Antigenic variation and the African trypanosome genome

Acta Tropica 85 (2003) 391 /404 www.elsevier.com/locate/actatropica

Review article

Antigenic variation and the African trypanosome genome John E. Donelson * Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA Accepted 11 October 2002

Abstract African trypanosomes are protozoan parasites that reside in the mammalian bloodstream where they constantly confront the immune responses directed against them. They keep one-step-ahead of the immune system by continually switching from the expression of one variant surface glycoprotein (VSG) on their surface to the expression of another immunologically distinct VSG */a phenomenon called antigenic variation. About 1000 VSG genes (VSG s) and pseudoVSG s are scattered throughout the trypanosome genome, all of which are transcriptionally silent except for one. Usually, the active VSG has been recently duplicated and translocated to one of about 20 potential bloodstream VSG expression sites (B-ESs). Each of the 20 potential B-ESs is adjacent to a chromosomal telomere, but only one B-ES is actively transcribed in a given organism. Recent evidence suggests the active B-ES is situated in an extra-nucleolar body of the nucleus where it is transcribed by RNA polymerase I. Members of another group of about 20 telomere-linked VSG expression sites (the M-ESs) are expressed only during the metacyclic stage of the parasite in its tsetse fly vector. Progress in sequencing the African trypanosome genome has led to additional insights on the organization of genes within both groups of ESs that may ultimately suggest better ways to control or eliminate this deadly pathogen. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Trypanosome; Antigenic variation; Karyotype; Epigenetic; Transferrin

1. The history and biology of African trypanosomiasis African trypanosomes cause a fatal disease commonly called sleeping sickness in humans and nagana in domestic livestock. Human sleeping

 Presented at the Fifth Annual Conference on New and ReEmerging Infectious Diseases in the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign, Illinois, USA. * Tel.: /1-319-335-7934; fax: /1-319-353-4204 E-mail address: [email protected] (J.E. Donelson).

sickness is caused by two subspecies of Trypanosoma brucei . T. b. gambiense occurs in central and West Africa, and causes a chronic infection that can sometimes persist for months or even years before symptoms appear. T. b. rhodesiense occurs in southern and East Africa, and causes a highly virulent, acute infection whose symptoms of central nervous system involvement (confusion, poor coordination, sleep cycle disturbance) can emerge after only a few weeks. T. congolense and T. vivax are the main trypanosome species responsible for the livestock disease. Most African trypanosome species are transmitted to their mammalian hosts

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by tsetse flies, which inhabit the portion of the African continent that extends about 108 above and below the equator. More than 1/2 billion people currently live in this tsetse fly ‘belt’, a geographical area equivalent to the combined size of the United States, India and Western Europe. Human sleeping sickness has probably existed in Africa for many centuries, although the first clear descriptions of the disease came from European explorers and colonists in the 1800s and early 1900s (Hide, 1999). Three severe epidemics of sleeping sickness occurred in Africa during the last century. The first took place in 1896 /1906 and was confined primarily to Uganda and the Congo basin, where as many as a million people may have died. The second occurred in the 1920s in several African countries and was arrested because mobile teams systematically screened millions of people at risk and treated those with disease. The disease nearly disappeared in 1960 /1965, but re-emerged in the 1970s to become the third and on-going epidemic. The World Health Organization estimates that 300 000 new cases of human sleeping sickness may occur annually, primarily in Sudan, Central African Republic, Congo, Uganda and Angola, but the actual number is unknown because most infected persons live in areas with little or no medical care (Smith et al., 1998; World Health Organization, 1996). Furthermore, the greatest impact of the disease across the continent is on the nutrition of millions of people living in the most highly endemic areas and on the agricultural economies of their countries because it renders vast areas of semi-arid Savannah land in Africa unsuitable for raising livestock that are the source of dairy and meat products. A vaccine against African trypanosomes has not proven feasible to date because in the mammalian bloodstream they readily switch their surface coat, as described below, and the current anti-trypanosome drugs are costly and toxic. African trypanosomes cycle between the tsetse fly and the mammalian bloodstream. They undergo cell multiplication during three developmental stages of their life cycle */the non-infective procyclic and epimastigote forms in the tsetse fly, and the long slender bloodstream form in the mammalian host. They do not multiply during two

