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J C Boothroyd

Publications and source records attributed to J C Boothroyd.

At least 73 records · Page 4Linked to original sources
PubMed1993

Population biology of Toxoplasma: clonality, virulence, and speciation (or not).

The population biology of Toxoplasma gondii is reviewed in the context of how its complex life cycle might be responsible for the current picture and how a knowledge of the population structure may have an impact on the thinking of both clinician and researcher. Published results already clearly indicate that the population can be divided into two distinct groups that are genetically and biologically distinct. The more virulent group, as defined by their LD50 in mice, is apparently a single, clonal line. The biological and clinical implications of the existence of such a group are discussed.

Animals↗
PubMed1992

Virulent strains of Toxoplasma gondii comprise a single clonal lineage.

The protozoan Toxoplasma gondii is a prevalent parasite in wild and domestic animals worldwide, being transmitted through the food chain by carnivorous feeding and scavenging. Toxoplasma normally divides asexually to yield a haploid form that can infect virtually any vertebrate but it also has a well defined sexual cycle that occurs exclusively in cats. Toxoplasma has become important as an often fatal opportunistic pathogen in patients with AIDS, although the 15-85% of adult human populations that are chronically infected with T. gondii are typically asymptomatic. Infections in immunocompromised hosts have variable outcomes. For example, only 30 to 50% of AIDS patients that are chronically infected with the parasite develop toxoplasmic encephalitis and only about half of acute maternal infections result in congenital disease of the newborn. T. gondii strains differ in their virulence in animals, but the extent to which different strains are related has not been determined. Here we analyse 28 strains from a variety of hosts on five continents and find that the ten virulent strains have an essentially identical genotype, whereas the nonvirulent strains are moderately polymorphic. These data strongly suggest that virulent strains of T. gondii originated from a single lineage which has remained genetically homogeneous despite being globally widespread, and despite the ability of this organism to reproduce sexually.

Animals↗
PubMed1992

Genomic organization and context of a trypanosome variant surface glycoprotein gene family.

We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.

Animals↗
PubMed1992

Sequence divergence among members of a trypanosome variant surface glycoprotein gene family.

We have used analysis of DNA sequence data from four members of a Trypanosoma brucei variant surface glycoprotein gene family to investigate the molecular basis of the generation of antigenic diversity in African trypanosomes. Among these four sequences we find the greatest similarity in the untranslated sequences immediately upstream from the coding region. A complex pattern of nucleic acid and predicted amino acid sequence divergence appears starting at the coding sequence. Two related but highly divergent hydrophobic leaders are associated with different members of this gene family; both forms of these hydrophobic leaders appear to exist in other isolates of T. b. brucei. We find conservative replacements in the first 120 predicted amino acid residues of the mature protein; the following 80 predicted residues show less conservative replacements, and we suggest that this region may be hypervariable and exposed to the aqueous environment.

Amino Acid Sequence↗
PubMed1992

Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.

Animals↗
PubMed1992

Antigen-specific (p30) mouse CD8+ T cells are cytotoxic against Toxoplasma gondii-infected peritoneal macrophages.

The importance of CD8+ T cells in immunity against Toxoplasma gondii is now well recognized. The mechanism by which these CD8+ T cells are able to confer this immunity is not yet understood. To examine the Ag specificity of this response, immune splenocytes from mice immunized with p30, a major surface parasite Ag, were evaluated for their ability to lyse peritoneal macrophages infected with three different strains of T. gondii. Macrophages infected with either the RH or P wild-type strain tachyzoites were lysed at varying E:T ratios by nylon wool nonadherent immune splenocytes whereas macrophages infected with a p30-deficient mutant (B mutant) of the P strain were not. The gene encoding p30 for the wild type and B mutant were amplified by the polymerase chain reaction. This revealed a nonsense mutation in the B mutant such that its primary translation product is predicted to be about two-thirds the size of the wild-type p30 molecule. mAb depletion studies indicate that the cytotoxic effect of the immune splenocytes is mediated by the CD8+ T cell population. Peritoneal macrophages infected with the three different strains (RH, P wild type, B mutant) from mice genetically restricted were not lysed by the immune CD8+ effector cell population. A cloned line (C3) of p30 Ag-specific CD8+ T cells exhibited significant cytotoxicity against syngeneic peritoneal macrophages infected with either the RH or P strain tachyzoites. There was no macrophage lysis observed by these CD8+ effector cells of either syngeneic macrophages infected with the B mutant or nonsyngeneic macrophages infected with the three different tachyzoite strains.

Amino Acid Sequence↗
PubMed1992

Genes that allow yeast cells to grow in the absence of the HDEL receptor.

