SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore–microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase–anaphase transition.     

A highly conserved pocket on PP2A‐B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.     

Cloning,Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.     

First divergence time estimate of spiders,scorpions, mites and ticks (subphylum: Chelicerata) inferred from mitochondrial phylogeny

Spiders, scorpions, mites and ticks (chelicerates) form one of the most diverse groups of arthropods on land, but their origin and times of diversification are not yet established. We estimated, for the first time, the molecular divergence times for these chelicerates using complete mitochondrial sequences from 25 taxa. All mitochondrial genes were evaluated individually or after concatenation. Sequences belonging to three missing genes (ND3, 6, and tRNA-Asp) from three taxa, as well as the faster-evolving ribosomal RNAs (12S and 16S), tRNAs, and the third base of each codon from 11 protein-coding genes (PCGs) (COI-III, CYTB, ATP8, 6, ND1-2, 4L, and 4-5), were identified and removed. The remaining concatenated sequences from 11 PCGs produced a completely resolved phylogenetic tree and confirmed that all chelicerates are monophyletic. Removing the third base from each codon was essential to resolve the phylogeny, which allowed deep divergence times to be calculated using three nodes calibrated with upper and lower priors. Our estimates indicate that the orders and classes of spiders, scorpions, mites, and ticks diversified in the late Paleozoic, much earlier than previously reported from fossil date estimates. The divergence time estimated for ticks suggests that their first land hosts could have been amphibians rather than reptiles. Using molecular data, we separated the spider-scorpion clades and estimated their divergence times at 397 ± 23 million years ago. Algae, fungi, plants, and animals, including insects, were well established on land when these chelicerates diversified. Future analyses, involving mitochondrial sequences from additional chelicerate taxa and the inclusion of nuclear genes (or entire genomes) will provide a more complete picture of the evolution of the Chelicerata, the second most abundant group of animals on earth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae)

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.     

Structural basis for the carbohydrate specificities of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity

Artocarpin, a tetrameric lectin of molecular mass 65 kDa, is one of the two lectins extracted from the seeds of jackfruit. The structures of the complexes of artocarpin with mannotriose and mannopentose reported here, together with the structures of artocarpin and its complex with Me-alpha-mannose reported earlier, show that the lectin possesses a deep-seated binding site formed by three loops. The binding site can be considered as composed of two subsites; the primary site and the secondary site. Interactions at the primary site composed of two of the loops involve mainly hydrogen bonds, while those at the secondary site comprising the third loop are primarily van der Waals in nature. Mannotriose in its complex with the lectin interacts through all the three mannopyranosyl residues; mannopentose interacts with the protein using at least three of the five mannose residues. The complexes provide a structural explanation for the carbohydrate specificities of artocarpin. A detailed comparison with the sugar complexes of heltuba, the only other mannose-specific jacalin-like lectin with known three-dimensional structure in sugar-bound form, establishes the role of the sugar-binding loop constituting the secondary site, in conferring different specificities at the oligosaccharide level. This loop is four residues longer in artocarpin than in heltuba, providing an instance where variation in loop length is used as a strategy for generating carbohydrate specificity.     

Differential Adhesion and Infection of Nematodes by the Endoparasitic Fungus Meria coniospora (Deuteromycetes)

The conidia of the endoparasitic fungus Meria coniospora (Deuteromycetes) had different patterns of adhesion to the cuticles of the several nematode species tested; adhesion in some species was only to the head and tail regions, on others over the entire cuticle, whereas on others there was a complete lack of adhesion. After adhesion, the fungus usually infected the nematode. However, adhesion to third-stage larvae of five animal parasitic nematodes, all of which carry the cast cuticle from the previous molt, did not result in infection. M. coniospora infected animal parasitic nematodes when this protective sheath was removed. Seven preparations of sialic acid (N-acetylneuraminic acid) gave three types of response in adhesion-infection of nematodes: (i) a significant reduction in conidial adhesions; (ii) no interference with adhesion, but a 10-day delay in infection; and (iii) a delay in infection by 2 to 3 days. The current results support previous findings indicating involvement of sialic acids localized on nematode cuticles in recognition of prey by M. coniospora.     

Control of Root-knot Nematodes on Tomato by Lectins

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.     

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.