other developmental stages*/the infective metacyclic form, which is the final developmental stage in the tsetse fly salivary glands, and the short stumpy form in the mammalian bloodstream. The outer membrane of the procyclic and epimastigote forms is covered with an invariant glycoprotein coat composed of about 107 copies of two forms of a protein called EP-procyclin and GPEET-procyclin. The two forms are named for amino acid repeats in their C-termini: EP-procyclin has 22 /30 Glu /Pro repeats and GPEET-procyclin has 5 /6 Gly /Pro /Glu /Glu /Thr repeats followed by 3 EP repeats (Roditi and Clayton, 1999). Both forms are attached to the membrane via glycosylphosphatidylinositol (GPI) anchors (reviewed by Roditi et al., 1998; Hotz et al., 1998; Mehlert et al., 1998). When epimastigotes differentiate into the nondividing metacyclic form, the EP- and GPEETprocyclin coat is replaced with about 107 copies of a single variant surface glycoprotein (VSG) that is also GPI-anchored on the outside of the outer membrane. Although each metacyclic organism is coated with only one VSG, the metacyclic population as a whole in a tsetse fly collectively expresses 15/20 different VSG genes (VSG s) (Lenardo et al., 1984; Turner et al., 1998). After the trypanosomes enter the bloodstream of their host, they continue to express the metacyclic VSGs for as long as 7 days and then switch to the expression of non-metacyclic, bloodstream VSGs (Esser et al., 1982).

2. The molecular biology of antigenic variation When T. brucei is in the bloodstream of its host, it is in constant contact with the immune system and has evolved complicated molecular mechanisms to evade the immune responses. The best characterized of these mechanisms, and the one for which African trypanosomes have become famous among molecular biologists, is antigenic variation, a phenomenon whereby bloodstream trypanosomes switch from one VSG on their surface to another at a rate of 10 2 to 10 7 switches/ doubling time of 5 /10 h (Turner and Barry, 1989; Davies et al., 1997; Turner, 1997). The only known function of the VSG is to serve as a

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protective barrier against the attack of the immune system on the other outer membrane constituents. Thus, antigenic variation permits the trypanosome population as a whole to keep ‘one step ahead’ of the immune response. The three-dimensional structures of the N-terminal two-thirds of two VSGs have been determined by X-ray crystallography and found to be very similar rod-like shapes despite having quite different amino acid sequences (Blum et al., 1993). These rod-like structures allow the homogeneous population of a single VSG to form a densely packed array on the surface, and suggest that all VSGs share a similar backbone structure from which emerge distinct epitopes derived from different groups of amino acid side chains. Southern blots probed with VSG cDNAs under low hybridization stringency indicate that the African trypanosome genome contains about 1000 different VSG s and pseudo-VSG s (Van der Ploeg et al., 1982). Under normal circumstances one and only one VSG is expressed at a time in a given bloodstream parasite, although rare exceptions have been noted (Mun˜oz-Jorda´n et al., 1996; Esser and Schoenbechler, 1985; Baltz et al., 1986). The unexpressed VSG s are scattered among different chromosomes, including small minichromosomes (see below), but all expressed VSG s studied to date are located near the telomeres (reviewed by Borst and Ulbert, 2001; Pays and Nolan, 1998; Cross et al., 1998). The telomere-linked VSG expression sites (ESs) have been defined as the sequences that extend from the VSG promoters to the telomeric repeats of (TTAGGG)n located downstream of the VSG s, i.e., the sequences encoding the primary VSG transcription unit and its immediate flanking regions (Pays and Nolan, 1998; Pedram and Donelson, 1999). The sequences of several bloodstream VSG ESs (B-ESs) and metacyclic VSG ESs (M-ESs) have been determined and the organization of their sequences is summarized in Fig. 1. Fig. 1A shows the B-ESs whose complete or partial sequences have been reported. Each of these telomere-linked B-ESs possesses a polycistronic transcription unit spanning 45 /60 kb. The transcription units begin at a promoter (the flags pointing right in Fig. 1) and are preceded by 20/50

