. Eight species (Trichaptum biforme, Bjerkandera adusta, Trametes hirsuta, Phellinus tremulae, Fomes fomentarius, Irpex lacteus, Fomitopsis ochracea and Antrodia serialis) made up 67 % of occurrences. Four polyporoid species (Funalia trogii, Pycnoporellus fulgens, Rigidoporus crocatus and Skeletocutis chrysella) are potentially rare and/or threatened in the Lake States. Non-metric multidimensional scaling and rarefaction curves demonstrated that small diameter substrata (especially those <5 cm) most strongly influenced polyporoid species occurrences. Aspen-dominated systems show great potential for biomass production, but these forests also support a species-rich community of polyporoid fungi, including potentially rare species.     

On the Interplay of Telomeres,Nevi and the Risk of Melanoma

The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.     

Role of human CD36 in bacterial recognition, phagocytosis, and pathogen-induced JNK-mediated signaling

Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6 signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacterium, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus, and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both gram-positive and gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36 transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with gram-positive bacteria and lipoteichoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40-50% in human fibroblasts induced by both gram-negative and gram-positive bacteria as well as LPS. Of all MAPK signaling cascade inhibitors tested, only the inhibitor of JNK, a stress-activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-kappaB inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an alpha-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner.     

Evaluation of Cruzia americana, Turgida turgida, and Didelphostrongylus hayesi infection in the Virginia opossum (Didelphis virginiana) and risk factors along the California coast

Three nematodes, Turgida turgida, Cruzia americana, and Didelphostrongylus hayesi, have been documented to cause morbidity and mortality in the Virginia opossum (Didelphis virginiana). The present study was designed to determine the frequency of infection of these nematodes in opossums at 2 study sites in California and to determine if there are risk factors associated with shedding of eggs or larvae in the feces. Turgida turgida and C. americana adults were found in 84.4% (stomach; n = 45) and 62.5% (intestinal wash and feces; n = 16) of sampled opossums. Eggs were present in opossum feces (n = 105) less frequently (40% T. turgida and 35.2% C. americana). Didelphostrongylus hayesi larvae were found in 79.0% of opossum feces examined (n = 105). Adult age and wet season (December through April) were significant predictive factors for the presence of T. turgida eggs, whereas the dry season (May through November) was significantly associated with the presence of C. americana eggs in feces. Adult opossums were more likely to have eggs and larvae from all 3 nematodes in the feces.     

COLONY SIZE OF PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE) AS INFLUENCED BY ZOOPLANKTON GRAZERS1

The haptophyte Phaeocystis antarctica G. Karst. is a dominant phytoplankton species in the Ross Sea, Antarctica, and exists as solitary cells and mucilaginous colonies that differ by several orders of magnitude in size. Recent studies with Phaeocystis globosa suggest that colony formation and enlargement are defense mechanisms against small grazers. To test if a similar grazer‐induced morphological response exists in P. antarctica, we conducted incubation experiments during the austral summer using natural P. antarctica and zooplankton assemblages. Dialysis bags that allowed exchange of dissolved chemicals were used to separate P. antarctica and zooplankton during incubations. Geometric mean colony size decreased by 35% in the control, but increased by 30% in the presence of grazers (even without physical contact) over the 15 d incubation. The estimated colonial‐to‐solitary cell carbon ratio was significantly higher in the grazing treatment. These results suggest that P. antarctica colonies would grow larger in the presence of indigenous zooplankton and skew the carbon partitioning significantly toward the colonial phase. While these observations show that the colony size of P. antarctica was affected by a chemical signal related to grazers, the detailed nature and ecological significance of this signal remain unknown.