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K Stuart

Publications and source records attributed to K Stuart.

At least 127 records · Page 7Linked to original sources
PubMed1985

The two mechanisms for antigenic variation in Trypanosoma brucei are independent processes.

Antigenic switching in Trypanosoma brucei can occur either by the production of a telomeric copy of a variant surface glycoprotein (VSG) gene through a gene conversion mechanism or by the nonduplicative activation of a telomeric VSG gene. The 5 VSG gene telomeric copy that is expressed in IsTaR 1 variant antigenic type (VAT) 5 is retained in an inactive state following an antigenic switch to VAT A5. This inactive telomeric 5 VSG gene copy is absent following independent single antigenic switches to VATs 1A5 and 11A5. The inactive 5 VSG gene does not appear to have been replaced with the newly expressed VSG gene. Thus, inactive telomeric VSG genes that are capable of being expressed can be lost, presumably through gene conversion to new VSG genes. These results suggest that gene conversion of an inactive VSG gene does not obligately activate the new VSG gene. We conclude that the gene conversion and telomeric activation mechanisms for antigenic switching are separate and independent processes.

Animals↗
PubMed1985

Identification of mitochondrial genes in Trypanosoma brucei and homology to cytochrome c oxidase II in two different reading frames.

We have determined the nucleotide sequence of a 3.3 kilobase segment of the kDNA maxicircle of Trypanosoma brucei brucei 164. The nucleotide sequence and its predicted translated sequence have homology to cytochrome c oxidase subunits I and II (CO I and II) and mammalian unidentified reading frame 1 (URF 1). Amino acid homology to CO II extends for 170 residues from the amino terminus in one reading frame and then continues in another reading frame for 39 residues to the carboxyl terminus. Similar results have been obtained for Leishmania tarentolae [de la Cruz, V.F., Neckelmann, N. and Simpson, L. (1984) J. Biol. Chem., in press] and T. brucei 427 [Hensgens, L.A.M., Brackenhoff, J., De Vries, B.F., Sloof, P., Tromp, M.C., Van Boom, J.H. and Benne, R. (1984) Nucleic Acids Res. 12, 7327-7344]. This may indicate that novel events are required for expression of this gene. Amino acid homology to URF 1 exists predominantly at the amino terminal end although no corresponding AUG codon occurs in this area. An alternative initiation codon may therefore be utilized by trypanosome mitochondria. Two other open reading frames (ORFs) were detected and these are discussed with reference to transcripts from this region. ORFs corresponding to transcripts are organized compactly and are distributed more equally on both strands compared to ORFs of other mitochondrial systems.

Amino Acid Sequence↗
PubMed1985

Differential expression of mitochondrial genes between life cycle stages of Trypanosoma brucei.

The mitochondrial respiratory system is differentially produced during the life cycle of the parasitic protozoan, Trypanosoma brucei. We have found 14 transcripts that are derived from a maxicircle (mitochondrial DNA) region that contains sequences homologous to cytochrome c oxidase subunits I and II, unassigned reading frame 1 from mitochondrial DNA of other organisms, and two other open reading frames. Ten of these transcripts occur as pairs that differ in size by approximately equal to 200 nucleotides. While most of these transcripts occur in both life cycle forms, four transcripts are differentially expressed. Of these, two are more abundant in bloodstream forms, one is more abundant in procyclic forms, and one is present in procyclic but not bloodstream forms. These results indicate that the differential production of the mitochondrial respiratory system in T. brucei entails regulation of mitochondrial transcriptional and/or posttranscriptional processes.

Animals↗
PubMed1985

Antigenic variation in clones of Trypanosoma brucei grown in immune-deficient mice.

