Stable transfection and continuous expression of heterologous genes in Entamoeba invadens.

Abstract:

:Amoebiasis is spread by the ingestion of dormant Entamoeba histolytica cysts. Intervention of encystation could break the transmission cycle, thereby reducing disease burden. The model system used to study trophozoite to cyst differentiation is Entamoeba invadens. Here we describe an electroporation-based method for stable transfection of E. invadens with a plasmid pEiNEO-LUC containing the neomycin phosphotransferase gene under the control of E. invadens ribosomal protein gene S10 promoter. The plasmid also contains luciferase reporter gene expressed from the promoter of ribosomal protein gene L3. After electroporation, cells receiving the plasmid were selected by growth in 10μgml(-1) G418 and stable lines were obtained in four to five weeks. The plasmid was replicated episomally to ∼10 copies per haploid genome. In the absence of drug selection 50% of the plasmid copies were lost in 9-10 days. In cells growing under drug selection the reporter gene was continuously expressed throughout the differentiation process from trophozoite to cyst and back, making this system suitable for analysis of genes involved in differentiation.

journal_name

Mol Biochem Parasitol

authors

Singh N,Ojha S,Bhattacharya A,Bhattacharya S

doi

10.1016/j.molbiopara.2012.02.012

subject

Has Abstract

pub_date

2012-07-01 00:00:00

pages

9-12

issue

1

eissn

0166-6851

issn

1872-9428

pii

S0166-6851(12)00054-0

journal_volume

184

pub_type

杂志文章
  • Sequencing and characterization of an Echinococcus multilocularis DNA probe and its use in the polymerase chain reaction.

    abstract::The nucleotide sequence of the cloned Echinococcus multilocularis DNA probe pAL1 was determined in order to simplify and improve the sensitivity of a diagnostic assay through the application of the polymerase chain reaction (PCR). The insert-specific oligonucleotides BG1 and BG2 define a 2.6-kb fragment in the genomic...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(91)90004-p

    authors: Gottstein B,Mowatt MR

    更新日期:1991-02-01 00:00:00

  • Role of 3'-untranslated regions in the regulation of hexose transporter mRNAs in Trypanosoma brucei.

    abstract::Trypanosoma brucei is a unicellular parasite that is transmitted from one mammalian host to the next by tsetse flies. The expression of many trypanosome genes is regulated during the life cycle but there is no evidence for developmental control of transcription by RNA polymerase II. T. brucei expresses at least two he...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(95)02503-0

    authors: Hotz HR,Lorenz P,Fischer R,Krieger S,Clayton C

    更新日期:1995-12-01 00:00:00

  • An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum.

    abstract::Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. T...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(88)90173-9

    authors: Epping RJ,Goldstone SD,Ingram LT,Upcroft JA,Ramasamy R,Cooper JA,Bushell GR,Geysen HM

    更新日期:1988-02-01 00:00:00

  • In vitro selection of halofantrine resistance in Plasmodium falciparum is not associated with increased expression of Pgh1.

    abstract::Recent investigations into quinoline and phenanthrene methanol resistance in Plasmodium falciparum have described a linkage between amplification of the mdr homologue pfmdr1 and decreased susceptibility to mefloquine (MQ) and halofantrine (HF). We have examined the current theories on cross-resistance patterns and pfm...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/s0166-6851(96)02746-6

    authors: Ritchie GY,Mungthin M,Green JE,Bray PG,Hawley SR,Ward SA

    更新日期:1996-12-02 00:00:00

  • Effects of curcumin on the parasite Schistosoma mansoni: a transcriptomic approach.

    abstract::Schistosomiasis remains a severe problem of public health in developing countries. Several reports show that praziquantel, the drug of choice for treating schistosomiasis, can select Schistosoma mansoni strains resistant to the drug. Thus, developing new drugs against this parasitosis is a highly desirable goal. Curcu...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2012.11.006

    authors: Morais ER,Oliveira KC,Magalhães LG,Moreira EB,Verjovski-Almeida S,Rodrigues V

