Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei

The genome of Trypanosoma brucei carries over a hundred genes coding for different variants of the major surface glycoprotein. Activation of some of these genes is accompanied by a duplication and transposition of the gene (the basic copy) to another region in the genome where it is transcribed. We present here physical maps of the basic and transposition-activated genes for two surface glycoproteins of Trypanosoma brucei, stock 427. In both cases the transposed segment starts 1-2 kb in front of the coding region and ends within the 3'-terminal region of the gene. The DNA segments flanking both transposed genes are indistinguishable and share a 6-kb stretch upstream and a 8-kb stretch downstream of the transposed segment not cut by several restriction endonucleases. The 5' borders of the two transposed segments are homologous and contain sequences present in many copies in the genome. A different repeated sequence has previously been found at the 3' edge of the transposed segment. The replicative transposition may, therefore, involve a unidirectional gene conversion initiated by base pairing between the edges of the transposed sequence and a single expression site elsewhere in the genome.     

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.     

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.