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kb of 50-bp repeats that are not transcribed. The promoters of several B-ESs have been identified by promoter trap assays using transiently transfected plasmids bearing reporter genes and were found to be highly similar sequences of about 70 bp (Zomerdijk et al., 1990; Kim and Donelson, 1997). After the promoter, B-ESs possess several ES-associated genes (ESAG s), 5 /20 kb of a 70bp repeat and the VSG , followed by sub-telomeric and telomeric repeats. The polycistronic precursor RNA is processed into mature monocistronic mRNAs of the individual ESAG s and VSG , each of which contains a 39-nucleotide spliced leader at its 5? end and a poly(A) tail at its 3? end (Tschudi and Ullu, 2002). Southern blot hybridization data suggest that about 20 such B-ESs occur in the T. brucei genome (Pays et al., 1989; Zomerdijk et al., 1990; Navarro and Cross, 1996), and it is likely that a similar number M-ESs exist (Lenardo et al., 1986; Turner et al., 1998), making a total of about 40 ESs. VSG s are introduced into, and removed from, the B-ESs by different molecular mechanisms (reviewed by Borst and Ulbert, 2001; Pays and Nolan, 1998; Cross et al., 1998). The best-studied switching mechanism is gene conversion, or ‘duplicative transposition’, in which the VSG in an active B-ES is replaced with a duplicated copy of an unexpressed ‘donor’ VSG . The duplicated VSG was formerly called an expression linked copy, but this term has fallen out of use in recent years. Sometimes the 70-bp repeats serve as the upstream boundary of the gene duplication; other times they do not (McCulloch et al., 1997). Since most unexpressed donor VSG s are not at the telomeres (Van der Ploeg et al., 1982), this gene conversion mechanism appears to be the most common pathway for VSG switching (Robinson et al., 1999). Other switching mechanisms include the duplicative conversion of an entire telomere plus its adjacent VSG to another chromosomal end (telomere conversion), and reciprocal exchange of two telomeres and their associated VSG s (telomere exchange). In addition, transcription of one B-ES can switch to another B-ES in situ without any associated DNA rearrangements (in situ activation). In contrast, there is no evidence that DNA rearrangements at M-ESs are associated with their

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Fig. 1. Comparison of the known VSG expression sites (ESs) of T. brucei . Grey and black rectangles above the lines are ESAGs and VSGs , respectively. Cross-hatched rectangles above the lines are pseudo-genes (c). Barred and cross-hatched rectangles on the lines denote 50- and 70-bp repeats, respectively. Black circles are telomeres. Black flags indicate promoters for bloodstream ESs (B-ESs). Open flags indicate promoters for metacyclic ESs (M-ESs) (A.) Known conventional B-ESs: VSG 10.1 ES (LaCount et al., 2001); AnTat 1.3 ES (Lips et al., 1993); VSG 221 ES, VSG VO2 ES and VSG Bn-2 ES (Berriman et al., 2002). Only partial sequences of the VSG VO2 and VSG Bn-2 ESs are known because the inserts of the sequenced BACs (bacterial artificial chromosomes) terminate within the ES. Lines drawn between the ESs emphasize the relative positions of members of the same ESAG family. (B.) Known MESs: metacyclic VSG MVAT4 ES and MVAT7 ES (Donelson et al., 1998); metacyclic VSG 1.61 and 1.22 ES (Barry et al., 1998). (C.) Unusual B-ESs: VSG ETat 1.2 CR (Xong et al., 1998); bloodstream VSG MVAT4 ES (Alarcon et al., 1994).

activation or expression. At the metacyclic stage the M-ESs appear to be only activated by an in situ mechanism (Lenardo et al., 1986). Very little is known about the trypanosome genes that regulate antigenic variation, or encode the enzymes that catalyze the switch from one VSG to another. One study examined whether RAD51, a highly conserved enzyme known to participate in DNA break repair and genetic

exchange in many organisms, plays a role in antigenic variation (McCullock and Barry, 1999). When both copies of the RAD51 gene in the trypanosome genome were disrupted, antigenic variation still occurred, but at a reduced rate. Although the results were somewhat variable, both in situ activation of new B-ESs and duplicative transposition of new VSG s into an active B-ES were impaired, but not eliminated. Thus, RAD51

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Fig. 1 (Continued)

might contribute to antigenic variation, but in its absence, other trypanosome factors provide for the VSG switching.

