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I Roditi

Publications and source records attributed to I Roditi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Contributions of the procyclin 3' untranslated region and coding region to the regulation of expression in bloodstream forms of Trypanosoma brucei.

When bloodstream forms of Trypanosoma brucei differentiate into procyclic forms they rapidly synthesise a new surface coat composed of procyclins. Procyclin genes are transcribed in bloodstream forms at approximately one-tenth of the rate in procyclic forms, but little, if any, mRNA can be detected, indicating that further down-regulation must occur post-transcriptionally. We have examined the role of the 297 bp procyclin 3' untranslated region (UTR) in regulating expression in bloodstream forms and have identified three discrete elements: a dominant, negative element between positions 101 and 173, and two positive elements. When chloramphenicol acetyl transferase (CAT) was used as the reporter gene, deletion of the negative element caused a approximately 6-fold increase in the level of steady state mRNA and > 30-fold increase in CAT activity, suggesting that both RNA stability and translation were affected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating that the 3' UTR acts independently of the coding region. In contrast, when trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3' UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernatants. These results imply that the procyclin coding region exerts yet another layer of control which prevents inappropriate expression of the protein in the mammalian host.

Amino Acid Sequence↗
PubMed1997

Antagonistic effects of IL-4 and interferon-gamma (IFN-gamma) on inducible nitric oxide synthase expression in bovine macrophages exposed to gram-positive bacteria.

Cytokine-mediated modulation of nitric oxide (NO) production by bacteria-stimulated bovine macrophages was studied. When Salmonella dublin, as a prototypic gram-negative organism, was used, NO generation was barely enhanced by recombinant bovine and ovine IFN-gamma, but was suppressed by IL-4. Salmonella dublin-induced NO generation was not influenced by a panel of nine other cytokines. The panel included IL-1, tumour necrosis factor (TNF) and IFN-alpha, which are active in a similar mouse macrophage model. The tested cytokines were either homologous or known to interact with bovine cytokine receptors. Recombinant bovine and ovine IFN-gamma were the only cytokines which strongly enhanced NO synthesis by macrophages exposed to the gram-positive organism, Listeria monocytogenes. Listeria-induced NO generation was strongly suppressed by recombinant human and bovine IL-4, but not by IL-10 and transforming-growth-factor-beta. Thus, two cytokines characterizing a Th1 and a Th2 response up- and down-regulate, respectively, bacteria-induced NO generation in bovine macrophages, whereas nine other cytokines had little activity in this regard. This modulation was reflected in changes in the steady state levels of mRNA coding for inducible nitric oxide synthase. Combinations of IFN-gamma and IL-4 suggested that the relative proportion of these cytokines determined whether bacteria-induced NO generation was up- or down-regulated. At saturating IL-4 concentrations, stimulation of bacteria-induced NO generation in macrophages by T cell supernatants was solely dependent on IFN-gamma. This was shown by antibody neutralization experiments and by a close correlation between the capacity of supernatants to stimulate NO generation and the IFN-gamma content, as determined by immunoassay.

Animals↗
PubMed1997

Elements in the 3' untranslated region of procyclin mRNA regulate expression in insect forms of Trypanosoma brucei by modulating RNA stability and translation.

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3' untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3' UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3' UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an approximately 2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t(1/2)) of the mRNA was reduced from approximately 50 min (the t(1/2) of the wild-type 3' UTR) to < 15 min, whereas removal of the LII element resulted in an increased t(1/2) of approximately 100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.

Animals↗
PubMed1996

Discrimination of Neospora caninum from Toxoplasma gondii and other apicomplexan parasites by hybridization and PCR.

Neospora caninum is a protozoan parasite which causes neurological problems in dogs and abortion in cattle. As N. caninum is difficult to distinguish morphologically from Toxoplasma gondii, we developed a molecular tool capable of discriminating between the two parasites. Genomic DNA was isolated from in vitro cultured N. caninum tachyzoites and cloned into a plasmid vector. Resulting colonies were subsequently screened by differential hybridization using N. caninum and T. gondii DNA. Two clones were characterized in detail: one clone, termed pNc5, was found to be specific for N. caninum whereas the second clone, pNc1, hybridized with DNA from both parasites. The sequence of pNc5 was determined and different oligonucleotide primers were designed for use in the polymerase chain reaction (PCR). A 944 bp fragment was specifically amplified from N. caninum DNA, but not from DNA extracted from T. gondii or different Sarcocystis species. Positive signals in PCR were obtained with as little as 100 pg parasite template DNA. In addition, dual PCR with primer pairs specific for N. caninum and T. gondii allowed the detection of either parasite in mixed samples.

Animals↗
PubMed1995

Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker.

Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface. Although GARP shows no sequence similarity to procyclins from T. brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes. Since T. brucei and T. congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments. As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T. brucei in which the GARP coding region has been integrated into a procyclin expression site. GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T. brucei rather than in T. congolense. The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection. We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.

Animals↗
PubMed1994

Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site: a dual role for polypyrimidine tracts in trypanosomes?

The procyclin-associated genes (PAGs) of Trypanosoma brucei are located downstream of tandemly repeated procyclin genes and belong to the same alpha-amanitin-resistant polycistronic transcription units. In procyclic form trypanosomes the PAG 1 pre-mRNA is alternatively spliced to give rise to three transcripts of 2.7 kb, 1.8 kb and 1.3 kb. The two larger transcripts contain additional short open reading frames (ORFs) upstream of the major ORF. Trans-splicing to generate these transcripts occurs downstream of three different polypyrimidine tracts. A minor population of procyclin mRNAs is also generated by alternative splicing at a polypyrimidine tract that begins 524 bp upstream of the major splice acceptor site of the procyclin beta-gene. The same polypyrimidine tract is also required for accurate polyadenylation of mRNAs from the upstream procyclin alpha-gene (1). Alternatively polyadenylated forms of PAG 1 mRNAs can also be detected. All polyadenylation sites are found at a similar distance upstream of splice-acceptor sites, in each case with a polypyrimidine tract between them. Our results point to a dual role for polypyrimidine tracts in the maturation of trypanosome mRNAs.

Alternative Splicing↗
PubMed1994

A conserved stem-loop structure in the 3' untranslated region of procyclin mRNAs regulates expression in Trypanosoma brucei.

African trypanosomes that cycle between mammalian hosts and the tsetse fly vector must be poised to survive in different environments. The control of stage-specific gene expression is undoubtedly one of the keys to successful adaptation, but no regulatory elements have been defined to date. Procyclins (also known as procyclic acidic repetitive proteins) are specifically expressed on the surface of procyclic and epimastigote forms in the fly. Procyclin genes are already transcribed in bloodstream forms, but stable mRNA, and later the protein, are first detected when the parasites begin to differentiate into procyclic forms. We have now identified a region of 16 bases that forms part of a predicted stem-loop structure in the 3' untranslated regions of different procyclin mRNAs; both the sequence and the secondary structure of this 16-mer appear to be required for efficient translation of a reporter gene in procyclic forms. The level of steady-state mRNA, its polyadenylylation, and its distribution in the cell are all unaffected by the presence or absence of this element. Deletion of the 16-mer alone reduces expression more than removal or reversal of the entire 3' untranslated region and flanking region, suggesting that there are additional negative regulatory elements in the same 3' untranslated region.

Animals↗
PubMed1994

Virus-like particles in Eimeria nieschulzi are associated with multiple RNA segments.

RNA preparations from sporulated oocysts of Eimeria nieschulzi were found to contain 2 double-stranded RNA segments of 5.0 kb and 5.7 kb that were not present in other species of Eimeria. Treatment of crude lysates with RNase A revealed that in addition to these two segments, 3 other segments of 0.57 kb, 0.72 kb and 11.5 kb were protected from digestion, suggesting that they were enclosed within particles. Virus-like particles with a diameter of approximately 39 nm were purified by caesium chloride buoyant density centrifugation. Four of the five RNA segments copurified with these particles. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this new isolate ENV 1. The largest RNA segment does not cosediment with ENV 1 particles and may be derived from another RNA-protein complex that is unstable under the conditions used. The particle size and genome structure of ENV 1 both differ from that of the Eimeria stiedae virus (ESV), which is the only other virus to have been isolated from Eimeria to date. Short cDNA clones derived from ENV 1 show significant homology to a region of the Leishmania virus (LRV 1) genome that encodes an RNA-dependent RNA polymerase. The polymerase sequences from ENV 1 and LRV 1 are more closely related to each other than to any other protein sequences in the GenEMBL Database. This raises intriguing questions about the origins of the two viruses, since Eimeria and Leishmania normally infect different hosts and also show different cell tropisms within these hosts.

Amino Acid Sequence↗
PubMed1994

The regulation of procyclin expression in Trypanosoma bruceli: making or breaking the rules?

The identification of procyclins as stage-specific coat proteins of procyclic forms of Trypanosoma brucei has not only provided a convenient molecular marker for the differentiation of bloodstream-form trypanosomes into procyclic forms, but has also allowed some important insights into gene regulation in trypanosomes. Here, Adrian Hehl and Isabel Roditi summarize what has been learnt in the past few years about the control mechanisms that may contribute to the stage-specific expression of procyclins.

Journal Article↗
PubMed1994

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.

