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.     

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.     

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.     

The IsTat 1.3 VSG multigene family in Trypanosoma brucei: retention of the expression linked copy through multiple antigenic switches.

In the IsTaR 1 serodeme of T. brucei the 3 variant surface glycoprotein (VSG) gene family contains about 10 members, one of which has a telomeric location on a minichromosome. The expression linked copy (ELC) of the 3 VSG gene which occurs in an antigenic variant expressing the 3 VSG, also has a telomeric location but unlike the minichromosomal 3 VSG gene has restriction sites upstream from the 5' barren region. This ELC is retained on the same telomere in a subsequent variant that expresses a telomeric 7 VSG ELC and in relapse variants and procyclic forms derived from variant antigenic types (VATs) 3 and 7. The 7 ELC has a restriction map upstream from the 5' barren region that differs from, but is similar to, that of the 3 ELC. These data indicate that the 3 and 7 ELCs are on different telomeres when expressed.     

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.     

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.     

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 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.     

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.     

Telomeric co-localization of the modified base J and contingency genes in the protozoan parasite Trypanosoma cruzi

Base J or beta-d-glucosylhydroxymethyluracil is a modification of thymine residues within the genome of kinetoplastid parasites. In organisms known to contain the modified base, J is located mainly within the telomeric repeats. However, in Trypanosoma brucei, a small fraction of J is also located within the silent subtelomeric variant surface glycoprotein (VSG) gene expression sites, but not in the active expression site, suggesting a role for J in regulating telomeric genes involved in pathogenesis. With the identification of surface glycoprotein genes adjacent to telomeres in the South American Trypanosome, Trypanosoma cruzi, we became interested in the telomeric distribution of base J. Analysis of J and telomeric repeat sequences by J immunoblots and Southern blots following DNA digestion, reveals approximately 25% of J outside the telomeric repeat sequences. Moreover, the analysis of DNA sequences immunoprecipitated with J antiserum, localized J within subtelomeric regions rich in life-stage-specific surface glycoprotein genes involved in pathogenesis. Interestingly, the pattern of J within these regions is developmentally regulated. These studies provide a framework to characterize the role of base J in the regulation of telomeric gene expression/diversity in T. cruzi.     

Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus (opaE1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5' region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.