3. The gene organization of B-ESs and M-ESs The known B-ESs differ substantially in the number and order of their ESAG s (Fig. 1A). Each ESAG is a member of its own gene family and different B-ESs contain similar, but non-identical, members of these families (Vanhamme and Pays, 1998). In addition, at least a few members of most ESAG families lie outside the telomere-linked BESs and can be expressed independent of the specific B-ES being expressed (Alarcon et al., 1999; Ansorge et al., 1999). ESAG 7 and ESAG 6 family members occur immediately after the promoters of the B-ESs sequenced to date. These are

the only two ESAG families to be represented in every polycistronic B-ES examined to date, although as can be seen in Fig. 1A, complete sequences are available for only three B-ESs so more information is necessary before the essential ESAG s of B-ESs can be deduced. Some B-ESs have more than one member of a specific ESAG family. For example, the VSG 10.1 and AnTat 1.3 B-ESs have two non-identical ESAG 8 members, whereas the VSG 221 B-ES has three. The VSG 221 B-ES also has two copies each of ESAG s 3 and 4. Each B-ES also contains one or more pseudogenes, i.e., sequences that are similar to the coding regions of other genes but have mutations that destroy or truncate the open reading frames. For example, the VSG 10.1 B-ES contains pseudo versions of ESAG 3, ESAG 9 and two VSG s. Several other B-ESs have a pseudo-ESAG 5. One B-ES has a pseudo-ESAG 4. The presence of

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multiple ESAG family members and pseudoESAG s suggests that B-ESs are in a dynamic state, undergoing periodic rearrangements, mutations and recombinations. Consistent with this possibility is the fact that the AnTat 1.3 B-ES and the Bn-2 B-ES each have two promoters, separated by an ESAG 10, and the VO2 B-ES has two adjacent ESAG 7 copies that may have arisen via a tandem duplication event. Furthermore, since the ESAG family members are highly similar in sequence, the B-ESs themselves share substantial sequence similarity over their 45/60 kb, favoring recombination events. The locations and functions of some of the ESAG protein products can be inferred from their sequences (reviewed by Vanhamme and Pays,

1998) (Fig. 2). Nascent ESAG1 proteins [ /320 amino acids (aa)] have an N-terminal signal peptide and a potential transmembrane region, suggesting they are integral membrane components. Nascent ESAG2 proteins (/450 aa) have both an N-terminal signal peptide and a Cterminal GPI anchor addition signal. Mature ESAG2 proteins are located in the flagellar pocket and appear to be glycoproteins. ESAG3 proteins (/370 aa) have a signal peptide, but no GPI addition signal or transmembrane region, and their function and location is unknown. ESAG4 proteins (/1270 aa) are homologous to adenylyl cyclases and can complement their activity in yeast mutants. They are located on the flagellum and may be involved in the burst of adenylyl cyclase

Fig. 2. The putative cellular locations and activities of the ESAG protein products (adapted from Pays and Nolan, 1998). A question mark means the deduced sequence of the protein is not similar to any known protein, but it likely has one or more of the following domains: SP, signal peptide; TM, transmembrane domain; GPI, C-terminal GPI addition signal.

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activity during differentiation from slender bloodstream forms to stumpy forms, and stumpy to procyclic forms. ESAG5 proteins (/420 aa) have two transmembrane regions and an unknown function. ESAG6 (/400 aa) and ESAG7 (/340 aa) proteins are closely related in sequence and heterodimerize to form a transferrin receptor in the flagellar pocket. Transferrin is a protein that complexes with iron in the blood and is the source of iron for bloodstream trypanosomes. The ESAG6 portion of the transferrin receptor has a GPI anchor, whereas nascent ESAG7 lacks the Cterminal GPI anchor signal, which accounts for its smaller size. ESAG8 proteins (/630 aa) contains potential protein/protein interaction signals (a RING finger and a very long leucine-rich repeat domain), which are separated by a putative nuclear localization signal. The ESAG8 protein (/630 aa) occurs in both the nucleolus and the cytoplasm and interacts with a protein involved in mRNA stability (Hoek and Cross, 2001; Hoek et al., 2002). ESAG9 (/320 aa) and ESAG11 (/ 370 aa) proteins have putative signal peptides and GPI addition signals, suggesting they are membrane proteins of unknown functions. ESAG10 (/690 aa) proteins have a signal peptide, 10 transmembrane domains and homology to biopterin transporters. Finally, it is not clear that all ESAG s have been discovered. Since ESAG s are defined as the non-VSG that occur in ESs, it is possible that as the sequences of more B-ESs become available, additional ESAG s will be identified. The sequences of several telomere-linked M-ESs have also been determined (Barry et al., 1998; Pedram and Donelson, 1999; Alarcon et al., 1999), and found to differ in several important respects from the B-ESs (Fig. 1B). The M-ESs are organized as short (3 /5 kb) monocistronic transcription units and generally lack multiple copies of the upstream 50-bp repeats and interior 70-bp repeats of B-ESs. Their 70-bp promoters bear some resemblance to the 70-bp promoters of B-ESs, but they are more similar to themselves than to the B-ES promoters (Kim and Donelson, 1997). Perhaps most dramatically, the M-ESs do not possess ESAG s between their promoter and the VSG. ESAG s can occur upstream of the M-ESs,

