Translocation alters the activation rate of a trypanosome surface antigen gene.

We report here the characterization of the gene coding for AnTat 1.13, a very late variable antigen type (VAT) from Trypanosoma b. brucei. This gene is chromosome-internal and it is activated by the duplicative mechanism. Like in another case of late VAT expression (1), its expression-linked copy (ELC) is flanked by "companion" sequences. It was possible to convert the late expression of this VAT into an early one, by changing the location of the gene in the genome. This has been achieved by selecting an AnTat 1.6 clone among heterotypes arising in the AnTat 1.13 cloned population. Indeed, this particular derivation leads to the conservation of the AnTat 1.13 ELC as a new telomeric member of the gene family, and this conserved ELC (or ex-ELC) appears to be preferentially activable. The telomeric position and other factors possibly involved in early or late antigen gene expression are discussed; in this respect, we propose that some antigen genes are rarely activated because their duplicative transposition requires the presence, in the expression site, of "companion" sequences only shared by a limited number of other genes.     

Gene conversion as a mechanism for antigenic variation in trypanosomes

Expression of the gene coding for the trypanosome AnTat 1.1 surface antigen is linked to the duplicative transposition of a basic copy (BC) of this gene to an expression site. In two trypanosome clones successively derived from AnTat 1.1 (AnTat 1.10 and AnTat 1.1B) we found evidence that gene conversions are involved in the transformation of the AnTat 1.1 transposed element into the two new surface antigen coding sequences. Although the three resultant mRNAs--AnTat 1.1, 1.10, and 1.1B--are different, they still share large homologies. Two of them, AnTat 1.1 and 1.1B, code for surface coats that are indistinguishable by conventional serological techniques, whereas AnTat 1.10 has been found different by the same methods. The three genomic rearrangements involve two of the five members of the AnTat 1.1 gene family. These two members are both located in unstable telomeric regions similar to the expression site, each in a different orientation with respect to the DNA terminus. We have concluded that the duplicative transposition is achieved by a gene conversion that may affect variable lengths of the same silent genes, and that different members of the same surface antigen gene family can contribute to the diversification of the antigen repertoire.     

The use of incomplete genes for the construction of a Trypanosoma equiperdum variant surface glycoprotein gene.

The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction.     

Inactivation and reactivation of a variant-specific antigen gene in cyclically transmitted Trypanosoma brucei.

In Trypanosoma brucei, the activation of the variant-specific antigen gene AnTat 1.1 proceeds by the synthesis of an additional gene copy, the AnTat 1.1 ELC, which is transposed to a new location, the expression site, where it is transcribed. Using the AnTat 1.1 variant to infect flies, we investigated the fate of the AnTat 1.1 ELC during cyclic transmission of T. brucei. We show here that the AnTat 1.1 ELC is conserved in procyclic trypanosomes, obtained either from the midgut of infected Glossina or from cultures, and in metacyclic trypanosomes, although the AnTat 1.1 serotype is not detected among metacyclic antigen types. This same AnTat 1.1 ELC, which is thus silent as the parasite develops in the insect vector, can be reactivated without duplication during the first parasitemia wave following cyclical transmission. This re-expression of the conserved ELC accounts for the early appearance of the 'ingested' antigenic type after passage through the fly.     

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.     

Telomere conversion in trypanosomes.

Activation of the gene coding for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei proceeds via a duplicative transposition to a telomeric expression site. The resulting active expression-linked extra copy (ELC) is usually flanked by DNA that lacks sites for most restriction enzymes and that is thought to interfere with the cloning of the ELC as recombinant DNA in Escherichia coli. We have circumvented this problem by cloning an aberrant 118 ELC gene, flanked at the 3'-side by at least 1 kb DNA, that contains restriction enzyme sites. Our analysis shows that this DNA and the 3'-end of the 118 ELC gene are derived from another VSG gene (1.1006) that is permanently located at a telomeric position. We propose that the 3'-end of the 1.1006 gene and (all of) its 3' flanking sequence moved to the expression site by a telomere conversion. Such a telomere conversion can also account for the appearance of an extra copy of the 1.1006 gene detected in a sub-population of our trypanosome strain.     

