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J C Boothroyd

Publications and source records attributed to J C Boothroyd.

At least 55 records · Page 3Linked to original sources
PubMed1997

Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro.

A microtiter assay for drug evaluation has been developed with a strain of Toxoplasma gondii that expresses bacterial beta-galactosidase. By using chlorophenol red-beta-D-galactopyranoside (CPRG) as the substrate for beta-galactosidase, the efficacy of a drug against the parasite can be determined with a colorimetric readout. Drugs known to have activity against T. gondii (specifically, pyrimethamine, sulfadiazine, atovaquone, and clindamycin) were tested, and efficacies were determined by CPRG cleavage. The 50% inhibitory concentrations determined by the CPRG-based colorimetric assay were similar to those determined by the traditional radiolabelled uracil incorporation assay. Since CPRG is nontoxic to the parasite, viable drug-treated parasites can be obtained at the conclusion of the assay for further evaluation if desired. This assay provides a high-throughput and nonradioactive alternative for the identification of anti-T. gondii compounds.

Animals↗
PubMed1996

Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii.

We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.

Animals↗
PubMed1996

Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: coding region hypervariability and downstream recombinogenic repeats.

The surface of the parasitic protozoan Trypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome of T. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 bp downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy.

Amino Acid Sequence↗
PubMed1996

Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins.

Identification of surface-exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.

Africa↗
PubMed1995

Toxoplasma gondii: stable complementation of sag1 (p30) mutants using SAG1 transfection and fluorescence-activated cell sorting.

Toxoplasma gondii and the related Apicomplexan protozoan pathogens, Plasmodium, Cryptosporidium, and Eimeria, are obligate intracellular parasites which cause severe disease in their hosts. The recent development of transient transfection of Toxoplasma permits the development of strategies utilizing "reverse genetics" to identify molecules critical to parasite survival within the host. We have utilized transfection of Toxoplasma tachyzoites to stably complement a sag1 (or p30) mutant that does not make detectable SAG1. Transfection of mutants with the wild-type SAG1 gene resulted in transient expression of SAG1 in approximately 15-20% of the transfected population. Stable transformants were enriched by repeated sorting of live parasites using a fluorescein-labeled monoclonal antibody specific for SAG1. Cloned recombinant parasites expressed SAG1 at wild-type levels and maintained expression for over 5 months after transfection (approximately 300 divisions). Cloned transformants (which proved to be siblings) carried both the mutated gene and one copy of the transfected gene which had inserted randomly into the Toxoplasma genome.

Animals↗
PubMed1995

Trypanosoma brucei: molecular cloning of homologues of small GTP-binding proteins involved in vesicle trafficking.

Members of the rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes. Individual rab proteins have a remarkable degree of specific subcellular localization. As a first approach to the study of these proteins in the protozoan parasite Trypanosoma brucei we have undertaken a survey of the rab subfamily using a strategy based on degenerate polymerase chain reaction utilizing the miniexon and the highly conserved WDTAGQE box which comprises part of the GTP-binding site. A number of T. brucei partial cDNAs were obtained from procyclic stage cDNA, and seven of these clones (designated rtb 1, 3-7, 9) were clearly members of the rab family. Northern analysis of poly(A)-selected RNA indicates that rtb1, 3, 4, and 7 are constitutively expressed at low levels in both life-stages of T. brucei. By Southern analysis of trypanosome genomic DNA and specific PCR from an isolated genomic clone, we show that two of the genes, RTB1 and RTB7, are adjacent or at least closely linked in the T. brucei genome, while the other five are dispersed. These data provide important molecular reagents for dissecting the unusual secretory pathway in this organism.

Amino Acid Sequence↗
PubMed1995

Interconnection between organellar functions, development and drug resistance in the protozoan parasite, Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii causes severe disease in animals and humans. In AIDS patients, for example, the encephalitis it produces is a major cause of death. Part of the very successful strategy adopted by the parasite centers on its ability to differentiate from the actively growing tachyzoite form to a chronic, almost latent state called the bradyzoite. The molecular signals and precise triggers involved in this differentiation process are not known. Drugs for treating toxoplasmosis are not capable of clearing the infection apparently because of their inability to eradicate the bradyzoites. Recently, as part of our efforts to understand the mode of action of a promising new drug, atovaquone, we have generated and analysed a mutant that is resistant to this drug. Surprisingly, we found that this mutant is predisposed to spontaneously differentiate from the tachyzoite to bradyzoite form in vitro (Tomavo & Boothroyd, submitted). Given that atovaquone is believed to act on the parasite mitochondria, we were interested to explore the relationship between mitochondrial function and differentiation. We find that atovaquone and a number of other drugs targeted to mitochondria will cause wild type parasites to differentiate from tachyzoites to bradyzoites suggesting some sort of adaptive response to a decrease in mitochondrial activities. The fact that atovaquone-resistant mutants are hypersensitive to clindamycin, a drug believed to work on the putative plastid of these parasites, suggests a model for how the mitochondrion and plastid interact and how they may be tied into the process and state of differentiation. This model is presented and discussed.

AIDS-Related Opportunistic Infections↗
PubMed1995

Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii.

This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BamHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NotI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and beta-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.

Animals↗
PubMed1995

Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection.

