Engineering evolution to study speciation in yeasts

The Saccharomyces 'sensu stricto' yeasts are a group of species that will mate with one another, but interspecific pairings produce sterile hybrids. A retrospective analysis of their genomes revealed that translocations between the chromosomes of these species do not correlate with the group's sequence-based phylogeny (that is, translocations do not drive the process of speciation). However, that analysis was unable to infer what contribution such rearrangements make to reproductive isolation between these organisms. Here, we report experiments that take an interventionist, rather than a retrospective approach to studying speciation, by reconfiguring the Saccharomyces cerevisiae genome so that it is collinear with that of Saccharomyces mikatae. We demonstrate that this imposed genomic collinearity allows the generation of interspecific hybrids that produce a large proportion of spores that are viable, but extensively aneuploid. We obtained similar results in crosses between wild-type S. cerevisiae and the naturally collinear species Saccharomyces paradoxus, but not with non-collinear crosses. This controlled comparison of the effect of chromosomal translocation on species barriers suggests a mechanism for the generation of redundancy in the S. cerevisiae genome.     

RecBCD enzyme is a bipolar DNA helicase

Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination. For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme. Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity. We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation. These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex. This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function.     

Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels

Haem is essential for living organisms, functioning as a crucial element in the redox-sensitive reaction centre in haemproteins. During the biogenesis of these proteins, the haem cofactor is typically incorporated enzymatically into the haem pockets of the apo-haemprotein as the functionally indispensable prosthetic group. A class of ion channel, the large-conductance calcium-dependent Slo1 BK channels, possesses a conserved haem-binding sequence motif. Here we present electrophysiological and structural evidence showing that haem directly regulates cloned human Slo1 channels and wild-type BK channels in rat brain. Both oxidized and reduced haem binds to the hSlo1 channel protein and profoundly inhibits transmembrane K+ currents by decreasing the frequency of channel opening. This direct regulation of the BK channel identifies a previously unknown role of haem as an acute signalling molecule.     

Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression

Reactive oxygen species are involved in many cellular metabolic and signalling processes and are thought to have a role in disease, particularly in carcinogenesis and ageing. We have generated mice with targeted inactivation of Prdx1, a member of the peroxiredoxin family of antioxidant enzymes. Here we show that mice lacking Prdx1 are viable and fertile but have a shortened lifespan owing to the development beginning at about 9 months of severe haemolytic anaemia and several malignant cancers, both of which are also observed at increased frequency in heterozygotes. The haemolytic anaemia is characterized by an increase in erythrocyte reactive oxygen species, leading to protein oxidation, haemoglobin instability, Heinz body formation and decreased erythrocyte lifespan. The malignancies include lymphomas, sarcomas and carcinomas, and are frequently associated with loss of Prdx1 expression in heterozygotes, which suggests that this protein functions as a tumour suppressor. Prdx1-deficient fibroblasts show decreased proliferation and increased sensitivity to oxidative DNA damage, whereas Prdx1-null mice have abnormalities in numbers, phenotype and function of natural killer cells. Our results implicate Prdx1 as an important defence against oxidants in ageing mice.     

Arrangement of nucleosomes in condensed chromatin fibres

Summary Condensed chromatin shows globules of 300 Å formed by 8 to 10 nucleosomes. Each globule might be an uncoiled turn of a supercoil. This supercoil forms major coils along the fibre.This work was supported by grants from Brazilian CNPq, FAPESP and FEDIB.We thank Dr A. Brunner Jr for the permission to use the electron microscope.     

Leibniz and the post-Copernican universe. Koyré revisited

This paper employs the revised conception of Leibniz emerging from recent research to reassess critically the ‘radical spiritual revolution’ which, according to Alexandre Koyré’s landmark book, From the closed world to the infinite universe (1957) was precipitated in the seventeenth century by the revolutions in physics, astronomy, and cosmology. While conceding that the cosmological revolution necessitated a reassessment of the place of value-concepts within cosmology, it argues that this reassessment did not entail a spiritual revolution of the kind assumed by Koyré, in which ‘value-concepts, such as perfection, harmony, meaning and aim’ were shed from the conception of the structure of the universe altogether. On the contrary, thanks to his pioneering intuition of the distinction between physical and metaphysical levels of explanation, Leibniz saw with great clarity that a scientific explanation of the universe which rejected the ‘closed world’ typical of Aristotelian cosmology did not necessarily require the abandonment of key metaphysical doctrines underlying the Aristotelian conception of the universe. Indeed the canon of value-concepts mentioned by Koyré—meaning, aim, perfection and harmony—reads like a list of the most important concepts underlying the Leibnizian conception of the metaphysical structure of the universe. Moreover, Leibniz’s universe, far from being a universe without God—because, as Clarke insinuated, it does not need intervention from God—is a universe which in its deepest ontological fabric is interwoven with the presence of God.     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.     

Regulatory functions of γδ T cells

γδ T cells share characteristics of innate and adaptive immune cells and are involved in a broad spectrum of pro-inflammatory functions. Nonetheless, there is accumulating evidence that γδ T cells also exhibit regulatory functions. In this review, we describe the different phenotypes of regulatory γδ T cells in correlation with the identified mechanisms of suppression.