We have produced monoclonal antibodies against six variant surface glycoproteins from early variant antigen types (VATs) of the IsTaR 1 serodeme of Trypanosoma brucei brucei. We have used these in fixed cell immunofluorescence assays to follow the VAT composition of populations of each early VAT when passaged through irradiated mice. The IsTat 1.A and 1.7a populations were stable for more than 30 days (approximately 150 generations), but 1.1a, 1.3a, 1.5a, and 1.11a all changed to 1.A within this time. The time and rate of this antigenic switch were characteristic for each VAT. Growth rates of the VATs were determined when they were both grown separately and grown with 1.A. It appeared that the order of growth rates was 1.7a greater than 1.A = 1.1a greater than 1.11a greater than 1.5a greater than 1.3a. We have generated theoretical curves for the replacement of one VAT by another based on differences in their growth rates and the rate at which one VAT switches to another (switch frequency). These curves closely match those derived experimentally. We postulate that the differences in growth rates between VATs and the different switch frequencies for VATs may be sufficient to generate the loosely defined sequence of VATs seen in chronic infections.

Animals↗
PubMed1985

Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei.

Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei.

Animals↗
PubMed1984

The IsTat 1.3 VSG multigene family in Trypanosoma brucei: retention of the expression linked copy through multiple antigenic switches.

In the IsTaR 1 serodeme of T. brucei the 3 variant surface glycoprotein (VSG) gene family contains about 10 members, one of which has a telomeric location on a minichromosome. The expression linked copy (ELC) of the 3 VSG gene which occurs in an antigenic variant expressing the 3 VSG, also has a telomeric location but unlike the minichromosomal 3 VSG gene has restriction sites upstream from the 5' barren region. This ELC is retained on the same telomere in a subsequent variant that expresses a telomeric 7 VSG ELC and in relapse variants and procyclic forms derived from variant antigenic types (VATs) 3 and 7. The 7 ELC has a restriction map upstream from the 5' barren region that differs from, but is similar to, that of the 3 ELC. These data indicate that the 3 and 7 ELCs are on different telomeres when expressed.

Animals↗
PubMed1984

Antigenic variation in African trypanosomes by gene replacement or activation of alternate telomeres.

We have analyzed antigenic variants with a known lineage and show that there are several telomeres on which variant surface glycoprotein (VSG) genes can be expressed. These telomeres have similar restriction maps 5' to the barren region. In addition, the same VSG gene was expressed on different telomeres. Some antigenic switches in the lineage were accomplished by duplicative replacement of one VSG gene with another. Other switches occurred without duplication by transcriptional activation of an alternate telomeric VSG gene. We call the latter process telomeric activation and propose that these two processes can occur independently. We further propose that antigenic switching by telomeric activation is mediated by the regulatory system that controls which telomere is transcriptionally active, while the duplicative mechanism does not.

Animals↗
PubMed1984

Expression of a Trypanosoma brucei brucei variant antigen in Escherichia coli.

A cDNA library derived from antigenically homogeneous bloodstream stage Trypanosoma brucei brucei was screened with an antiserum directed against the variant surface antigen (VSA) using an enzyme-linked filter immunoassay. Several recombinant clones were detected and the clone giving the most intense reaction was further analyzed. It contained a VSA-specific cDNA insert and synthesized a protein of the expected molecular weight bearing VSA determinants. The nucleotide sequence of the insert was determined and shown to have the unusual codon bias characteristic of T. brucei VSAs, frequently employing codons specifying tRNAs rare in Escherichia coli. These results indicate that a codon bias very different from that of E. coli does not preclude the expression of a cloned sequence to detectable levels in this heterologous host.

Amino Acid Sequence↗
PubMed1984

Variant antigen genes of Trypanosoma brucei: genomic alteration of a spliced leader orphon and retention of expression-linked copies during differentiation.

Variant surface glycoprotein (VSG) gene expression in Trypanosoma brucei involves not only the sequential activation of individual VSG genes during mammalian bloodstream stage antigenic variation but also the regulation of gene expression during cyclic transmission through alternate mammalian and insect hosts. In the bloodstream stage, transcriptional activation of many VSG genes is correlated with the appearance of an additional copy of the gene in a novel genomic location, the expression-linked copy. The parasite loses the ability to synthesize VSG during differentiation from mammalian bloodstream to insect procyclic stage. Five different bloodstream populations were individually converted to procyclic forms. In each case, the procyclic cells retained the expression-linked copy of the bloodstream parent, and it remained in the same immediate genomic context. Transcripts homologous to the VSG structural gene exon were found in bloodstream stage RNA but not in procyclic RNA. Nevertheless, transcripts containing sequences homologous to the VSG mRNA spliced leader were abundant in both procyclic and bloodstream stage cells. When the genomic organization of sequences homologous to the VSG leader was examined, a specific alteration correlated with procylic differentiation was found. These data are discussed in light of biological studies on antigenic variation.