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particularly ESAG 1 members, but ESAG s are not located within the transcription unit derived from the M-ES promoter, and the upstream ESAG 1s are probably not transcribed. Thus, metacyclic trypanosomes can get along without the expression of ESAG s from M-ESs, although they may utilize ESAG members from non-M-ES regions located elsewhere in the genome. The M-ESs also appear to be much more stable than B-ESs (Lenardo et al., 1986). Only occasionally do changes occur in the metacyclic VSG repertoire of a T. brucei cell line (Barry et al., 1983). The introduction of a new VSG into a M-ES has not been experimentally observed as it has with B-ESs, and when a given bloodstream trypanosome line is passaged through tsetse flies, the same repertoire of 15 /20 VSG s is repeatedly expressed by the population of metacyclic trypanosomes in the salivary glands of the tsetse fly (Esser et al., 1982; Barry et al., 1983). Thus, each metacyclic trypanosome has the ability to express one of only 15/20 VSG s and the M-ESs designed for this purpose are much simpler than B-ESs. However, there are several examples of a telomere-linked MES providing the donor VSG for a gene duplication event that places a metacyclic VSG into a BES, where it is expressed in bloodstream trypanosomes (Lu et al., 1993; LaCount, et al., 2001; Donelson et al., 1998). So, although M-ESs are seldom, if ever, recipients of gene conversions, they can be the donors of gene conversions. Two examples of unusual B-ESs deserve mention (Fig. 1C). The B-ES for the ETat1.2CR VSG is a small B-ES that contains only three conventional ESAG s */ESAG s 7, 6 and 5*/and a fourth ESAG called serum resistance-associated gene (SRA) surrounded by 70-bp repeats. SRA s occur only in T. b. rhodesiense , the T. brucei subspecies that causes the acute form of human sleeping sickness in southern and East Africa. The nascent SRA product (/410 aa) resembles a truncated VSG and has an N-terminal signal peptide and a C-terminal GPI anchor signal (Xong et al., 1998). Its presence appears to prevent lysis of T. b. rhodesiense in human serum and transfection of this gene into non-resistant T. b. brucei confers resistance to this subspecies. The mechanism by which SRA contributes the resistance is not known

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and more investigation of this interesting phenomenon is needed. Another unexpected observation is that occasionally a M-ES can serve as a B-ES. Using monoclonal antibodies directed against the MVAT4 VSG, bloodstream trypanosome clones have been identified that express the MVAT4 VSG (Alarcon et al., 1994). In these bloodstream organisms the MVAT4 VSG is expressed from the same ES that it is in metacyclic organisms. As indicated in Fig. 1B and C, no differences were detected in sequence of this ES or its flanking regions as a result of its activation in bloodstream organisms. The bloodstream trypanosomes expressing the MVAT4 VSG have ESAG mRNAs, as detected in a cDNA library of MVAT4 organisms, so they must be derived from ESAG s located elsewhere in the genome (Alarcon et al., 1999). In contrast, when bloodstream trypanosomes expressing MVAT5 VSG were identified using MVAT5 VSG monoclonal antibodies, the MVAT5 VSG was found to have been duplicatively translocated to a conventional B-ES, an example of a M-ES providing the donor VSG in a duplication to a BES (Lu et al., 1993). Multiple M-ESs in the same genome may be necessary to generate sufficient VSG heterogeneity in a population of metacyclic organisms from a single tsetse fly to ensure that a few organisms survive long enough to switch to a bloodstream VSG not recognized immediately by the host immune response. Less clear is why multiple BESs are required, but Borst and colleagues have proposed the interesting model that multiple BESs provide different assortments of ESAG proteins suitable for maximal growth in different environments, i.e., the bloodstreams of different mammalian species (Bitter et al., 1998; Gerrits et al., 2002). This host adaptation model predicts that different B-ESs would be favored by T. brucei in different mammalian hosts. Some evidence for the model exists. The transferrin receptors encoded by ESAG s 6 and 7 in different B-ESs have differing affinities for the transferrin molecules of different mammalian hosts (Bitter et al., 1998). Furthermore, bloodstream trypanosomes appear to switch away from expression of B-ESs encoding a low-affinity receptor for transferrin of the animal serum in which they are cultured in vitro (Gerrits

et al., 2002). Thus, the 20 B-ESs potentially provide 20 unique transferrin receptors for the trypanosome to use in its search for the most efficient uptake of iron in a given mammalian bloodstream. The protein products of the other ESAG s in a specific B-ES might also help the trypanosome adapt to a given bloodstream environment (Pays et al., 2001).

