Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed. Received: 1 March 1997 / Accepted: 13 May 1997     

The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans     

Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells

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Validation of the first step of the heuristic refinement method for the derivation of solution structures of proteins from NMR data

A new method for the analysis of NMR data in terms of the solution structure of proteins has been developed. The method consists of two steps: first a systematic search of the conformational space to define the region allowed by the initial set of experimental constraints, and second, the narrowing of this region by the introduction of additional constraints and optional refinement procedures. The search of the conformational space is guided by heuristics to make it computationally feasible. The method is therefore called the heuristic refinement method and is coded in an expert system called PROTEAN. The paper describes the validation of the first step of the method using an artificial NMR data set generated from the known crystal structure of sperm whale carbon monoxymyoglobin. It is shown that the initial search procedure yields a low-resolution structure of the myoglobin molecule, accurately reproducing its main topological features, and that the precision of the structure depends on the quality of the initial data set.     

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC,composed of human Argonaute 2 and a guide RNA

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. [BMB Reports 2015; 48(12): 643-644]     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

The nuclear pores of early meiotic prophase nuclei of plants

Summary Pollen mother cells at early meiotic prophase fromFritillaria lanceolata, F. mutica, Tulbaghia violacea, the lily “Formobel”,Triticum aegilopoides, T. dicoccoides, T. aestivum and synaptic and asynaptic forms ofT. durum were studied in thin sections with the electron microscope (a) in relation to distribution of nuclear pores (b) in respect of fine structure of the pore complex in those of the first four. The pores were distributed in random clusters during leptotene to pachytene in all plants, except in the two forms ofT. durum where there were either no pores or so few that they were not detectable. Probably correlated with this, the two membranes of the nuclear envelope were often widely separated and frequently sacculated. No pores were seen at leptotene in the part of the envelope to which, in theFritillarias and lily, the nucleolus was adpressed at this time. Evidence supporting a recent model which proposes that annuli are composed of three rings of eight granular subunits was obtained. These subunits as well as a dense central element, observed in most pores, were composed of filaments about 3 nm in diameter and evidently protein in character. There was evidence of a continuity between filaments in the central element and those in the rings of subunits which encircle the pore aperture at both the nuclear and cytoplasmic sides of the pore. In profiles of pores knobbed filaments were sometimes seen extending laterally from the pore wall into the perinuclear space at two sides. Questions concerning the role of the annulus are discussed. The author wish to thank Mr. R. F. Scott for construction to the model.     

Tungsten-based material as promising new lead-free gamma radiation shielding material in nuclear medicine

PurposeThe main objective of this study was to evaluate the efficacy of tungsten carbide as new lead-free radiation shielding material in nuclear medicine by evaluating the attenuation properties.Materials and methodsThe elemental composition of tungsten carbide was analysed using Field-Emission Scanning Electron Microscopy (FESEM) with energy dispersive X-ray (EDX). The purity of tungsten carbide was 99.9%, APS: 40–50 µm. Three discs of tungsten carbide was fabricated with thickness of 0.1 cm, 0.5 cm and 1.0 cm. Three lead discs with similar thickness were used to compare the attenuation properties with tungsten carbide discs. Energy calibration of gamma spectroscopy was performed by using ¹²³I, ¹³³Ba, ¹⁵²Eu, and ¹³⁷Cs. Gamma radiation from these sources were irradiated on both materials at energies ranging from 0.160 MeV to 0.779 MeV. The experimental attenuation coefficients of lead and tungsten carbide were compared with theoretical attenuation coefficients of both materials from NIST database. The half value layer and mean free path of both materials were also evaluated in this study.ResultsThis study found that the peaks obtained from gamma spectroscopy have linear relationship with all energies used in this study. The relative differences between the measured and theoretical mass attenuation coefficients are within 0.19–5.11% for both materials. Tungsten carbide has low half value layer and mean free path compared to lead for all thickness at different energies.ConclusionThis study shows that tungsten carbide has high potential to replace lead as new lead-free radiation shielding material in nuclear medicine.