Animals↗
PubMed1984

The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme.

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.

Animals↗
PubMed1983

Mitochondrial DNA of an African trypanosome.

The maxicircles of African trypanosome kDNA are the genetic equivalent of other mitochondrial DNAs, but the function of minicircles is unknown. The maxicircle of Trypanosoma brucei 164 encodes conventional mitochondrial gene products and is largely but not completely transcribed. Nucleotide sequence analysis of a region not found to be transcribed revealed numerous translation termination codons in all three reading frames of both strands and numerous inverted repeats, suggesting that this segment does not have polypeptide-coding function. This segment may encode a t-RNA and has a sequence resembling a consensus sequence found in mitochondrial introns, thus implying that transcript processing occurs in trypanosome mitochondria. While several cloned minicircles had distinct restriction maps reflecting T brucei minicircle heterogeneity, one segment of the minicircle contained a sequence that was conserved by minicircles from other trypanosome strains and species. Of nine mutants unable to grow as the respiring procyclic forms, seven were devoid of kDNA. The other two mutants retained normal amounts of all maxicircle restriction fragments and normal amounts of those minicircle sequences tested. Minicircle alterations probably occur in these mutants, since the kDNA does not stain with Giemsa and bands at an altered density in cesium chloride/ethidium bromide density gradients.

Animals↗
PubMed1983

Genomic organization of variant surface glycoprotein genes in Trypanosoma brucei procyclic culture forms.

The production of the variant surface glycoprotein coat of bloodstream form African trypanosomes ceases after conversion to the procyclic form. In the bloodstream stage alternate expression of different variant surface glycoprotein genes is responsible for the antigenic variation that occurs during relapse infections in the mammalian host. We have examined procyclic stage populations, derived from different bloodstream variant antigen types, for the two types of genomic alterations associated with variant surface glycoprotein genes in the bloodstream stage. Transcriptional activation of some variant antigen genes is accompanied by the generation of a new copy of the gene, the expression-linked copy. We find that the expression-linked copy is maintained after conversion to procyclic form, indicating that the presence of an expression-linked copy is not sufficient for the expression of a surface coat. Sequences 3' to other variant surface glycoprotein genes show expression-independent variation in bloodstream stage trypanosomes. The same genes showed variation between procyclic populations of different origin, and between procyclics and their bloodstream parent. These data are discussed in light of observations on the sequence of variant antigen expression after cyclic transmission.

Animals↗
PubMed1983

Sequences homologous to the variant antigen mRNA spliced leader are located in tandem repeats and variable orphons in trypanosoma brucei.

We have examined the organization of genomic sequences homologous to the spliced leader of Trypanosoma brucei variant surface glycoprotein (VSG) mRNA, using a synthetic oligodeoxynucleotide probe. These sequences are highly reiterated in the trypanosome genome and most are located in 1.4 kb units arranged in a direct tandem repeat. However, some of the 1.4 kb sequences are dispersed from the cluster(s) of tandem repeats and are flanked by non-repeat DNA. The number and arrangement of these leader sequence orphons varies among different T. brucei stocks. Within the IsTat serodeme, the arrangement of three of four spliced leader orphons observed with Eco RV digestion was stable during a chronic infection and cyclic transmission through the insect vector. The fourth Eco RV orphon, however, undergoes rearrangement during antigenic variation and life-cycle differentiation.

Animals↗
PubMed1983

Molecular characterization of initial variants from the IsTat I serodeme of Trypanosoma brucei.

