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Biomedical subjects

G A Cross

Publications and source records attributed to G A Cross.

At least 19 recordsLinked to original sources
PubMed2001

Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei.

Inositol acylation is an obligatory step in glycosylphosphatidylinositol (GPI) biosynthesis whereas mature GPI anchors often lack this modification. The GPI anchors of Trypanosoma brucei variant surface glycoproteins (VSGs) undergo rounds of inositol acylation and deacylation during GPI biosynthesis and the deacylation reactions are inhibited by diisopropylfluorophosphate (DFP). Inositol deacylase was affinity labelled with [3H]DFP and purified. Peptide sequencing was used to clone GPIdeAc, which encodes a protein with significant sequence and hydropathy similarity to mammalian acyloxyacyl hydrolase, an enzyme that removes fatty acids from bacterial lipopolysaccharide. Both contain a signal sequence followed by a saposin domain and a GDSL-lipase domain. GPIdeAc(-/-) trypanosomes were viable in vitro and in animals. Affinity-purified HA-tagged GPIdeAc was shown to have inositol deacylase activity. However, total inositol deacylase activity was only reduced in GPIdeAc(-/-) trypanosomes and the VSG GPI anchor was indistinguishable from wild type. These results suggest that there is redundancy in T.brucei inositol deacylase activity and that there is another enzyme whose sequence is not recognizably related to GPIdeAc.

3' Untranslated Regions↗
PubMed2001

African trypanosomes in the 21st century: what is their future in science and in health?

The African trypanosomes remain well recognised for their role as an interesting model eukaryote for basic science, but are loosing ground in their ability to contribute to understanding common cellular mechanisms. At the same time, the diseases they cause remain as prevalent as ever, but appear increasingly irrelevant in their wider medical, social, economic and political context. What can be done to keep trypanosome biology relevant and vigorous in the 21st century?

Africa South of the Sahara↗
PubMed2001

t-loops at trypanosome telomeres.

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang. Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.

Animals↗
PubMed2001

Telomere shortening and cell cycle arrest in Trypanosoma brucei expressing human telomeric repeat factor TRF1.

Trypanosoma brucei has telomeres composed of 15 kb tracts of TTAGGG repeats that end in 3' overhangs and form t-loops. This structure is also present in mammalian cells and is thought to reflect the presence of telomere-binding proteins. The human TTAGGG repeat-binding factor TRF1 binds to telomeres and regulates their length. We attempted to interfere with the normal function of trypanosome telomeres by expressing human TRF1 in T. brucei. TRF1 localized to telomeres in cultured procyclic (midgut-stage) trypanosomes with great fidelity, but not in bloodstream-stage trypanosomes. Procyclic trypanosomes expressing high levels of TRF1 for extended periods of time exhibited shortening and increased size heterogeneity of their telomeres and the cell cycle was arrested in G1-S. These effects were not detected in cells expressing a TRF1 mutant incapable of binding to TTAGGG repeats. We argue that TRF1 displaces putative endogenous trypanosome telomere-binding proteins, not yet identified, and affects telomeres in ways that reflect its role as a negative regulator of telomere length in human cells.

Amino Acid Sequence↗
PubMed2000

Trypanosoma cruzi: cloning and characterization of a RAB7 gene.

The small monomeric GTP-binding proteins of the RAB subfamily are key regulatory elements of the machinery that controls membrane traffic in eukaryotic cells. These proteins have been localized to many different intracellular organelles, on both endocytic and exocytic compartments, suggesting that each step of vesicular traffic can involve a specific RAB protein. The presence of conserved amino acid domains in these proteins has allowed the cloning of their genes from several organisms, including yeast, plants, humans, and parasites. In this work we describe the identification, cloning, and characterization of a RAB7 gene homologue in Trypanosoma cruzi (TcRAB7). Our data indicate that this gene is present as a single copy in the T. cruzi genome, located on a 2.25-Mb chromosomal DNA. TcRAB7 is expressed in T. cruzi epimastigotes, metacyclic trypomastigotes, and spheromastigotes. We established transformed cell lines that express two versions of an epitope-tagged TcRAB7 protein: one wild type (pTAG) and one deleted at the C-terminal cysteines (pDeltaCXC). Wild-type TcRAB7 protein (pTAG) appears to be localized exclusively in the membrane fraction, while the mutated TcRAB7 protein (pDeltaCXC) loses the ability to associate with the membrane, showing only cytosolic localization. Also, we produced the recombinant TcRAB7 protein and demonstrated that it binds GTP. The identification of exo- and endocytic machinery components in T. cruzi and their function would provide specific markers of these subcellular compartments, thereby unveiling important aspects of vesicular traffic in this parasite.