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.     

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.     

The inactivation and reactivation of an expression-linked gene copy for a variant surface glycoprotein in Trypanosoma brucei.

We have previously shown that the gene for variant surface glycoprotein 118 of Trypanosoma brucei (strain 427) is activated by a duplicative transposition to a telomeric expression site. In chronically-infected animals, this expression-linked copy is lost when the 118 gene is replaced at the expression site by another variant surface glycoprotein gene. We show here that expression of the 118 gene can also be switched off without loss of the extra expression-linked copy. In two variants, called 1.8b and 1.8c, we find expression of the variant surface glycoprotein 1.8 gene, notwithstanding the continued presence of the 118 expression-linked copy. The 1.8 gene activated has a telomeric location, like the 118 expression-linked copy. In variant 1.8b, activation is accompanied by duplication of the 1.8 gene, resulting in an extra telomeric gene copy; in variant 1.8c it is not. Variants 1.8b and 1.8c both switch back preferentially to expression of the 118 gene. The 5'-flanking regions of the active, inactive and reactivated versions of the 118 expression-linked copy are indistinguishable by restriction mapping up to 28 kb. We conclude that there are at least two separate telomeric expression sites in our T. brucei strain. How these are switched on and off is unclear. The ability to retain expression-linked copies in inactive form may allow the trypanosome to re-programme the order in which variant surface glycoprotein genes are expressed.     

Comparison of the expression-linked extra copy (ELC) and basic copy (BC) genes of a trypanosome surface antigen

A recombinant clone of an expression-linked extra copy (ELC) gene of a trypanosome-variable surface glycoprotein was sequenced. In addition the sequences of the corresponding cDNA and portions of the two basic copy genes were determined. Comparison of these sequences reveals that the 5' boundary of the ELC-transposed segment (2.2 kb) occurs within a repetitive sequence about 700 bp upstream from the start codon of the coding sequence. This sequence does not contain internal symmetries and is not homologous with the repetitive sequence at the 3' boundary. The first 35 nucleotides of the cDNA are different than the corresponding ELC sequence and presumably were transcribed from another genomic location. A restriction fragment containing predominantly sequences outside of the 5' boundary hybridizes to a Pst I fragment whose length is variable in different trypanosome clones. This hybridization pattern is similar to that observed using probes for surface glycoprotein genes that are expressed via the nonduplication-associated (NDA) mechanism rather than the ELC mechanism. This indicates that there is a sequence correlation between these two DNA rearrangement mechanism.     

Characterization of the expression-linked gene copies of variant surface glycoprotein 118 in two independently isolated clones of Trypanosoma brucei.

We have analysed the gene for variant surface glycoprotein 118 in eight independent clones of Trypanosoma brucei, two of which express the 118 gene. Expression of this gene is strictly coupled to the presence of an extra copy of the gene. In both clones the expression-linked copy is transposed to the same (or a very similar) expression site elsewhere in the genome, but the length of the sequences flanking the transposed segment in the expression site differs markedly. By means of S1 nuclease protection experiments we demonstrate that the 3'-ends of the messenger RNAs for variant surface glycoproteins 118a and 118b are different, in agreement with the hypothesis that the generation of an expression-linked copy involves a recombination between the 3' segment of the basic gene copy and a homologous region present in the expression site.     

Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein

The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so-called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site-transposon junction, and the 5' half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co-transposed segment 5' to the coding sequence is 430 bp in length. The extreme 5' end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.     

Frequent independent duplicative transpositions activate a single VSG gene.

