The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

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Abstract

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb ‘intermediate’ chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.

Introduction

Trypanosoma brucei effectively evades the immune response of the mammals that it infects by continuously changing a homogeneous Variant Surface Glycoprotein (VSG) coat. T. brucei has hundreds of VSG genes and pseudogenes, but only one VSG is expressed at a time, from one of several telomeric transcription units known as VSG expression sites (ESs). Changing the active VSG frequently involves gene conversion, whereby a copy of a silent VSG is transposed into the active ES, displacing the existing VSG. Alternatively, VSG switching can be achieved by switching from one ES to another (reviewed in: [1], [2], [3], [4]).

VSG ESs are large polycistronic transcription units varying in size from about 30 to 60 kb [5], [6], [7]. In addition to the telomeric VSG, each ES contains several classes of ES-associated genes (ESAGs) (reviewed in [2], [8]). The function of only a few ESAGs is known. ESAG6 and ESAG7 encode the subunits of a heterodimeric transferrin receptor, allowing the trypanosome to obtain iron in a form that has been sequestered by the host [9], [10]. ESAG4 encodes an adenylate cyclase, which can rescue adenylate cyclase deficient mutants in yeast [11]. ESAG10 is homologous to the BT1 biopterin transporter of Leishmania [12]. The serum resistance associated (SRA) gene, which confers human infectivity to T. brucei through an unknown mechanism, is also ES-associated in the one strain in which it has been characterised [5].

Sequence polymorphisms in ESAG6 and 7 affect the affinities of the transferrin receptors for the transferrin molecules from different mammalian hosts [13]. As T. brucei can infect many mammalian species, this could provide a reason for the existence of multiple ESs, which then requires a mechanism to ensure mutually exclusive VSG expression [14]. The role of the SRA gene in human infectivity [5] supports the idea that ESAGs could play a role in host adaptation. However, the function of ESAGs other than 4, 6, 7 and 10 is more speculative, and based on recognisable protein motifs. It is also unclear which ESAGs are essential ES components. Some ESAGs (1, 3 and 4, for example) are members of large gene families that are also present in non-ES locations [15], [16], [17]. ESAG8 appears to be exclusively ES-located, but does not appear to be an essential gene under the laboratory conditions tested [5], [18]. If the host adaptation hypothesis is correct, it is possible that some ESAGs will be essential or advantageous only in some host environments.

The sequence of the T. brucei 927 genome is currently being determined. However, ES sequences are highly underrepresented in standard large-insert libraries. Determining the sequence of telomeric ESs will require specific cloning efforts. Little is known about the extent of ES polymorphism. In order to get more insight into this variability, we have determined the contiguous DNA sequences of three BAC clones containing sequences from three T. brucei 427 bloodstream-form ESs. These sequences included flanking regions extending for up to one hundred kilobases upstream of the ES promoters. These data allowed us to evaluate the overall architecture of six T. brucei ESs, four of which are complete. There is an overall conservation of ES architecture, but individual ESs may contain different numbers of functional ESAGs and pseudogenes.

Section snippets

Bacterial artificial chromosome ES clones

ES clones were isolated from BAC libraries (P. de Jong, Children's Hospital Oakland Research Institute: http://www.chori.org/bacpac/) made from clones of T. brucei strain 427, variant 221a [19], [20] into which specific ES tags had been introduced. BAC H25N7 (containing the 221 VSG ES on a 3.2 Mb T. brucei chromosome-VIa [20]) and BAC N19B2 (containing part of the VO2 VSG ES on a 330 kb chromosome [21]) were isolated from BAC library RPCI-97, which was made in the vector pBACe3.6 [22] with

Results

BACs provide an efficient means of cloning DNA inserts of up to 300 kilobases [35], [36]. Since it is difficult to distinguish between different ESs, BAC libraries were made from T. brucei lines in which single-copy drug-resistance genes had been inserted immediately downstream of the promoters of specific ESs, and BAC clones were isolated using the marker genes as probes. ES BACs were about ten-fold underrepresented compared with BACs containing chromosome-internal genes.

The 55-kb 221 ES is

Discussion

The AnTat 1.3A ES has long been considered the ‘canonical’ VSG ES [42], and appears to be highly similar to the AnTat 11.17 ES [48]. However, T. brucei ESs are polymorphic in size and structure, and can range from the truncated ETat1.2CR ES [5] to the extensive 221 ES described here, with its ESAG duplications and triplications [6]. An overview of all currently sequenced T. brucei ESs (Fig. 4) shows considerable diversity in the number and order of ESAGs. Only ESAGs 6 and 7 appear to have

Acknowledgements

We are grateful to P. de Jong (Children's Hospital Oakland Research Institute) for constructing the BAC libraries used in this study. We thank Professor Keith Gull for stimulating discussions. This work was funded by the Wellcome Trust through its Beowulf genomics initiative (grant number 059213), a Wellcome Senior Fellowship in the Basic Biomedical Sciences to G.R., a grant to P.B. from the Netherlands Foundation for Chemical Research (CW) with financial support of the Netherlands Organisation

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    Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the accession numbers: AL671259, AL671256, AL670322.

    1

    Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.

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