A Genetic Screen for Attenuated Growth Identifies Genes Crucial for Intraerythrocytic Development of Plasmodium falciparum

A majority of the Plasmodium falciparum genome codes for genes with unknown functions, which presents a major challenge to understanding the parasite's biology. Large-scale functional analysis of the parasite genome is essential to pave the way for novel therapeutic intervention strategies against the disease and yet difficulties in genetic manipulation of this deadly human malaria parasite have been a major hindrance for functional analysis of its genome. Here, we used a forward functional genomic approach to study P. falciparum and identify genes important for optimal parasite development in the disease-causing, intraerythrocytic stages. We analyzed 123 piggyBac insertion mutants of P. falciparum for proliferation efficiency in the intraerythrocytic stages, in vitro. Almost 50% of the analyzed mutants showed significant reduction in proliferation efficiency, with 20% displaying severe defects. Functional categorization of genes in the severely attenuated mutants revealed significant enrichment for RNA binding proteins, suggesting the significance of post-transcriptional gene regulation in parasite development and emphasizing its importance as an antimalarial target. This study demonstrates the feasibility of much needed forward genetics approaches for P. falciparum to better characterize its genome and accelerate drug and vaccine development

The Mechanism for RNA Recognition by ANTAR Regulators of Gene Expression

ANTAR proteins are widespread bacterial regulatory proteins that have RNA–binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR–associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism

The Mitotic Arrest Deficient Protein MAD2B Interacts with the Small GTPase RAN throughout the Cell Cycle

Contains fulltext : 81260.pdf (publisher's version ) (Open Access)BACKGROUND: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed. CONCLUSIONS/SIGNIFICANCE: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase

The Mitotic Arrest Deficient Protein MAD2B Interacts with the Clathrin Light Chain A during Mitosis

Contains fulltext : 87811.pdf (publisher's version ) (Open Access)BACKGROUND: Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes. CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis

piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome

<p>Abstract</p> <p>Background</p> <p>Much of the <it>Plasmodium falciparum </it>genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the <it>Plasmodium </it>genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the <it>Plasmodium </it>genome.</p> <p>Results</p> <p>In this study, we investigated the lepidopteran transposon, <it>piggyBac</it>, as a molecular genetic tool for functional characterization of the <it>Plasmodium falciparum </it>genome. Through multiple transfections, we generated 177 unique <it>P. falciparum </it>mutant clones with mostly single <it>piggyBac </it>insertions in their genomes. Analysis of <it>piggyBac </it>insertion sites revealed random insertions into the <it>P. falciparum </it>genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in <it>P. falciparum </it>with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.</p> <p>Conclusion</p> <p>Our results clearly demonstrate that <it>piggyBac </it>is a novel, indispensable tool for forward functional genomics in <it>P. falciparum </it>that will help better understand parasite biology and accelerate drug and vaccine development.</p

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

How do firms comply with international sustainability standards? Processes and consequences of adopting the global reporting initiative

This paper addresses the issue of the influence of global governance institutions, particularly international sustainability standards, on a firm’s intra-organizational practices. More precisely, we provide an exploratory empirical view of the impact of the Global Reporting Initiative (GRI) on a multinational corporation’s (MNC) corporate social responsibility (CSR) management practices. We investigate standard compliance by comparing the stated intention of the use of the GRI with its actual use and the consequent effects within the firm. Based on an in-depth case study, our findings illustrate the processes and consequences of the translation of the GRI within the organization. We show that substantive standard adoption can lead to unintended consequences on CSR management practices, specifically it can influence the management structure and CSR committee function; the choice of CSR activities, the relationships between subsidiaries, the temporal dimension of CSR management, and the interpretation of CSR performance. We also highlight the need to look at the relationship dynamics (or lack of) between standards. Finally we illustrate and discuss the role of reporting and its influence on management in order to better understand the internal issues arising from compliance with standards

Computer Aided Design of Centrifugal Pump

The design of centrihgal pump involves a large number of empirical data derived from experimental evaluation of pumps. For any small change in input details, it requires a complete rework of design procedure. Even after following this detailed, time consuming procedure, it was very difficult to explain the geometry of the pump elements. This paper discusses the optimisation and computerisation of the design procedure of single stage centrifUga1 pumps. The design procedure was developed using Graphical User Interface software with Visual Basic 5.0 and interfaced with a specially developed AutoLISP program in AutoCAD R13 environment. An algorithm for generating the Cartesian co-ordinates of the designed pump elements was evolved and incorporated in the program, which are used to show the geometrical shapes of the pump elements and also used as an input data for the Auto LISP program for generating three-dimensional views of the pump components. The options and utilities made available through a single program enables the user to design, view, redesign and optimize the pump geometry till it reaches a satisfactory value. The developed software is user-friendly and can be used as a design tool to configure an optimum pump geometry for a given duty like discharge and head

A view on the role of epigenetics in the biology of malaria parasites

Cells and unicellular organisms are similar to their progenitors because information is transmitted from one generation to the next. The information is mainly transmitted in the primary sequence of the genome (genetic information), but there are heritable traits that are transmitted by other mechanisms. Epigenetics studies these alternative modes of inheritance. According to classic definitions, epigenetics refers to heritable differences between cells or organisms that occur without changes in DNA sequence, and do not depend on different external conditions