The ROP1 gene of Toxoplasma gondii encodes a rhoptry protein that has been implicated in host cell invasion by this obligate intracellular protozoan. To further explore the function of this protein, we created a ROP1 deletion mutant by transfection with a plasmid encoding the bacterial chloramphenicol acetyltransferase (cat) gene flanked by ROP1 genomic sequences. Selection for chloramphenicol resistance yielded the desired ROP1-deleted or 'knock-out' mutant. Analysis of this mutant both in vitro and in vivo shows no significant alterations in growth rate, host specificity, invasiveness or virulence and thus the ROP1 gene is not obligatory for the RH strain, at least under the conditions tested. However, electron microscopy reveals that the mutant strain's rhoptries are altered in ultrastructure; they are thinner and homogeneously electron-dense compared with the thicker and normally mottled or honeycombed appearance of wild-type rhoptries. The knock-out mutant was rescued using co-transfection of a cosmid carrying the ROP1 gene together with a plasmid encoding a new selectable marker for T. gondii, the bleomycin resistance gene (ble) from Streptoalloteichus. Southern blot analysis showed that both DNAs were stably integrated into the Toxoplasma genome, although not into the ROPI locus. The resulting strain showed wild-type levels of ROP1 expression and rescue of the ultrastructural phenotype (i.e., the rhoptries returned to their normal, mottled appearance), thus establishing a cause/effect relationship between the absence of ROP1 and the electron-opacity. These results demonstrate the utility of the reverse genetic approach in the study of Toxoplasma gene function and provide a further selectable marker for such manipulations.

Animals↗
PubMed1995

A selector of transcription initiation in the protozoan parasite Toxoplasma gondii.

The recent development of an efficient transfection system for the apicomplexan Toxoplasma gondii allows a comprehensive dissection of the elements involved in gene transcription in this obligate intracellular parasite. We demonstrate here that for the SAG1 gene, a stretch of six repeated sequences in the region 35 to 190 bp upstream of the first of two transcription start sites is essential for efficient and accurate transcription initiation. This repeat element shows characteristics of a selector in determining the position of the transcription start sites.

Animals↗
PubMed1994

Conformationally appropriate expression of the Toxoplasma antigen SAG1 (p30) in CHO cells.

The Toxoplasma gondii major surface antigen, called SAG1 or p30, is a highly immunogenic protein which has generated great interest as a diagnostic reagent, as a potential subunit vaccine, and for its role in invasion. Unfortunately, bacterial recombinant protein is grossly misfolded so that, for example, it is not effectively recognized by antibodies to native SAG1. To overcome this, we have turned to expression in CHO cells, using cotransfection of the SAG1 gene and the mouse dihydrofolate reductase (DHFR) gene into CHO cells that are DHFR-. SAG1 expression was amplified by methotrexate coselection of CHO cells in combination with fluorescence-activated cell sorting for SAG1 expression. The resulting population expressed recombinant SAG1 that is recognized by antiserum specific for natural, nonreduced SAG1, indicating that, unlike in bacteria, expression in CHO cells results in proper folding. Processing was at least partially correct in that, like natural SAG1, recombinant SAG1 was attached to the plasma membrane via a glycolipid anchor, although tunicamycin treatment was necessary to prevent N-glycosylation (SAG1 is not glycosylated in the parasite but does have a consensus N-linked site). Finally, purified recombinant SAG1 was recognized by human sera known to be reactive to toxoplasma proteins, indicating that this material has potential as a diagnostic reagent and possibly as a component of a subunit vaccine.

Animals↗
PubMed1993

Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker.

A system for stable transformation of Toxoplasma gondii tachyzoites was developed that exploited the susceptibility of Toxoplasma to chloramphenicol. Introduction of the chloramphenicol acetyltransferase (CAT) gene fused to Toxoplasma flanking sequences followed by chloramphenicol selection resulted in parasites stably expressing CAT. A construct incorporating the tandemly repeated gene, B1, targeted efficiently to its homologous chromosomal locus. Knockout of the single-copy gene, ROP1, was also successful. Stable transformation should permit the identification and analysis of Toxoplasma genes important in the interaction of this opportunistic parasite with its host.

Animals↗
PubMed1993

Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan pathogen that produces severe disease in humans and animals. This obligate intracellular parasite provides an excellent model for the study of how such pathogens are able to invade, survive, and replicate intracellularly. DNA encoding chloramphenicol acetyltransferase was introduced into T. gondii and transiently expressed with the use of three vectors based on different Toxoplasma genes. The ability to introduce genes and have them efficiently and faithfully expressed is an essential tool for understanding the structure-function relation of genes and their products.

Animals↗
PubMed1993

Development of genetic systems for Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii has recently emerged as an important opportunistic pathogen in humans. Toxoplasma also shares a number of biological features with Plasmodium and Eimeria, which are important pathogens of humans and animals. Because o f the ease o f experimental use, David Sibley, Elmer Pfefferkom and John Boothroyd have undertaken the development of genetics in Toxoplasma as a model intracellular parasite. Toxoplasma is presently the only parasitic protozoan where both classical and molecular genetics are feasible. The recent advances in this system are highlighted here, along with potential applications of genetics for understanding intracellular parasitism.

Journal Article↗
PubMed1993

Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote.

Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

Amino Acid Sequence↗