Amino Acid Sequence↗
PubMed2000

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427.

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427, clone 221a. This cloned stock is most commonly used in research laboratories in genetic manipulation experiments and in studies of antigenic variation. Using 116 previously characterised chromosome-specific markers, we identify 11 diploid pairs of megabase chromosomes and detect no loss of synteny in EST and gene marker distribution between this stock and the genome project reference stock TREU 927/4. Nevertheless, the chromosomes of 427 are all larger than their homologues in 927, except chromosomes IIa and IXa. The greatest size variation is seen in chromosome I, the smallest of which is 1.1 Mb (927-Ia) and the largest 3.6 Mb (427-Ib). The total nuclear DNA content of both stocks has been estimated by comparison of the mobility of T. brucei and yeast chromosomes. Trypanosomes of stock 427 contain approximately 16.5 Mb more megabase chromosomal DNA than those of stock 927. We have detected the presence of bloodstream-form expression-site-associated sequences on eight or more megabase chromosomes. These sequences are not found on the same chromosomes in each stock. We have determined the chromosomal band location of nine characterised variant surface glycoprotein genes, including the currently expressed VSG 221. Our results demonstrate both the stability of the T. brucei genome, as illustrated by the conservation of syntenic groups of genes in the two stocks, and the polymorphic nature of the genomic regions involved in antigenic variation. We propose that the chromosomes of stock 427 be numbered to correspond to their homologues in the genome project reference stock TREU 927/4.

Animals↗
PubMed2000

Genetic manipulation indicates that ARD1 is an essential N(alpha)-acetyltransferase in Trypanosoma brucei.

N(alpha)-acetylation, the most common protein modification, involves the transfer of an acetyl group from acetyl-coenzyme A to the N-terminus of a protein or peptide. The major N(alpha)-acetyltransferase in Saccharomyces cerevisiae is the ARDI-NATI complex. To investigate N(alpha) -acetylation in Trypanosoma brucei we have cloned and characterised genes encoding putative homologues of ARD1 and NAT1. Both genes are single copy and ARD1, the putative catalytic component, is expressed in both bloodstream-form and insect-stage cells. In either of these life-cycle stages, disruption of both ARD1 alleles was only possible when another copy was generated via gene duplication or when ARD1 was expressed from elsewhere in the genome. These genetic manipulations demonstrate that, unlike the situation in S. cerevisiae, ARD1 is an essential gene in T. brucei. We propose that protein modification by ARD1 is essential for viability in mammalian and insect-stage T. brucei cells.

Acetylation↗
PubMed2000

Expression-site-associated gene 8 (ESAG8) of Trypanosoma brucei is apparently essential and accumulates in the nucleolus.

Trypanosoma brucei variant surface glycoprotein expression sites are interesting examples of genomic loci under complex epigenetic control. In the infectious bloodstream stage, only one of about 20 expression sites is actively transcribed. In the Tsetse midgut (procyclic) stage, chromatin remodeling silences all expression sites. We have begun to explore the function of one of the expression-site-associated genes, ESAG8. Gene knockout experiments implied that ESAG8 is essential. ESAG8 is present at a very low level and apparently accumulates in the nucleolus. A 32-amino-acid domain, which contains a putative bipartite nuclear localization signal (NLS), is both necessary and sufficient to target fusions of ESAG8, with Aequorea victoria green fluorescent protein, to the trypanosome nucleolus. This same sequence functioned only as an NLS in mammalian cells, supporting the idea that nucleolar accumulation requires specific interactions. These results have implications for models of ESAG8 function.

Amino Acid Sequence↗
PubMed1999

Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei.

