Design principles for site-selective hydroxylation by a Rieske oxygenase

Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.

Hidden genetic variation in plasticity increases the potential to adapt to novel environments

Adaptive plasticity increases population persistence, but can slow adaptation to changing environments by hiding the effects of different alleles on fitness. However, if plastic responses are no longer adaptive in novel environments, then differences among alleles can emerge and increase genetic variation in fitness that allows rapid adaptation. We tested this hypothesis by transplanting cuttings and seeds of a Sicilian daisy within and outside its native range, and quantifying variation in morphology, physiology, gene expression and fitness. We show that genetic variance in plasticity increases the potential for rapid adaptation to novel environments. Genetic variation in fitness was low across native environments where plasticity effectively tracked familiar environments. In the novel environment however, genetic variation in fitness increased threefold, and correlated with genetic variation in plasticity. Furthermore, genetic variation that can increase fitness in the novel environment had the lowest fitness at the native site, suggesting that adaptation to novel environments relies on genetic variation in plasticity that is selected against in native environments.

Repositioning the Sm-Binding Site in Saccharomyces cerevisiae Telomerase RNA Reveals RNP Organizational Flexibility and Sm-Directed 3′-End Formation

Telomerase RNA contains a template for synthesizing telomeric DNA and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase RNA, TLC1, is bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. However, it remains unclear (1) whether Sm7 retains this function when its binding site is repositioned within TLC1, as has been shown for other TLC1-binding telomerase subunits, and (2) how Sm7 stabilizes poly(A)– TLC1. Here, we first show that Sm7 can stabilize poly(A)– TLC1 even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we show that when an Sm site is inserted 5′ of its native position and the native site is mutated, Sm7 stabilizes shorter forms of poly(A)– TLC1 in a manner corresponding to how far upstream the new site was inserted, providing strong evidence that Sm7 binding to TLC1 controls where the mature poly(A)– 3′ is formed by directing a 3′-to-5′ processing mechanism. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

The formation of the population of the fern Matteuccia struthiopteris in the University Grove of Tomsk University

Matteuccia struthiopteris (L.) Tod. – Ostrich fern (Onocleaceae) is the circumboreal species widely distributed in the temperate zone of the northern hemisphere. In the paper, we presented the results of the observations on the developing population of M. struthiopteris on the man-made lawn situated in the University Grove of Tomsk State University (Tomsk, Russian Federation). In the original making of the lawn, the ground was replaced by new rich soil, which was taken in early summer of 2007 from a native site situated 6.65 km towards south-west from Tomsk University. The formation of the plant community on the new lawn occurred mostly naturally, and sporophytes of this fern appeared naturally. The first sporophytes on the new lawn were detected in 2009, occurring initially in the significant numbers, recorded here. During the time of observation (2009–2018) the number of sporophytes decreased from an original 263 to 96, i.e. 63.5% of sporophytes died. The initial population in 2009 included only juvenile sporophytes. No spore-bearing sporophytes appear during the period of observation. For detection of the source of spores, from which the population on the lawn was formed, we selected three populations having fertile sporophytes, and conducted a molecular-genetic analysis. Two of the analyzed source populations were found to be situated at 160–365 m distance from the developing population and one population occurs near the place where the soil for new lawn making was taken. We used the ISSR method for analyzing the genetic diversity of populations and processed the results by software STRUCTURE Version 2.3.4. The result show, that new population, and population from the soil taking point belong to the same group. The coefficient of Nei’s genetic identity between these populations is high (I = 0.931). This confirms the origin of the new population from spores contained in the soil used for the lawn construction.

The formation of the population of the fern Matteuccia struthiopteris in the University Grove of Tomsk University

Matteuccia struthiopteris (L.) Tod. – Ostrich fern (Onocleaceae) is the circumboreal species widely distributed in the temperate zone of the northern hemisphere. In the paper, we presented the results of the observations on the developing population of M. struthiopteris on the man-made lawn situated in the University Grove of Tomsk State University (Tomsk, Russian Federation). In the original making of the lawn, the ground was replaced by new rich soil, which was taken in early summer of 2007 from a native site situated 6.65 km towards south-west from Tomsk University. The formation of the plant community on the new lawn occurred mostly naturally, and sporophytes of this fern appeared naturally. The first sporophytes on the new lawn were detected in 2009, occurring initially in the significant numbers, recorded here. During the time of observation (2009–2018) the number of sporophytes decreased from an original 263 to 96, i.e. 63.5% of sporophytes died. The initial population in 2009 included only juvenile sporophytes. No spore-bearing sporophytes appear during the period of observation. For detection of the source of spores, from which the population on the lawn was formed, we selected three populations having fertile sporophytes, and conducted a molecular-genetic analysis. Two of the analyzed source populations were found to be situated at 160–365 m distance from the developing population and one population occurs near the place where the soil for new lawn making was taken. We used the ISSR method for analyzing the genetic diversity of populations and processed the results by software STRUCTURE Version 2.3.4. The result show, that new population, and population from the soil taking point belong to the same group. The coefficient of Nei’s genetic identity between these populations is high (I = 0.931). This confirms the origin of the new population from spores contained in the soil used for the lawn construction.

