Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Kinetic Partitioning Modulates Human Telomere DNA G-Quadruplex Structural Polymorphism

Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T₂AG₃)_n, which is predominantly double-stranded but terminates with a 3’ single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs) that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET), we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K⁺ ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ structures.     

Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress

The constitutive and drought-induced activities of the Arabidopsis thaliana RD29A and RD29B promoters were monitored in soybean (Glycine max (L.) Merr.] via fusions with the visual marker gene β-glucuronidase (GUS). Physiological responses of soybean plants were monitored over 9 days of water deprivation under greenhouse conditions. Data were used to select appropriate time points to monitor the activities of the respective promoter elements. Qualitative and quantitative assays for GUS expression were conducted in root and leaf tissues, from plants under well-watered and dry-down conditions. Both RD29A and RD29B promoters were significantly activated in soybean plants subjected to dry-down conditions. However, a low level of constitutive promoter activity was also observed in both root and leaves of plants under well-watered conditions. GUS expression was notably higher in roots than in leaves. These observations suggest that the respective drought-responsive regulatory elements present in the RD29X promoters may be useful in controlling targeted transgenes to mitigate abiotic stress in soybean, provided the transgene under control of these promoters does not invoke agronomic penalties with leaky expression when no abiotic stress is imposed.     

Book reviews

John B. Boles (ed.), MASTERS AND SLAVES IN THE HOUSE OF THE LORD: RACE AND RELIGION IN THE AMERICAN SOUTH, 1740–1870, Lexington, KY: The University of Kentucky Press, 1988, 257 pp.

David E. Swift, BLACK PROPHETS OF JUSTICE: ACTIVIST CLERGY BEFORE THE CIVIL WAR, Baton Rouge, Louisiana, LA: Louisiana State University Press, 1989, xv + 384 pp., £31.50.

Alexander B. Murphy, THE REGIONAL DYNAMICS OF LANGUAGE DIFFERENTIATION IN BELGIUM: A STUDY IN CULTURAL‐POLITICAL GEOGRAPHY, Chicago, IL: University of Chicago, Geography Research Paper No. 227, 1988, xiii + 249 pp., $12.00.

Pierre L. van den Berghe, STRANGER IN THEIR MIDST, Denver, CO: University Press of Colorado, 1989, xvii + 300 pp., $24.95.     

Teaching ethnic and racial relations: some comments on Michael Banton

Banton's critique of race and ethnic relations highlights differences between the U.K. and the U.S.A. The interplay between ethnic politics and the teaching of the subject in the universities reflects differing reactions to the demise of Marxism, the impact of globalization and the growth of transnationalism. In America, anthropology, economics, cultural studies and conflict resolution have had a mixed impact on the field, but an examination of textbooks over the past thirty years - as well as Banton's prolific scholarship - suggests that most of the core problems remain the same.     

Identification of the N-Terminal Domain of the Influenza Virus PA Responsible for the Suppression of Host Protein Synthesis

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA''s reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.     

Deforestation of watersheds of Panama: nutrient retention and export to streams

A series of eight watersheds on the Pacific coast of Panama where conversion of mature lowland wet forest to pastures by artisanal burning provided watershed-scale experimental units with a wide range of forest cover (23, 29, 47, 56, 66, 73, 73, 91, and 92 %). We used these watersheds as a landscape-scale experiment to assess effects of degree of deforestation on within-watershed retention and hydrological export of atmospheric inputs of nutrients. Retention was estimated by comparing rainfall nutrient concentrations (volume-weighted to allow for evapotranspiration) to concentrations in freshwater reaches of receiving streams. Retention of rain-derived nutrients in these Panama watersheds averaged 77, 85, 80, and 62 % for nitrate, ammonium, dissolved organic N, and phosphate, respectively. Retention of rain-derived inorganic nitrogen, however, depended on watershed cover: retention of nitrate and ammonium in pasture-dominated watersheds was 95 and 98 %, while fully forested watersheds retained 65 and 80 % of atmospheric nitrate and ammonium inputs. Watershed forest cover did not affect retention of dissolved organic nitrogen and phosphate. Exports from more forested watersheds yielded DIN/P near 16, while pasture-dominated watersheds exported N/P near 2. The differences in magnitude of exports and ratios suggest that deforestation in these Panamanian forests results in exports that affect growth of plants and algae in the receiving stream and estuarine ecosystems. Watershed retention of dissolved inorganic nitrogen calculated from wet plus dry atmospheric deposition varied from 90 % in pasture- to 65 % in forest-dominated watersheds, respectively. Discharges of DIN to receiving waters from the watersheds therefore rose from 10 % of atmospheric inputs for pasture-dominated watersheds, to about 35 % of atmospheric inputs for fully forested watersheds. These results from watersheds with no agriculture or urbanization, but different conversion of forest to pasture by burning, show significant, deforestation-dependent retention within tropical watersheds, but also ecologically significant, and deforestation-dependent, exports that are biologically significant because of the paucity of nutrients in receiving tropical stream and coastal waters.     

The taxonomic identity and physiological ecology of Chlamydomonas hedleyi sp. nov. algal flagellate symbiont from the foraminifer Archaias angulatus

The fine structure of the symbiotic alga isolated from the formainiferan Archaias angulatus (Fichtel et Moll) DeMontfort is typical of the Chlorophyceae of the volvocalean and chlorococcalean lines. Spherical non-motile cells, 10–14 μm in diameter, characterise the dominant life cycle phase. Long oval motile forms with truncated apices are present 3–5 days after transfer to fresh medium. The pyrenoids are embedded anteriorly in the singly bilobed chloroplast and are surrounded by a sheath of starch platelets. In spite of the non-motile state of cells in older cultures (which is perhaps a reflection of its normally symbiotic condition), the alga is identified as a species of the volvocalean genus Chlamydomonas and is named C. hedleyi sp. nov.

The symbiont has no vitamin or organic requirements but growth is increased threefold in the presence of thiamine, and twofold in the presence of 1 μm glutamic acid, histidine and methionine. Urea was the best nitrogen source tested. Purines and pyrimidines did not serve as nitrogen sources. Chlamydomonas hedleyi grows well in a salinity range of 6->52‰. and a pH range of 6–8·5, 7·04 × 10^-7 m carbon h^-1 g^-1 was fixed by the symbiont, 57% being released into the medium as a chromatographically homogeneous organic molecule provisionally identified as mannitol.     

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.