Trypanosoma brucei glycosylphosphatidylinositol phospholipase C (GPIPLC) is expressed in the bloodstream stage of the life cycle, but not in the procyclic form. It is capable of hydrolyzing GPI-anchored proteins and phosphatidylinositol (PI) in vitro. Several roles have been proposed for GPIPLC in vivo, in the release of variant surface glycoprotein during differentiation or in the regulation of GPI and PI levels, but none has been substantiated. To explore GPIPLC function in vivo, tetracycline-inducible GPIPLC gene (GPIPLC) conditional knock-out bloodstream form and tetracycline-inducible GPIPLC-expressing procyclic cell lines were constructed. We were unable to generate GPIPLC null mutants. Cleavage of GPI-anchored proteins was abolished in extracts from uninduced conditional knock-outs and was restored upon induction. Despite the barely detectable level of GPIPLC activity in uninduced conditional knock-out bloodstream forms, their growth was not affected. GPI-protein cleavage activity could be induced in procyclic cell extracts, up to wild-type bloodstream levels. Myo-[3H]inositol incorporation into [3H]inositol monophosphate was about 14-fold lower in GPIPLC conditional knock-out bloodstream forms than in the wild type. Procyclic cells expressing GPIPLC showed a 28-fold increase in myo-[3H]inositol incorporation into [3H]inositol monophosphate and a 1.5-fold increase in [3H]inositol trisphosphate levels, suggesting that GPIPLC may regulate levels of inositol phosphates, by cleavage of PI and phosphatidylinositol 4,5-bisphosphate.

Animals↗
PubMed1999

Trypanosoma brucei variant surface glycoprotein regulation involves coupled activation/inactivation and chromatin remodeling of expression sites.

Trypanosoma brucei is an extracellular protozoan parasite that cycles between mammalian hosts and the tsetse vector. In bloodstream-form trypanosomes, only one variant surface glycoprotein gene (VSG) expression site (ES) is active at any time. Transcriptional switching between ESs results in antigenic variation. No VSG is transcribed in the insect procyclic stage. We have used bacteriophage T7 RNA polymerase (T7RNAP) to study the transcriptional accessibility of ES chromatin in vivo. We show that T7RNAP-mediated transcription from chromosomally integrated T7 promoters is repressed along the entire length of the ES in the procyclic form, but not in the bloodstream form, suggesting that the accessible chromatin of inactive bloodstream-form ESs is remodeled upon differentiation to yield a structure that is no longer permissive for T7RNAP-mediated transcription. In the bloodstream form, replacing the active ES promoter with a T7 promoter, which is incapable of sustaining high-level transcription of the entire ES, prompts an ES switch. These data suggest two distinct mechanisms for ES regulation: a chromatin-mediated developmental silencing of the ES in the procyclic form and a rapid coupled mechanism for ES activation and inactivation in the bloodstream form.

Animals↗
PubMed1999

A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.

First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.

Animals↗
PubMed1998

Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei.

Inability of T7 RNA polymerase to processively transcribe higher eukaryotic chromatin is interpreted as a correlate of its reported inhibition by nucleosomes on reconstituted templates in vitro . We used chromosomally integrated reporter cassettes to examine features of T7 transcription in a lower eukaryotic system. Luciferase reporters were targeted to rDNA in transgenic Trypanosoma brucei stably expressing the phage polymerase. Because trypanosome mRNAs are capped by RNA splicing in trans , T7 transcription could be gauged by luciferase activity. In contrast to findings from higher eukaryotes, T7 transcription is vigorous and processive on chromatin templates in T.brucei , surpassing levels achieved with endogenous promoters, including those recruiting RNA polymerase I. This may be a reflection of intrinsic differences in chromatin structure between differently evolved eukaryotes or of an integration site that is exceptionally permissive for T7 transcription due to a local accessible chromatin conformation. T7 transcription could be manipulated to achieve different levels of constitutive expression, through the use of promoter mutations. Moreover, T7 initiation could be regulated by the prokaryotic Tet repressor and elongation halted by T7 terminator sequences. We have exploited these features to construct a robust inducible expression system, whose utility potentially extends to other trans -splicing organisms.

Animals↗
PubMed1998

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei.