The Reconstruction of Buddhist Landscape Planning of Kagyu Samye Ling Monastery in Scotland

By comparing the differences in the Buddhist landscape between Scottish Kagyu Samye Ling Monastery and the Samye Ling Temple in Tibet, the article points out the former has three problems such as an inadequate Buddha-Hall, unbalanced distribution of Buddhist landscapes and incomplete Mandala landscape pattern. It is proposed that to enlarge Buddha-Hall and build out corridors for more circumambulations, and to construct new landscape in four directions to form a complete Buddhist pattern including Dvipa and Eight Small Continents. The conclusion is the reconstruction of Kagyu Samye Ling Monastery in Scotland brings new spatial distinction, which will enhance its influence and sustainable development.So the modernity method of Tibetan Buddhist temples overseas is based on the inheriting the traditional Tibetan Mandala Buddhist pattern, and then to develop the Five-Holy style Buddhist landscape pattern which inculturation with the native site situation.

Pythium spp. Associated with Rooibos Seedlings, and Their Pathogenicity Toward Rooibos, Lupin, and Oat

Rooibos (Aspalathus linearis) is an important indigenous crop in South Africa. Oomycetes are a common problem in rooibos nurseries, causing serious losses, but limited information is available on the species involved. Molecular and morphological analyses of 117 oomycete isolates from 19 rooibos nurseries and 33 isolates from 11 native rooibos sites revealed the presence of several Pythium spp., including Pythium acanthicum, P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, P. cederbergense, and Pythium RB II, and Phytophthora cinnamomi (native site). Most of the species were identified in nurseries and native rooibos, with Pythium irregulare being the most common species occurring in all nurseries and 46% of the native sites. Phylogenetic analyses of the internal transcribed spacer region of the P. irregulare isolates showed that isolates within this species complex fit into three subclades, of which only two have previously been reported. On rooibos, all species except P. acanthicum and the previously characterized P. cederbergense and Pythium RB II were pathogenic and highly virulent. On lupin and oat, rotation crops in nurseries, the three aforementioned species were also nonpathogenic. All the other oomycete species were pathogenic on lupin but less so than on rooibos. On oat, only P. irregulare, P. myriotylum, and P. pyrilobum were pathogenic. This is the first report of P. mamillatum, P. pyrilobum, and P. myriotylum as pathogens of lupin, and P. irregulare and P. pyrilobum as pathogens of oat. The three nonpathogenic Pythium spp. were able to significantly reduce disease caused by pathogenic species in the less susceptible lupin and oat but not on rooibos. On lupin, the nonpathogenic species enhanced the virulence of Phytophthora cinnamomi.

The Chromosomal Basis of Cancer

Conventional genetic theories have failed to explain why cancer (1) is not heritable and thus extremely rare in newborns, (2) is caused by non-mutagenic carcinogens, (3) develops only years to decades after initiation by carcinogens, (4) follows pre-neoplastic aneuploidy, (5) is aneuploid, (6) is chromosomally and phenotypically “unstable”, (7) carries specific aneusomies, (8) generates much more complex phenotypes than conventional mutation such as multidrug resistance, (9) generates nonselective phenotypes such as metastasis (no benefit at native site) and “immortality” (not necessary for tumorigenesis), and (10) does not contain carcinogenic mutations. We propose, instead, that cancer is a chromosomal disease. Accordingly carcinogenesis is initiated by random aneuploidies, which are induced by carcinogens or spontaneously. Since aneuploidy unbalances 1000s of genes, it corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is therefore a steady source of chromosomal variations from which, in classical Darwinian terms, selection encourages the evolution and malignant progression of cancer cells. The rates of specific chromosomal variations can exceed conventional mutations by 4–11 orders of magnitude, depending on the degrees of aneuploidy. Based on their chromosomal constitution cancer cells are new cell “species” with specific aneusomies, but unstable karyotypes. The cancer-specific aneusomies generate complex, malignant phenotypes through the abnormal dosages of 1000s of genes, just as trisomy 21 generates Down syndrome. In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy. Thus, cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) non-heritable cancer because aneuploidy is not heritable, (2) non-mutagenic carcinogens as aneuploidogens, (3) long neoplastic latencies by the low probability of evolving new species, (4) nonselective phenotypes via genes hitchhiking with selective chromosomes, and (5) immortality because, through their cellular heterogeneity, cancers survive negative mutations and cytotoxic drugs via resistant subspecies.

A convenient synthesis of labelled rhodopsin and studies on its active site

Digitonin solutions of labelled rhodopsin, containing 3H in the retinyl moiety, were prepared by two related methods. Labelled rhodopsin was also prepared for the first time in cetyltrimethylammonium bromide and purified by column chromatography. It was shown that only certain rhodopsin preparations on denaturation in the dark and the reduction with sodium borohydride gave up to 60% of the radioactivity in a fraction characterized as N-retinylphosphatidylethanolamine. Such preparations also gave a lipid-linked retinyl moiety at the metarhodopsin-I stage, but, as expected, a protein-linked retinyl moiety at the metarhodopsin-II stage. Other preparations however, gave exclusively protein-bound radioactivity at the native-rhodopsin, metarhodopsin-I and metarhodopsin-II stages. It is therefore conceivable that the formation of N-retinylphosphatidylethanolamine is due to a non-enzymic reaction resulting from the transfer of the retinyl moiety from its native site to an amino group of a favourably oriented phospholipid molecule. The only firmly established aspect of the rhodopsin active site remains the demonstration in our previous work that at the metarhodopsin-II stage the retinyl moiety is linked to an ∈-amino group of lysine. On the basis of chemical reactivity it is argued that the light-induced conversion of rhodopsin into metarhodopsin II involves a profound conformational change resulting in the dislocation of the retinylideneiminium chromophore from a non-polar environment in rhodopsin to a polar environment in metarhodopsin II.