Animals↗
PubMed1993

Eimeria tenella: characterization of a 5S ribosomal RNA repeat unit and its use as a species-specific probe.

The oocysts of Eimeria tenella, one of the most pathogenic of several species causing chicken coccidiosis, are difficult to distinguish microscopically from several other infective Eimeria species. One copy of a gene coding for 5S ribosomal RNA has been cloned from E. tenella and sequenced. A coding region of 120 nucleotides and an intergenic region of 608 nucleotides together make up a 5S rRNA repeat unit, of which there are many copies tandemly repeated in the genome. The intergenic region is species-specific and sequences derived from it can be used for species identification by polymerase chain reaction (PCR). The PCR procedure described here is particularly sensitive, with fewer than 10 oocysts sufficing to give a positive result. This method may also be useful for the identification by PCR of other cyst-forming parasites.

Animals↗
PubMed1993

Identification and characterization of an acidic major surface glycoprotein from procyclic stage Trypanosoma congolense.

Monoclonal antibodies (mAbs) were derived against the procyclic culture form of Trypanosoma congolense and 14 were selected which bound to the surface of living procyclics in immunofluorescence assays. These antibodies bound to procyclics and epimastigotes of T. congolense (both savannah-type and Kilifi-type) and procyclics of Trypanosoma simiae, but not to procyclics of other species of trypanosomes, to bloodstream forms of several species of trypanosomes or to Leishmania, and were thus life cycle stage- and subgenus-specific. Fluorescence-activated cell sorter analysis with these antibodies showed that the kinetics of expression of the surface antigen during transformation from bloodstream to procyclic forms was similar to that of procyclin or procyclic acidic repetitive protein (PARP) of T. brucei spp. appearing at the cell surface as early as 8 h after initiating transformation. All fourteen antibodies detected broad bands of 40-44 and 28-32 kDa in immunoblot analysis of whole procyclic lysates and were specific for carbohydrate epitopes. The antigen was purified by cation-exchange chromatography and gel electrophoresis, and was shown to be an acidic glycoprotein. Amino acid microanalysis of the purified antigen showed an abundance of glutamic acid/glutamine and alanine. Sequences of peptides produced by cyanogen bromide cleavage matched amino acid sequences predicted by the nucleotide sequence of a gene described in the accompanying paper by Bayne et al. [26]. No sequence similarity to T. brucei procyclin/PARP or to any other protein was found. However, its stage and subgenus specificity, surface disposition, immunodominance, acidity and kinetics of expression during transformation from bloodstream to procyclic forms indicate that the molecule is an analog of procyclin/PARP described in T. brucei spp.

Amino Acid Sequence↗
PubMed1992

A procyclin-associated gene in Trypanosoma brucei encodes a polypeptide related to ESAG 6 and 7 proteins.

The procyclin genes of Trypanosoma brucei encode a family of glycoproteins expressed on the surface of procyclic forms of the parasite. These genes are present at different loci in tandem arrays of two or three copies depending on the strain. It has previously been shown that procyclin genes are transcribed from a promotor immediately upstream of the first procyclin gene in each cluster by an RNA polymerase that is resistant to high levels of alpha-amanitin. Here we show that additional genes, which we term procyclin-associated genes (PAGs), are located downstream of the procyclin genes and belong to the same alpha-amanitin-resistant polycistronic transcription units. A gene in the pro A locus, PAG 1, encodes a polypeptide that is related to the ESAG 6 and 7 proteins encoded in the VSG expression site. An unexpected feature of PAG 1 is that the major open reading frame of 405 amino acids only starts at position 1283 in the cDNA sequence and extends to the poly(A) tail. Sequences related to the 5' untranslated region of PAG 1 are also found downstream of procyclin genes in other loci, but the 3' coding region is unique to Pro A. This suggests that there are related PAGs which are coordinately transcribed with procyclin genes from different loci.

Amino Acid Sequence↗
PubMed1991

Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues.

The complete amino acid sequences for nine variant specific glycoproteins (VSGs) of Trypanosoma brucei are presented. These have more than doubled the size of the VSG sequence data base and have enabled a new and more rigorous comparison to be made between amino acid sequences of different VSGs. Each VSG can be defined as a combination of an N-terminal domain type and a C-terminal domain type, based on the distribution of cysteine residues within the molecule. This identifies three N-terminal domain types and at least four C-terminal domain types. Different combinations of N and C-terminal domains can be formed; for example, in the sequences presented here, two different N-terminal domains are found in association with each of three different C-terminal domains. The biological context of the domain structure of VSGs is discussed.

Amino Acid Sequence↗
PubMed1990

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei.

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.

Amino Acid Sequence↗