The ERD2 gene of Saccharomyces cerevisiae encodes the HDEL receptor that sorts ER proteins; it is essential for growth. In the absence of Erd2p the Golgi apparatus is both functionally and morphologically perturbed. Here we describe the isolation of four SED genes (suppressors of the erd2-deletion) which, when present in multiple copies, allow cells to grow in the absence of ERD2. The suppressed strains secrete the ER protein BiP and their internal membranes show a variety of morphological abnormalities. Sequence analysis indicates that all these SED genes encode membrane proteins: SED1 encodes a probable cell surface glycoprotein; SED2 is identical to SEC12, a gene required for the formation of ER-derived transport vesicles; SED4 encodes a protein whose cytoplasmic domain is 45% identical to that of Sec12p; SED3 is DPM1, the structural gene for dolichol-P-mannose synthase. We suggest that the absence of ERD2 causes an imbalance between membrane flow into and out of the Golgi apparatus, and that the SED gene products can compensate for this either by slowing transport from the ER or by stimulating vesicle budding from Golgi membranes.

Amino Acid Sequence↗
PubMed1992

Construction of a molecular karyotype for Toxoplasma gondii.

As an important step in developing genetic systems for the protozoan parasite Toxoplasma gondii, we have constructed a molecular karyotype based on separation of chromosomes by pulsed field gel electrophoresis and assignment of linkage groups by hybridization. Toxoplasma chromosomes were separated using transverse alternating field electrophoresis (TAFE) gels into 9 distinct bands that defined a minimum of 10 physical linkage groups with apparent sizes that range from approximately 2 Mb to more than 6 Mb. Individual chromosome sizes were stable with prolonged mitotic passage of a single strain but varied by approximately 15% for chromosomes III and V between three different strains of Toxoplasma. Preliminary physical linkage groups were defined by mapping 57 single or low copy number probes to specific chromosomes by hybridization. The majority of these probes consist of random DNA segments; however, a number of cDNAs encoding important structural and antigenic components were also mapped to specific linkage groups. Assuming random distribution, this set of probes should provide approximately 1 marker every 1.0-1.5 Mb over the 80 Mb haploid genome and should greatly aid in using genetics to study the biology, drug resistance, and virulence of this important opportunistic pathogen.

Animals↗
PubMed1992

A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry.

The monoclonal antibody Tg49 both recognizes a Toxoplasma gondii rhoptry protein (ROP1) and inhibits penetration enhancing factor. The latter is a proteinaceous factor found in Toxoplasma lysates or conditioned media that increases the efficiency with which parasites invade host cells. Tg49 was used to screen a lambda gt11 cDNA library and the clone obtained was identified as the cognate gene for ROP1 by several criteria: (1) recombinant protein reacted with the monoclonal; (2) antiserum against the recombinant reacted with the same bands on Western blots as did Tg49; and (3) antiserum against the recombinant recognized a protein in the rhoptries. The ROP1 gene is a single copy gene with a message of approximately 2.1 kb. The predicted polypeptide sequence of ROP1 shows an unusual charge and amino acid asymmetry. There is a highly acidic, proline-rich domain in the amino-terminal portion of the predicted protein, followed by a strongly basic carboxy-terminal domain. An octapeptide repeat is found almost midway through the peptide sequence toward the end of the acidic domain. The ROP1 gene was expressed in a bacterial system, and the resulting polypeptide exhibited anomalous migration on polyacrylamide gel electrophoresis. Given that Tg49 inhibits penetration enhancing factor, it seems likely that the ROP1 protein is a component of that factor, and that the unusual sequence of this protein plays some role in host cell penetration by T. gondii.

Amino Acid Sequence↗
PubMed1992

Generation of a restriction fragment length polymorphism linkage map for Toxoplasma gondii.

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.

Animals↗
PubMed1991

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.

Amino Acid Sequence↗
PubMed1991

Protection of mice from fatal Toxoplasma gondii infection by immunization with p30 antigen in liposomes.

Using liposomes as adjuvant, a purified membrane Ag from Toxoplasma gondii (p30) has been tested for its protective effect in mice. Immunization with p30 in liposomes resulted in only one in 15 mice dying from a challenge that killed 11 of 15 control mice (receiving only buffer or liposomes without p30). The p30 Ag alone gave intermediate levels of protection, with 5 of 15 animals dying. The source of the p30 Ag was the rapidly growing, laboratory-adapted strain of T. gondii, RH; challenge was with the recently isolated C strain which still has all the properties of a wild-type strain. To assess the validity of this combination, the amino acid sequence of p30 from these two strains (as predicted from the corresponding gene sequence) was compared and found to differ in only eight residues. This minimal variation argued that RH was a valid source of material for a subunit vaccine, as subsequently confirmed by the protection studies. These results indicate that p30 is an appropriate Ag for development into a subunit vaccine for immunization of humans and/or domestic livestock, which are a major source of human infection.

Amino Acid Sequence↗
PubMed1991

Inhibition of protein synthesis results in super-induction of procyclin (PARP) RNA levels.