    更新日期:2013-02-01 00:00:00

  • Chemical modification of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

    abstract::Chemical modifications of Class I aldolases from Trypanosoma brucei, rabbit muscle and Staphylococcus aureus with carboxypeptidase A, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. Aldolase from S. aureus was not affected by any of the...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(91)90143-t

    authors: Callens M,Opperdoes FR

    更新日期:1991-07-01 00:00:00

  • Trichomonas vaginalis ribosomal RNA: identification and characterisation of the transcription promoter and terminator sequences.

    abstract::Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription ...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2012.05.004

    authors: Franco B,Hernández R,López-Villaseñor I

    更新日期:2012-09-01 00:00:00

  • Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis.

    abstract::A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into tw...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(96)02721-1

    authors: Poole CB,Hornstra LJ,Benner JS,Fink JR,McReynolds LA

    更新日期:1996-11-12 00:00:00

  • Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation.

    abstract::Babesia bovis, an intraerythrocytic, protozoal parasite of cattle, undergoes clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91-98). This ability could provide a mechanism by which the parasite escapes host immune defenses to establish chronic infection. Previous work identif...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/s0166-6851(97)00125-4

    authors: O'Connor RM,Lane TJ,Stroup SE,Allred DR

    更新日期:1997-11-01 00:00:00

  • Accurate transcription of a cloned gene from Babesia bovis in Saccharomyces cerevisiae.

    abstract::Two cloned chromosomal segments of the hemoprotozoan Babesa bovis bearing genes from the polymorphic locus designated BabR were inserted into plasmid vector YCp19, cloned in Escherichia coli and then introduced into Saccharomyces cerevisiae. Both genes were transcribed producing poly(A+) RNAs. In at least one case, th...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(84)90044-6

    authors: Kemp DJ,Easton KE,Cowman AF

    更新日期:1984-05-01 00:00:00

  • Identification by extrachromosomal amplification and overexpression of a zeta-crystallin/NADPH-oxidoreductase homologue constitutively expressed in Leishmania spp.

    abstract::A gene which overexpresses a 36-kDa protein (p36) in tunicamycin-resistant Leishmania was mapped by transfection and overexpression to the upstream region of the drug maker in the extrachromosomal amplicon. Complete sequencing of this region revealed a single open reading frame of about 1 kb. Authenticity of the clone...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(94)90147-3

    authors: Liu X,Chang KP

    更新日期:1994-08-01 00:00:00

  • Characterization of protein kinase CK2 from Trypanosoma brucei.

    abstract::CK2 is a ubiquitous but enigmatic kinase. The difficulty in assigning a role to CK2 centers on the fact that, to date, no biologically relevant modulator of its function has been identified. One common theme revolves around a constellation of known substrates involved in growth control, compatible with its concentrati...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2006.10.002

    authors: Jensen BC,Kifer CT,Brekken DL,Randall AC,Wang Q,Drees BL,Parsons M

    更新日期:2007-01-01 00:00:00

  • Distinct effects of HIV protease inhibitors and ERAD inhibitors on zygote to ookinete transition of the malaria parasite.

    abstract::In an effort to eradicate malaria, new interventions are proposed to include compound/vaccine development against pre-erythrocytic, erythrocytic and mosquito stages of Plasmodium. Drug repurposing might be an alternative approach to new antimalarials reducing the cost and the time required for drug development. Previo...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2017.12.003

    authors: Goulielmaki E,Kaforou S,Venugopal K,Loukeris TG,Siden-Kiamos I,Koussis K

    更新日期:2018-03-01 00:00:00

  • Purification and characterization of three larval excretory-secretory proteins of Dirofilaria immitis.

    abstract::Two proteins were previously described in the excretory-secretory products (ES) collected from Dirofilaria immitis during the molt from the third stage to the fourth stage in vitro. The two proteins were purified using cation exchange and reverse phase HPLC. During the purification of these two proteins, a third prote...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(95)02533-2

    authors: Frank GR,Grieve RB

    更新日期:1996-01-01 00:00:00

  • Surface epitope localization and gene structure of a Babesia bovis 44-kilodalton variable merozoite surface antigen.

