Variant-specific Surface Glycoprotein Genes Research Articles

Variant Surface Glycoprotein from Trypanosoma evansi Is Partially Responsible for the Cross-Reaction between Trypanosoma evansi and Trypanosoma vivax

Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

Insertion of the promoter for a variant surface glycoprotein gene expression site in an RNA polymerase II transcription unit of procyclic Trypanosoma brucei

The variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei are invariably expressed near the ends of chromosomes (telomeres). We have targeted a VSG gene expression site (ES) promoter driving a selectable marker gene (neomycin phosphotransferase) into a chromosome-internal transcription unit, the tubulin gene array of procyclic trypanosomes. To avoid read through transcription of the marker gene from the tubulin promoter, we targeted the ES promoter in inverse orientation relative to tubulin gene transcription. The only correctly targeted transformant obtained contained the marker gene close to the border of the tubulin gene array, and expression of this gene was relatively low. Possible reasons for the low targeting efficiency and expression level are discussed.

Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes

Transcription of the predominant surface antigen genes in Trypanosoma brucei is unusual in its resistance to the RNA polymerase inhibitor alpha-amanitin, a property typical for rDNA transcription in eukaryotes. Transcription of most other protein-coding genes in trypanosomes is sensitive to alpha-amanitin. To investigate whether RNA polymerase I, the polymerase that transcribes rRNA genes, can give rise to functional mRNAs in trypanosomes, we have fused the putative promoter of the T.brucei rRNA genes to the chloramphenicol acetyl transferase (CAT) gene and determined CAT activity after transient expression of chimeric constructs in procyclic trypanosomes. We show here that the rRNA promoter yields the same high CAT activity as the promoters for the two predominant surface antigen genes of trypanosomes, the Variant-specific Surface Glycoprotein (VSG) gene of bloodstream trypanosomes and the procyclin gene of insect-form trypanosomes, both of which are also transcribed by an alpha-amanitin-insensitive RNA polymerase. RNA polymerase I of trypanosomes seems therefore able to synthesize pre-mRNAs that are effectively processed into translatable mRNAs. Dissection of the promoter segments showed the minimal elements for a VSG gene expression site promoter to be confined to a segment of -60 to +77 bp, overlapping the most 5' putative transcription start sites as determined in vivo by RNase protection experiments. For the ribosomal promoter region a segment of -258 to +200 bp relative to the putative transcription start site was sufficient for maximal CAT activity. There is a precise requirement for specific nucleotides at the rRNA transcription start site. We detect no homology between the sequences required for promoter function of the three alpha-amanitin-resistant transcription units, rRNA, VSG and procyclin (parp) genes. This suggests that the sequence-specific recognition of these promoters either occurs by common factors detecting sequence homologies that escape us, or by separate factors that bind to different DNA sequences but interact with a common alpha-amanitin-resistant RNA polymerase.

A novel DNA nucleotide inTrypanosoma bruceionly present in the mammalian phase of the life-cycle

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.

Control of surface antigen genes in African trypanosomes

Conference Article| October 01 1990 Control of surface antigen genes in African trypanosomes PIET BORST; PIET BORST *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar JANET H. GOMMERS-AMPT; JANET H. GOMMERS-AMPT *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar JAN LUTGERINK; JAN LUTGERINK †Department of Human Genetics, Free University Amsterdam, 1081 HV Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar MARC OUELLETTE; MARC OUELLETTE *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar ANNELOOR L. M. A. TEN ASBROEK; ANNELOOR L. M. A. TEN ASBROEK *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar JOOST C. B. M. ZOMERDIJK JOOST C. B. M. ZOMERDIJK *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1990) 18 (5): 741–742. https://doi.org/10.1042/bst0180741 Article history Received: April 30 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation PIET BORST, JANET H. GOMMERS-AMPT, JAN LUTGERINK, MARC OUELLETTE, ANNELOOR L. M. A. TEN ASBROEK, JOOST C. B. M. ZOMERDIJK; Control of surface antigen genes in African trypanosomes. Biochem Soc Trans 1 October 1990; 18 (5): 741–742. doi: https://doi.org/10.1042/bst0180741 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: VSG, variant-specific surface glycoprotein, ES, expression site, CAT, chloramphenicol acetyl transferase, PGK, phosphoglycerate kinase This content is only available as a PDF. © 1990 Biochemical Society1990 Article PDF first page preview Close Modal You do not currently have access to this content.

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Structure of a telomeric expression site for variant specific surface antigens in Trypanosoma brucei

We have studied the organization of the expression site, in which most chromosome-internal variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei strain 427 are expressed (the dominant expression site) and compared it to the previously characterized VSG 221 expression site [1]. With the exception of a 500 bp segment and a VSG pseudogene, which are absent from the dominant expression site, overall all major sequence elements of the two sites are organized similarly, as judged from their relative mapping positions by UV inactivation of transcription. Transcription is insensitive to 1 mg α-amanitin per ml, a characteristic property of VSG gene expression sites analyzed thus far. The sequence elements of the dominant expression site include at least one other expressed gene of unknown function [2] and homologues of at least two other open reading frames. The large internal duplications of the 60-kb 221 expression site appear to be missing from the dominant site, resulting in a shorter, 40-kb transcription unit. As judged from its relative sensitivity to UV inactivation of transcription, a subsidiary promoter, identified by other methods in the dominant expression site [3] appears fully dependent for its activity on the promoter located 40 kb upstream of the VSG gene. We conclude that all VSG gene expression sites may be similarly organized as large polygenic transcription units.

The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei

The variant specific surface glycoprotein (VSG) genes of T. brucei are expressed in telomeric expression sites. We have determined the structure of the active site in trypanosome variant 221a, which contains VSG gene 221, by analysis of cloned DNA segments that represent 65 kb of the 5′-flanking region of the VSG gene. In nuclear run-on experiments, 57 kb of adjacent sequences are cotranscribed with the VSG gene at approximately similar rates and in the α-amanitin-resistant manner characteristic of VSG genes. Besides the VSG mRNA, this expression site yields at least seven stable RNAs, suggesting that it is a multicistronic transcription unit. Our results also show that insertion of a transcriptional terminator is not the general mechanism of switching off expression sites.

Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene

We have used inactivation of transcription by UV irradiation to map transcription units in trypanosomes. The relative inactivation rate of the transcription of mini-exon, 5S, and rRNA genes was inversely proportional to the previously estimated lengths of these transcription units. The telomeric transcription unit containing the gene for variant-specific surface glycoprotein (VSG) 221 was inactivated as a single unit of 60 kb. This long transcription unit comprises at least one other protein-coding gene and yields seven other stable mRNAs. These data thus provide evidence for a multicistronic transcription unit for cellular genes in a eukaryote.