Variant surface glycoproteins (VSGs) were isolated from variant antigen types (VATs) of the IsTat 1 serodeme. Molecular weight and isoelectric focusing analysis demonstrate that seven early VSGs possess properties generally attributed to VSGs isolated from other trypanosome serodemes. Six of the seven VSGs characterized are distinct from one another, while two (D and 1) appear identical. The presence of VSG specific mRNA in corresponding VATs was demonstrated by in vitro translation of RNA from each of the VATs, followed by immunoprecipitation with homologous and heterologous antisera. Hybridization of VSG cDNA clones with RNA from each VAT confirm that VSG mRNA is present only in homologous VATs and verifies the transcriptional control of these VSG genes. The two VATs D and 1 express indistinguishable VSGs by a variety of biochemical criteria, as well as by reactivity with 24 monoclonal antibodies. The VSG mRNAs in VATs D and 1 also appear identical. However, this identity is not reflected at the genomic level. Data is presented which establishes that DNA rearrangements can occur around both expressed and non-expressed VSG genes without qualitatively affecting VSG gene expression.

Animals↗
PubMed1983

Genomic organization of Trypanosoma brucei variant antigen gene families in sequential parasitemias.

cDNA libraries were made from mRNA purified from each of seven sequentially isolated variant antigen types (VATs) of the IsTat 1 serodeme. Plasmids containing variant surface glycoprotein (VSG) sequences corresponding to each of the isolates were used in Southern analyses to examine the genomic organization of VSG nucleotide sequences. In most cases, cells expressing a given VSG were shown to have an extra copy of the corresponding VSG gene. In one case an expression-linked copy (ELC) was not detectable. VSG gene rearrangements not obviously correlated with the expression of homologous sequences were detected in four of six VSG gene families. Thus, even cDNAs which detected an ELC revealed additional genomic reorganization in regions flanking VSG sequences. The cells used to initiate the chronic infection expressed the same VSG as those isolated from the first parasitemia. The extent of genomic rearrangement observed between these two sequentially derived populations was comparable to that observed between any of the other serially derived VATs. Thus, within a short period of time and in the absence of detectable antigenic variation, the amount of genetic flux in sequences associated with VSG genes can be substantial.

Animals↗
PubMed1983

Kinetoplast DNA, mitochondrial DNA with a difference.

Kinetoplast DNA occurs in flagellated protozoa belonging to the order Kinetoplastida. Kinetoplast DNA contains tens of maxicircles and thousands of minicircles which are catenated into a single network in each cell. Maxicircles contain genetic information analogous to that in other mitochondrial DNAs. Maxicircles encode mitochondrial ribosomal RNAs and hybridize with mitochondrial gene sequences from other organisms. Minicircles evolve rapidly, may not be transcribed, and vary greatly in total complexity among genera. The functions of minicircles and the network structure are unknown.

Animals↗
PubMed1982

Activation of misonidazole by rat liver microsomes and purified NADPH-cytochrome c reductase.

Rat liver microsomes and purified NADPH-cytochrome c reductase metabolized [14C]misonidazole anaerobically to a reactive intermediate that covalently binds to tissue macromolecules. Air strongly inhibited the binding whereas carbon monoxide had no effect, indicating that misonidazole is activated via reduction and not by cytochrome P-450-dependent oxidation. Both systems showed an absolute requirement for NADPH and were stimulated by flavine (FAD) and paraquat. The apparent Km for misonidazole binding to microsomal protein was 0.74 mM the apparent Vmax was 0.64 nmole 14C bound . mg-1 . min-1. At a single substrate concentration, nitrofurantoin, nitrofurazone and desmethylmisonidazole inhibited the covalent binding of misonidazole to microsomal protein by 47, 26, and 38% respectively. The effect of nitrofurantoin on the kinetics of misonidazole binding gave a complex interaction indicative of uncompetitive inhibition. Glutathione reduced the binding of misonidazole to microsomal protein below the level observed for boiled microsomes while ascorbic acid had no effect. Compared to nitrofurantoin and paraquat, misonidazole was a poor stimulator of superoxide production as measured by adrenochrome formation.

Animals↗