Relationship between multiple copies of a T. brucei variable surface glycoprotein gene whose expression is not controlled by duplication

Unlike many other T. brucei variable surface glycoprotein (VSG) genes, the IITat 1.3 gene is not duplicated when it is expressed. Analysis of the multiple copies of this gene present in all IITaR 1 trypanosome clones by restriction enzyme mapping and sequencing shows that the expressed copy may have arisen by duplication and transposition to a telomeric site, as is observed for those VSG genes whose expression is linked to duplication. The existence of a mechanism selecting between a number of complete telomeric VSG gene copies for expression is implied by these results. Comparisons of the nontelomeric copies of the IITat 1.3 gene are consistent with involvement of gene duplication and mutational drift in the evolution of new VSG genes.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.     

Re-expression of an inactivated variable surface glycoprotein gene in Trypanosoma equiperdum

Variable surface glycoprotein (VSG) genes in African trypanosomes are often activated by the duplicative transposition of a silent basic copy (BC) gene into an unlinked telomerically located expression site, producing an active expression-linked copy (ELC) of that gene. However, some BC genes that are already linked to a telomere are activated without apparent duplication or transposition. We have recently shown that an active VSG ELC can be inactivated in situ, apparently without rearrangement. To explain these observations it has been suggested that VSG genes that are associated with chromosome telomeres are activated by chromosome end exchanges that occur at a considerable distance upstream from the genes themselves and place them cis to a unique VSG expression element. In an attempt to test this model we derived five VSG-1 expressing variants from BoTat-2, a VSG-2 expressing variant of Trypanosoma equiperdum which carries an inactive residual VSG-1 ELC (R-ELC) as well as the active VSG-2 ELC near unlinked chromosome telomeres. We examined the fates of the VSG-2 ELC and the VSG-1 R-ELC in these variants. All five had maintained the VSG-1 R-ELC; three in a reactivated form and two in an inactive state. The latter two variants carried new, active VSG-1 ELCs: one in the site that had previously contained the VSG-2 ELC and one in a previously unidentified site. The VSG-2 ELC was lost in all five of the variants. The results are not consistent with the simple chromosome end exchange model, which predicts that the VSG-2 ELC would be inactivated but not deleted when the VSG-1 R-ELC was reactivated.     

Trypanosome variant surface glycoprotein genes expressed early in infection

We have studied further the genes for trypanosomal variant surface glycoproteins expressed during a chronic infection of rabbits with Trypanosoma brucei, strain 427. We show that there are three closely related chromosomal-internal isogenes for VSG 121; expression of one of these genes is accompanied by the duplicate transposition of the gene to a telomeric expression site, also used by other chromosome-internal VSG genes. The 3' end of the 121 gene is replaced during transposition with another sequence, also found in the VSG mRNAs of two other variants. We infer that an incoming VSG gene duplicate recombines with the resident gene in the expression site and may exchange ends in this process. The extra expression-linked copy of the 121 gene is lost when another gene enters the expression site. However, when the telomeric VSG gene 221 is activated without duplication the extra 121 gene copy is inactivated without detectable alterations in or around the gene. We have also analysed the VSG genes expressed very early when trypanosomes are introduced into rats or tissue culture. The five genes identified in 24 independent switching events were all found to be telomeric genes and we calculate that the telomeric 1.8 gene has a 50% chance of being activated in this trypanosome strain when the trypanosome switches the VSG that is synthesized. We argue that the preferential expression of telomeric VSG genes is due to two factors: first, some telomeric genes reside in an inactive expression site, that can be reactivated; second, telomeric genes can enter an active expression site by a duplicative telomere conversion and this process occurs more frequently than the duplicative transposition of chromosome-internal genes to an expression site.     

Duplicative activation mechanisms of two trypanosome telomeric VSG genes with structurally simple 5'' flanks.

In the mammalian bloodstream, African trypanosomes express variant surface glycoprotein (VSG) genes from a family of long and complex telomeric expression sites. VSG switching generally occurs by the duplication of different VSG genes into these sites by gene conversion involving a series of 70 base pair (70bp) repeats in the 5' flank. In contrast, when VSG is first synthesised by trypanosomes in the tsetse fly at the metacyclic stage, a separate set of telomeric expression sites is activated. These latter telomeres appear not to act as recipients in gene conversion. We have found that the structure of two such expression sites is simple, with very short 70bp repeat regions and very little other sequence in common with bloodstream expression sites. However, the two telomeres readily act as donors in VSG gene conversion in the bloodstream and we show for one a consistent association of the conversion 5' end point with the short 70bp repeat region. These findings help explain why a very predictable set of VSGs is expressed in the tsetse fly and have implications for VSG gene conversion mechanisms.     

Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.     

Expression of whole and hybrid genes in Trypanosoma equiperdum antigenic variation.

A rapid technique involving the S1 nuclease resistance of RNA:DNA duplexes has been used to screen four Trypanosoma equiperdum variant surface glycoprotein (VSG) genes for evidence of hybrid gene structure in their transcribed regions. The results suggest that VSGs appearing early in a chronic infection each have a complete co-linear basic copy (BC) of their expressed gene while VSGs appearing later in infection are particularly associated with BC genes which are recombined before being expressed. The intensities of the S1-protected bands from hybrid VSGs indicate that the basic and expression linked copies are present in equivalent gene dosages. In addition, studies are presented on the expression of two additional VSG genes in T. equiperdum, VSG 4 and VSG 78, which (i) show that the basic copies are not located on telomeres even though one (VSG 4) is expressed early in infection and (ii) emphasize the role of a predominant expression site in T. equiperdum while nevertheless confirming the presence of multiple expression sites.     

Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules

Pulsed field gradient gel electrophoresis fractionates chromosome-sized DNA molecules from T. brucei. About 60% of the DNA remains in or close to the gel slot (large DNA). There are about three chromosomes of approximately 2 Mb, at least six chromosomes of 200-700 kb, and roughly a hundred mini-chromosomes of 50-150 kb. The basic copy genes for VSGs 118 and 221 reside in large DNA. Their activation by duplicative transposition leads to the appearance of an additional copy in the 2 Mb DNA, showing that activation involves an interchromosomal gene transposition. When gene 221 is activated without duplication, it remains in large DNA, proving that at least two sites for expression of VSG genes exist. In support of this, the mini-exons encoding the 5' 35 nucleotides of VSG messenger RNAs are in large and 2 Mb DNA. The mini-chromosomes hybridize strongly to VSG gene probes and are absent in C. fasciculata. We suggest that their main function is to provide a large pool of telomeric VSG genes.     

Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.     

Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.     

VSG 117 gene is conservatively present and early expressed in Trypanosma evansi YNB stock

African trypanosomes, including Trypanosoma brucei and the closely related species Trypanosoma evansi, are flagellated unicellular parasites that proliferate extracellularly in the mammalian bloodstream and tissue spaces. They evade host immune system by periodically switching their variant surface glycoprotein (VSG) coat. Each trypanosome possesses a vast archive of VSGs with distinct sequence identity and different strains contain different archive of VSGs. VSG 117 was reported as a widespread VSG detected in the genomes of all the T. brucei strains. In this study, the presence and expression of VSG 117 gene was observed in T. evansi YNB stock by RT-PCR with VSG-specific primers. We further confirmed that this VSG tends to be expressed in the early stage of T. evansi infections (on day 12-15) by immuno-screening the previously isolated infected blood samples. It is possible that the VSG 117 gene evolved and spread through the African trypanosome population via genetic exchange, before T. evansi lost its ability to infect tsetse fly. Our finding provided an evidence of the close evolutionary relationship between T. evansi and T. brucei, in the terms of VSG genes.     

Trypanosoma brucei: frequent loss of a telomeric variant surface glycoprotein gene

We have observed the loss of an inactive telomeric variant surface glycoprotein (VSG) gene that is located on a minichromosome in Trypanosoma brucei. If this is due to gene conversion, it is the third "silent" gene conversion (i.e., one that does not produce an antigenic switch) detected in 19 antigenic switches of the IsTaR 1 serodeme. This is surprisingly frequent since the immune response cannot select against the inactive gene. We estimate that 10(-1) to 10(-3) telomeric VSG gene conversions occur per generation, which is at least 100 times more frequent than antigenic switching. Since all three "silent" gene conversions involved an IsTat 5 VSG gene, the frequency may vary among telomeric VSG genes. However, the high gene conversion frequency for the 5 VSG gene does not ensure a higher antigenic switch frequency than other telomeric VSG genes for which we have probes. These results suggest that gene conversion rapidly alters the repertoire of telomeric VSG genes, possibly including those on minichromosomes, producing a continual variation in the VSG genes that are more likely to be expressed.