    abstract::Variation of Babesia bovis merozoite surface antigens occurs among geographic strains of the parasite. In this and a concurrent report, we investigate this variation at the gene and protein level. Using a monoclonal antibody (mAb 23/70.174), B. bovis gene sequences were identified that encoded a surface epitope of a 4...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(92)90128-7

    authors: Jasmer DP,Reduker DW,Hines SA,Perryman LE,McGuire TC

    更新日期:1992-10-01 00:00:00

  • Sequences required for the flagellar targeting of an integral membrane protein.

    abstract::Previous studies have established that the ISO1 glucose transporter of Leishmania enriettii resides primarily in the flagellar membrane, whereas the ISO2 glucose transporter is located in the pellicular plasma membrane surrounding the cell body. This pronounced difference in subcellular targeting is conferred by the N...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2004.01.009

    authors: Nasser MI,Landfear SM

    更新日期:2004-05-01 00:00:00

  • Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria.

    abstract::In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electro...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(80)90002-x

    authors: Köhler P,Bachmann R

    更新日期:1980-04-01 00:00:00

  • Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi.

    abstract::The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminis...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2003.10.005

    authors: DaRocha WD,Otsu K,Teixeira SM,Donelson JE

    更新日期:2004-02-01 00:00:00

  • Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences.

    abstract::Polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis is a simple, rapid and accurate method for molecular detection of various nematode species. The objective of the present study was, for the first time, to develop a PCR-HRM assay for the detection of various animal Trichostrongylus spp. A...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2020.111260

    authors: Arbabi M,Hooshyar H,Lotfinia M,Bakhshi MA

    更新日期:2020-03-01 00:00:00

  • Effects of oxygen concentration on the intermediary metabolism of Leishmania major promastigotes.

    abstract::Leishmania major promastigotes grown in late log phase were incubated with glucose as sole exogenous carbon source in the presence of 5% CO2 and the amounts of glucose consumed and of the major products formed--succinate, pyruvate, alanine, acetate, glycerol, and D-lactate--were measured as a function of pO2. Glucose ...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(90)90062-q

    authors: Keegan F,Blum JJ

    更新日期:1990-03-01 00:00:00

  • Expression of substrate-specific transporters encoded by Plasmodium falciparum in Xenopus laevis oocytes.

    abstract::When the malarial parasite Plasmodium falciparum multiplies in erythrocytes it dramatically increases uptake of essential metabolic precursors (nucleosides, nucleobases and glucose) and export of lactic acid by undefined mechanisms. The first evidence is provided here, by a detailed study in Xenopus laevis oocytes, th...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/s0166-6851(98)00024-3

    authors: Penny JI,Hall ST,Woodrow CJ,Cowan GM,Gero AM,Krishna S

    更新日期:1998-05-15 00:00:00

  • Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi.

    abstract::The major proteolytic activity of Trypanosoma cruzi is a cathepsin L-like cysteine protease expressed in all stages of the parasite. As an initial step in identifying possible functions of this enzyme in the life cycle of T. cruzi, and examining its potential as a target for rational drug design, two fluoromethyl keto...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(93)90086-d

    authors: Harth G,Andrews N,Mills AA,Engel JC,Smith R,McKerrow JH

    更新日期:1993-03-01 00:00:00

  • Species-specific inhibition of Giardia lamblia triosephosphate isomerase by localized perturbation of the homodimer.

    abstract::Giardia lamblia depends on glycolysis to obtain ATP, highlighting the suitability of glycolytic enzymes as targets for drug design. We studied triosephosphate isomerase from G. lamblia (GlTIM) as a potential species-specific drug target. Cysteine-reactive agents were used as probes, in order to test those regions near...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2007.10.013

    authors: Enriquez-Flores S,Rodriguez-Romero A,Hernandez-Alcantara G,De la Mora-De la Mora I,Gutierrez-Castrellon P,Carvajal K,Lopez-Velazquez G,Reyes-Vivas H