In Trypanosoma brucei, the active variant surface glycoprotein genes (vsg) are located at telomeric expression sites (ES), whose expression is highly regulated during the life cycle. In the procyclic form, all ESs are repressed. In the bloodstream form, where antigenic variation occurs, only one of approximately 20 ESs is active at a given time. We have investigated chromatin structure and DNA sequence around the ES promoter to identify cis-acting regulatory regions. A marker gene, inserted 1 kb downstream of the ES promoter, was used as a specific probe to map the position of nuclease hypersensitive sites. A prominent hypersensitive site was detected within the core promoter. This site was present in both active and inactive ES promoters, suggesting that a protein complex is bound to the promoter irrespective of its transcriptional state. However, none of the regions showed differential nuclease sensitivity between active and inactive transcriptional states. A systematic deletion analysis of the sequences surrounding the active ES promoter in situ confirmed the absence of cis-regulatory elements. We find that only 70 bp within the ES promoter are necessary to support ES regulation. Analysis of the reporter activities in an inactive bloodstream-form ES revealed the existence of an intermediate promoter activity in some clones, but we never observed full activation of more than one ES. The vsg mRNA from this intermediate ES was expressed less efficiently.

Animals↗
PubMed1998

Regulation of vsg expression site transcription and switching in Trypanosoma brucei.

Current understanding of expression-site transcription in Trypanosoma brucei, has been refined by recent results of promoter manipulations at vsg expression sites (ES) and examination of the behavior of ES promoters in ectopic locations both within the ES and at other loci. In summary, ES promoter sequences inserted into non-transcribed rRNA spacers are generally inactive, or have low activity, in bloodstream and procyclic forms. Some mechanism apparently operates to ensure full activation of a single ES in bloodstream-form trypanosomes and the inactivity of all ES promoters in procyclic forms. As previously shown, a rRNA promoter can replace an ES promoter. In bloodstream forms, the replacement rRNA promoter was down-regulated in a 'silent' ES but it was active in procyclic forms. In addition to manipulations of endogenous promoters, we have recently shown that, when an ES promoter is replaced by a T7 promoter, the T7 promoter is unregulated but transcription is attenuated before the vsg, and another ES switches on to maintain cell viability. However, T7 transcription is repressed in the context of core ES-promoter sequences in both stages, particularly in procyclic forms. These observations strongly argue that sequences in the vicinity of the ES core promoter play a role in ES control by nucleating critical events in silencing as well as in activation. Deletions of sequences surrounding the ES core promoter, in situ, did not affect its activity or regulation. In bloodstream forms, rRNA or ES promoters inserted adjacent to silent telomeres or to a non-telomeric 'basic-copy' vsg were > 98% repressed. After transformation to procyclic forms, the sub-telomeric rRNA promoter regained about 10% of its maximal activity but the 'basic-copy' rRNA promoter was fully active. Similarly-positioned ES promoters remained silent in procyclic forms. These results suggest that telomere-proximal or vsg-proximal sequences might mediate suppression of transcription via position-effects that could be sufficient to suppress the expression of chromosome-internal vsgs or telomeric metacyclic vsgs, in bloodstream-form trypanosomes. Recent experiments with T7 promoters indicate that sequences within the ES core promoter might be responsible for silencing ES promoters in procyclic forms. Precedents for regulatory mechanisms that modulate transcription over large chromatin domains are reviewed and possible models for ES regulation are presented.

Animals↗
PubMed1997

Position-dependent and promoter-specific regulation of gene expression in Trypanosoma brucei.

Trypanosoma brucei evades the mammalian immune response by a process of antigenic variation. This involves mutually exclusive and alternating expression of telomere-proximal variant surface glycoprotein genes (vsgs), which is controlled at the level of transcription. To examine transcription repression in T.brucei we inserted reporter genes, under the control of either rRNA or vsg expression site (ES) promoters, into various chromosomal loci. Position-dependent repression of both promoters was observed in the mammalian stage of the life cycle (bloodstream forms). Repression of promoters inserted into a silent ES was more pronounced closer to the telomere and was bi-directional. Transcription from both ES and rRNA promoters was also efficiently repressed at a non-telomeric vsg locus in bloodstream-form trypanosomes. In cultured tsetse fly midgut-stage (procyclic) trypanosomes, in which vsg is not normally expressed, all inserted rRNA promoters were derepressed but ES promoters remained silent. Our results suggest that vsg promoters and ectopic rRNA promoters in bloodstream-form T.brucei are restrained by position effects related to their proximity to vsgs or other features of the ES. Sequences present in rRNA promoters but absent from vsg ES promoters appear to be responsible for rRNA promoter-specific derepression in procyclic cells.

Animals↗