The great American biotic interchange and diversification history in Dynastes beetles (Scarabaeidae; Dynastinae)

Biotic interchange between geographic regions can promote rapid diversification. However, what are the important factors that determine the rate of diversification (e.g., trait‐dependent diversification) vary between study systems. The evolutionary history of Dynastes beetles, which can be found in both North and South Americas and exhibit two different altitudinal preferences (highland and lowland) is tested for the effects of biotic interchange between continents and different ecological preferences on the rate of species diversification. Additionally, the hypotheses of geological time‐dependent and lineage specific diversification rates are also tested. Results from this study indicate that in Dynastes beetles a pre‐landbridge dispersal hypothesis from South to North America is preferred and that the speciation rates estimated using BAMM are similar between lineages of different geographic origins and different altitudinal preferences (i.e., diversification rate is not trait‐dependent). On the other hand, my result from marcoevolutionary cohort analysis based on BAMM outputs suggests that the rate of speciation in Dynastes beetles is, instead of trait‐dependent (geographic and ecological), lineage specific. Furthermore, a steadily increasing speciation rate can be found in Pliocene and Pleistocene, which implies that geological and climatic events, i.e., colonizing North America, habitat reformation in the Amazonia, and forest contraction in Pleistocene, may have together shaped the current biodiversity pattern in Dynastes beetles.     

Investigation of structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115

Background

Alcohol dehydrogenases (ADHs) catalyze the reversible oxidation of alcohol using NAD⁺ or NADP⁺ as cofactor. Three ADH homologues have been identified in Komagataella phaffii GS115 (also named Pichia pastoris GS115), ADH1, ADH2 and ADH3, among which adh3 is the only gene responsible for consumption of ethanol in Komagataella phaffii GS115. However, the relationship between structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115 (KpADH3) is still not clear yet.

Cointegrate formation by IS50 requires multiple donor molecules

Summary The insertion sequence, IS50R, promotes cointegrate formation between a lambda::IS50R phage and the chromosome of Escherichia coli strain C. We show that formation of cointegrates mediated by IS50R between the non-replicating phage genome and the bacterial chromosome requires multiple donor molecules and depends on homologous recombination functions. We conclude that the two copies of IS50 present in the cointegrate originate in two different molecules. Thus, the existence of the cointegrate structure cannot be used as evidence for replication of IS50 sequences during IS50 transposition.     

Striatins Contain a Noncanonical Coiled Coil That Binds Protein Phosphatase 2A A Subunit to Form a 2:2 Heterotetrameric Core of Striatin-interacting Phosphatase and Kinase (STRIPAK) Complex

The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies.     

Binding of f-PIP, a pyrrole- and imidazole-containing triamide, to the inverted CCAAT box-2 of the topoisomerase IIalpha promoter and modulation of gene expression in cells

An N-formamido pyrrole- and imidazole-containing triamide (f-PIP) has been shown by DNase I footprinting, SPR, and CD studies to bind as a stacked dimer to its cognate sequences: 5'-TACGAT-3' (5'-flank of the inverted CCAAT box-2 of the human topoisomerase IIalpha promoter) and 5'-ATCGAT-3'. A gel shift experiment provided evidence for f-PIP to inhibit protein-DNA interaction at the ICB2 site. Western blot studies showed that expression of the topoisomerase IIalpha gene in confluent NIH 3T3 cells was induced by treatment with f-PIP. The results suggested that the triamide was able to enter the nucleus, interacted with the target site within ICB2, inhibited NF-Y binding, and activated gene expression.     

Preparation of site-specific T=mCG cis-syn cyclobutane dimer-containing template and its error-free bypass by yeast and human polymerase η