Procyclin is an abundant surface antigen found exclusively on the procyclic forms of African trypanosomes. We are interested in the induction of procyclin gene expression during differentiation from bloodstream forms. We find that increased levels of procyclin RNA are evident as early as 15 min after triggering differentiation. The increase in procyclin RNA levels requires the temperature shift from 37 degrees C to 27 degrees C and is aided by addition of the tricarboxylic acid cycle intermediate cis-aconitate. Maximal induction is observed with a combination of three triggers of differentiation: citrate, cis-aconitate and the temperature shift. Protein synthesis does not appear to be required for induction of procyclin RNA during differentiation. In fact, addition of protein synthesis inhibitors results in super-induction of procyclin RNA levels, even under conditions where no induction is normally observed (i.e., at 37 degrees C in the absence of citrate and cis-aconitate). This super-induction was observed with four different protein synthesis inhibitors that affect different stages of translation. Thus, the accumulation of procyclin transcripts may be under the control of a negative regulator whose effective levels are reduced during differentiation from bloodstream to procyclic forms.

Aconitic Acid↗
PubMed1990

An expression-site-associated gene family of trypanosomes is expressed in vivo and shows homology to a variant surface glycoprotein gene.

Utilizing first-strand cDNA from different stages, a gene family was identified that is expressed in bloodstream form trypanosomes but not in cultured procyclic forms. This family of 50-100 genes, termed bloodstream-specific 1 (BS1), shares a chromosomal distribution pattern similar to the variant surface glycoprotein (VSG) genes and the expression-site-associated genes (ESAGs). The BS1 genes are expressed in several variants of Trypanosoma brucei brucei and in Trypanosoma brucei gambiense. Sequence analysis of five members of this gene family reveals the recently described ESAG 6 and ESAG 7 genes as well as the ESAG X gene to be members of this family. We have been unable to localize the BS1 gene product in the cell but show that chronically infected rabbit serum recognizes recombinant BS1 protein arguing for expression in vivo. Finally we note that the derived protein sequence for the BS1 genes suggests an evolutionary relationship with at least one variant surface glycoprotein gene, and hence these studies may provide clues to understanding the molecular origins of antigenic variation in trypanosomes.

Amino Acid Sequence↗
PubMed1990

Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid.

Infection of pregnant women with Toxoplasma gondii places the developing fetus at risk for congenital infection. We report a prospective study of 43 documented cases of acute maternal Toxoplasma infections acquired during gestation in which the polymerase chain reaction (PCR) was evaluated for diagnosis of fetal infection and compared with the current standard methods. On the basis of direct lysis of pelleted amniotic fluid cells followed by amplification of a gene sequence specific for T. gondii, PCR correctly identified the presence of T. gondii in five of five samples of amniotic fluid from four proven cases of congenital infection. PCR also detected three of five positive cases from a nonprospective group. The two diagnostic methods of comparable speed, detection of specific immunoglobulin M from fetal blood and and inoculation of amniotic fluid into tissue culture, correctly identified only 3 and 4 of the 10 positive samples, respectively. The considerably more time-consuming methods of mouse inoculation of amniotic fluid and fetal blood both detected 7 of 10 positive samples. There were no false-positive diagnoses by any of the methods. Therefore, detection of T. gondii by PCR appears to be the most promising method for prenatal diagnosis of congenital Toxoplasma infection, since it is both extremely rapid and highly sensitive.

Amniocentesis↗
PubMed1990

Lack of introns in the ribosomal protein gene S14 of trypanosomes.

Introns are almost always present in ribosomal protein genes, even in organisms in which introns are rare. Although trans spliced, the trypanosome ribosomal protein gene S14 apparently does not have cis introns, which supports the notion that such introns are absent in this organism.

Amino Acid Sequence↗
PubMed1989

Heat-shock disruption of trans-splicing in trypanosomes: effect on Hsp70, Hsp85 and tubulin mRNA synthesis.

The synthesis of most, if not all, mRNA in trypanosomes involves the splicing in trans of two RNA molecules. One of these includes the common 5' mini-exon or spliced-leader sequence, while the other contains the complete protein-coding exon sequence for a given gene. We have recently shown that trans-splicing of tubulin transcripts is disrupted by heat shock, whereas trans-splicing of Hsp70 mRNA continues unabated, despite the increased expression of this latter gene [Muhich and Boothroyd, Mol. Cell Biol. 8 (1988) 3837-3846]. In this report we show that, in addition to being polyadenylated, the dimeric beta-alpha tubulin transcripts which accumulate as a result of heat shock have a correctly processed 5'-end (through mini-exon addition), and that the extent of disruption is proportional to the degree of heat shock. We further show that mRNA levels for two evolutionarily-conserved heat-shock proteins, Hsp70 and Hsp85, are increased under the splicing-disruptive conditions of heat shock.

Animals↗