The expression of several surface antigen genes in Trypanosoma brucei is mediated by the duplicative transposition of a basic-copy variant surface glycoprotein (VSG) gene into an expression site. We determined that the appearance of variant 118, in a parasitemia, resulted from at least four independent duplicative transpositions of the same VSG 118 gene. Variants 117 and 118 both appeared at specific periods but resulted from multiple independent activations. Antigenic variants thus occur in an ordered manner. We show that in the duplicative transpositions of VSG genes, the ends of the transposed segments were homologous between the basic copy and the expression site. Sequences other than the previously reported 70-base-pair (bp) repeats could be involved. In one variant, 118 clone 1, the homology was between a sequence previously transposed into the expression site and a sequence located 6 kilobases upstream of the VSG 118 gene. In variant 118b the homology was presumably in 70-bp repeat arrays, while in a third 118 variant yet another sequence was involved. The possibility that the 70-bp repeats are important in the initial steps of the recombinational events was illustrated by a rearrangement involving a 70-bp repeat array. The data provide strong evidence for the notion that gene conversion mediates the duplicative transposition of VSG genes. We discuss a model that explains how the process of duplicative transposition can occur at random and still produce an ordered appearance of variants.     

DNA rearrangements and antigenic variation in Trypanosoma equiperdum: multiple expression-linked sites in independent isolates of trypanosomes expressing the same antigen.

African trypanosomes resist the immune response of their mammalian hosts by varying the surface glycoprotein which constitutes their antigenic identity. The molecular mechanism of this antigenic variation involves the successive activation of a series of genes which code for different variant surface glycoproteins (VSGs). We have studied the expression of two VSG genes (those of VSG-1 and VSG-28) in Trypanosoma equiperdum, and we report the following findings. (i) The expression of both VSG genes is associated with the duplication and transposition of corresponding basic copy genes. (ii) The duplicated transposed copy appears to be the expressed copy. (iii) Although there are multiple genes which cross-hybridize with the VSG-1 cDNA probe, only one of these appears to be used as a template for the expression-linked copy in four independent BoTat-1 clones. (iv) Analysis of the genomic environments of the expressed VSG-1 genes from each of four independently derived BoTat-1 trypanosome clones revealed that there are at least three different sites into which the expression-linked copy can be inserted.     

Cloning and characterization of a genomic DNA fragment carrying the basic copy of the gene coding for variant surface antigen 118 of Trypanosoma brucei

It has been proposed (Hoeijmakers et al., 1980b) that variant surface antigen (VSA) gene expression in Trypanosoma brucei is accomplished by a gene re-arrangement involving the basic copy of the VSA gene to give the so-called expression-linked copy (which is present only in the strain expressing that particular antigen). In this publication, the basic and expression-linked copies of the gene have been visualized by Southern blot analysis of nuclear DNA and shown to be located on HindIII fragments of 4.5 and 10-12 kb, respectively. In addition, several other bands of weaker hybridization are seen, probably representing evolutionary relatives. Using a shotgun approach, HindIII gene banks have been constructed and recombinants isolated which carry the 4.5-kb HindIII fragment containing the VSA118 gene basic copy. Several clones containing evolutionary relatives were also found. The 4.5-kb HindIII fragment is able to hybridize to probes derived from both the 5' and 3' ends of the cDNA, while the relatives have homology only to the 3' end. A detailed comparison of the restriction map of VSA118 cDNA with that of the VSA118 basic copy showed no differences, demonstrating that the gene contains no introns. This result also indicates that the gene from which VSA118 mRNA is transcribed (whether this be the basic copy or the expression-linked copy) is identical to the basic copy over the region analysed.     

Coincident multiple activations of the same surface antigen gene in Trypanosoma brucei

Trypanosomes with a coat of variant surface glycoprotein (VSG) 118, consistently appear around day 20 when a rabbit is infected with Trypanosoma brucei strain 427. There is a single chromosome-internal gene for VSG 118 and this is activated by duplicative transposition to a telomeric expression site. We show here that the expression-linked extra copy of VSG gene 118 in a day 18 population of a chronic infection is heterogeneous, and we infer that the population is not monoclonal but is the result of multiple independent activations of the 118 gene. We show that the heterogeneity of expression-linked extra copies is also present in other trypanosome populations expressing chromosome-internal VSG genes. We present a model for the timing of VSG gene activation during chronic infection that emphasizes two features: the relative activation and inactivation frequencies of different expression sites, and the degree of homology of the sequences flanking VSG genes with expression sites.