    更新日期:2008-02-01 00:00:00

  • Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: proof of concept.

    abstract::The scarcity of molecular tools for genetic manipulation is a critical obstacle for functional genomics studies on Trypanosoma cruzi. The current study adapted an inducible site-specific recombination system based on Dimerizable CRE recombinase (DiCRE). Two vectors for stable transfection were created, a first one to ...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2015.01.001

    authors: Kangussu-Marcolino MM,Cunha AP,Avila AR,Herman JP,DaRocha WD

    更新日期:2014-12-01 00:00:00

  • Sequence preference of chloroquine binding to DNA and prevention of Z-DNA formation.

    abstract::Chloroquine is a critically important antimalarial drug and a well known intercalator into DNA. We now show that chloroquine binds more avidly to poly(dG-dC).poly(dG-dC) than to other synthetic polynucleotides and that this binding inhibits both salt- and cobalt-induced transitions to Z-DNA. These data are consistent ...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(90)90066-u

    authors: Kwakye-Berko F,Meshnick S

    更新日期:1990-03-01 00:00:00

  • The Leishmania mexicana proteasome.

    abstract::As a start to understanding the importance of intracellular proteolysis in the protozoon Leishmania mexicana, the parasite proteasome has been purified and characterised. The L. mexicana proteasome is similar to proteasomes from other eukaryotes. It is soluble, and the 20S form has a mass of around 670 kDa, composed o...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/s0166-6851(99)00110-3

    authors: Robertson CD

    更新日期:1999-09-20 00:00:00

  • Molecular cloning of GRA4, a Toxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies.

    abstract::Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(92)90172-g

    authors: Mevelec MN,Chardès T,Mercereau-Puijalon O,Bourguin I,Achbarou A,Dubremetz JF,Bout D

    更新日期:1992-12-01 00:00:00

  • Chaperoning a cellular upheaval in malaria: heat shock proteins in Plasmodium falciparum.

    abstract::In addition to their ability to help newly synthesized proteins to fold, molecular chaperones are also recognized for their participation in cellular processes ranging from protein trafficking, signal transduction, differentiation and development. Novel roles for this group of proteins have come to light through studi...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章,评审

    doi:10.1016/j.molbiopara.2007.01.009

    authors: Acharya P,Kumar R,Tatu U

    更新日期:2007-06-01 00:00:00

  • Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis.

    abstract::The cysteine proteinase B of Leishmania parasites is an important virulence factor. In this study we have expressed, isolated and characterized for the first time a recombinant CPB from Leishmania braziliensis, the causative agent of mucocutaneous leishmaniosis. The mature region of the recombinant CPB shares a high p...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/j.molbiopara.2008.06.005

    authors: Lanfranco MF,Loayza-Muro R,Clark D,Núñez R,Zavaleta AI,Jimenez M,Meldal M,Coombs GH,Mottram JC,Izidoro M,Juliano MA,Juliano L,Arévalo J

    更新日期:2008-10-01 00:00:00

  • Substrate specificities of 5'-deoxy-5'-methylthioadenosine phosphorylase from Trypanosoma brucei brucei and mammalian cells.

    abstract::The separation by chromatofocusing of two distinct purine nucleoside cleaving activities from crude extracts of Trypanosoma brucei brucei is described. One catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MeSAdo) and adenosine (Ado) and was designated an MeSAdo/Ado phosphorylase, while the o...

    journal_title:Molecular and biochemical parasitology

    pub_type: 杂志文章

    doi:10.1016/0166-6851(88)90030-8

    authors: Ghoda LY,Savarese TM,Northup CH,Parks RE Jr,Garofalo J,Katz L,Ellenbogen BB,Bacchi CJ

    更新日期:1988-01-15 00:00:00