C-to-T mutations are a hallmark of UV light and, in humans, occur preferentially at methylated Py(m)CG sites, which are also sites of preferential cyclobutane pyrimidine dimer (CPD) formation. In response, cells have evolved DNA damage bypass polymerases, of which polymerase η (pol η) appears to be specifically adapted to synthesize past cis-syn CPDs. Although T=T CPDs are stable, CPDs containing C or 5-methylcytosine ((m)C) are not and spontaneously deaminate to U or T at pH 7 and 37 °C over a period of hours or days, making their preparation and study difficult. Furthermore, there is evidence to suggest that, depending on solvent polarity, a C or an (m)C in a CPD can adopt three tautomeric forms, one of which could code as T. Although many in vitro studies have established that synthesis past T or U in a CPD by pol η occurs in a highly error-free manner, the only in vitro evidence that synthesis past C or (m)C in a CPD also occurs in an error-free manner is for an (m)C in the 5'-position of an (m)C=T CPD. Herein, we describe the preparation and characterization of an oligodeoxynucleotide containing a CPD of a T(m)CG site, one of the major sites of C methylation and C-to-T mutations found in the p53 gene of basal and squamous cell cancers. We also demonstrate that both yeast and human pol η synthesize past the 3'-(m)C CPD in a >99% error-free manner, consistent with the highly water-exposed nature of the active site.     

Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B₄ receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G_i2 protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human α₅ integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB₄ binding in the presence of the purified G protein Gα_i2. The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5′-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified Gα_i2β₁γ₂ protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.     

Cytotoxicity and DNA binding property of the dimers of triphenylethylene–coumarin hybrid with one amino side chain

Novel dimers of triphenylethylene–coumarin hybrid containing one amino side chain were designed and synthesized by the condensation of four dicarboxylic acids with the amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature. The adding order of the reactants had a significant effect on the condensation reaction when the malonic acid was used. The dimeric compounds 7a and 7b linked by the malonic acid, showed a broad-spectrum and good anti-proliferative activity against four tumor cells and low cytotoxicity in osteoblast. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that compounds 7b, 8b, 9b, and 10b had significant interactions with Ct-DNA by the intercalative mode of binding. Both the DNA binding properties and the anti-proliferative activities would be enhanced by dimerization of the monomeric hybrid with one amino side chain, and were significantly affected by the length of the linker (dicarboxylic acids).     

An NMR Study of the Polymorphous Behavior of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G) in Solution. The B,Z, and Hairpin Forms

Abstract

The polymorphism exhibited by the mismatched octamer d(m ⁵C-G-m⁵C-G-T-G-m⁵C-G), as a function of the temperature, DNA concentration and ionic strength, was investigated by means of NMR spectroscopy.

It is shown that this partly self-complementary DNA fragment, under conditions of low DNA concentration (0.4 mM) and low ionic strength, exclusively prefers to adopt a monomeric hairpin form, which consists of a stem of three Watson-Crick-type base pairs and a loop of only two residues. This in striking contrast with earlier intimations in literature, which postulated that in oligonucleotides loop formations containing only two residues are sterically impossible. Moreover, the hairpin form displays an unusual stability in comparison with previously reported hairpins. AT_m of 332 K and a ΔH° of—130 kj · mol^?1 were calculated for the hairpin to random coil transition.

At high DNA concentration (8 mM)and/or upon the addition of sodium chloride the hairpin form occurs in slow exchange with a B-DNA dimer structure (approximately 20% at 270 K, no added salt), which comprises two central GxT-mismatched base pairs with the bases as major tautomers.

At higher ionic strength (> 100 mM NaCI), or upon the addition of methanol, a third species appears, which is in slow exchange with both the B dimer and the hairpin form. This third species could be identified with a Z DNA form, comprising two GxT mismatches with the bases as major tautomers, with the guanine bases syn and the cytosine and thymine bases anti.     

Ultra-high resolution crystal structure of a dimeric defensin SPE10

Defensins are key players of the innate immune system known to act against bacteria, fungi and viruses. Here we report the 0.98-Å crystal structure of SPE10, a dimeric plant defensin. SPE10 associates as a dimer through a unique amino acid triplet involving residues R36–W42–R40. The helix from one subunit interacts with arginines R36 and R40 from the other subunit, forming a sheet-like dimer with a highly extended molecular surface. A conserved hydrophobic patch on the molecular head largely overlaps with the putative receptor-binding site previously reported for another defensin. Structural analysis and mutational studies indicate that the dimeric association of SPE10 is relevant to its function, and that the hydrophobic patch on the molecular head is required for its antifungal activity.

Structured summary

SPE10binds to SPE10 by X-ray crystallography(View interaction)