4. The chromosomal karyotypes of African trypanosomes African trypanosomes are diploid organisms and can undergo genetic exchange in the tsetse fly (Bingle et al., 2001; Sternberg and Tait, 1990; Turner et al., 1990). The techniques of pulsed field gel electrophoresis (PFGE), DNA renaturation and cytophotometry all indicate that the haploid nuclear DNA content of T. brucei is about 35 Mb, with as much as 25% variation among isolates (Van der Ploeg et al., 1984; Borst et al., 1982; Van der Ploeg et al., 1989; Gibson et al., 1992; Kanmogne et al., 1997; Hope et al., 1999). By analogy, the Saccharomyces cerevisiae genome contains 12 Mb and 6000 genes (Mewes et al., 1997), and the Caenorhabditis elegans genome has 97 Mb and 19 000 genes (The C. elegans Sequencing Consortium, 1998). Thus, the 35 Mb T. brucei nuclear genome probably harbors about 10 000 genes, of which about 1000 (10%) may be VSG s (Van der Ploeg et al., 1982). Many T. brucei genes have now been sequenced, and to date only two have been reported to possess an intron. An 11nucleotide intron occurs in a tRNA(tyr) gene (Schneider et al., 1994), and a 653-nucleotide intron occurs in the gene for poly(A) polymerase (Mair et al., 2000). Thus, the molecular machinery for excision of introns from both tRNA genes and protein-encoding genes must exist in trypanosomes. It remains to be determined how many T. brucei genes possess introns, but it is likely to be a small percentage. The nuclear chromosomes of T. brucei can be grouped into three general size classes based on their migrations in PFGE */megabase chromosomes (1 /6 Mb), intermediate chromosomes (200 /900 kb) and minichromosomes (50 /150

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kb). At least 11 pairs of megabase chromosomes exist that are numbered I to XI from smallest (/1 Mb) to largest (/6 Mb) as they occur in T. brucei stock TREU927/4 (Melville et al., 1998; Turner et al., 1997). Letters are used to distinguish the two chromosomal homologues, for example, chromosome Ia and Ib. The sizes of both the chromosomal pairs and the homologues within a pair vary greatly among T. brucei stocks. For example, in T. brucei TREU927/4 the chromosome III homologues are about 1.7 Mb, whereas in another T. brucei isolate they are larger than 4 Mb. The causes of these enormous size polymorphisms can only be determined unambiguously by genome sequence determinations, but are due at least in part to expansion/contraction of regions rich in retrotransposon-like INGI and RIME sequences (Melville et al., 1999). A joint project to determine the sequences of the megabase chromosomes of the African trypanosome clone GUTat 10.1 of the TREU 927/4 stock of T. b. brucei is underway at The Institute for Genome Research (TIGR) in the United States and the Sanger Centre in Britain. Since one of the sequencing approaches being used by both sequencing institutes is to sequence the ends of a large number of randomly sheared genomic fragments, it is likely that much sequence information will also be obtained on the intermediate chromosomes and minichromosomes. From this sequencing effort there is already a 95% probability that at least a portion of any given trypanosome gene is available in the public databases of sequences. These partial sequences can be accessed at the web sites of the two institutes conducting the sequencing: http://www.tigr.org/tdb/parasites/ (TIGR) and http://www.genedb.org/genedb/tryp/trypexample.jsp (Sanger Centre). The complete sequences of chromosome I (/ 1.0 Mb) and chromosome II ( /1.3 Mb) have been determined and are available at the web sites of the Sanger Centre and TIGR, respectively. Earlier hybridizations of PFGE-resolved chromosome I with various ES probes indicate that a B-ES is adjacent to one telomere and a M-ES is likely next to the other (Melville et al., 1999). A large interior region rich in INGI and RIME retrotransposons is responsible for most of the size polymorphism

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between chromosomes Ia and Ib. The interior portions of both chromosomes I and II appear to contain long polycistronic arrays of housekeeping and trypanosome-specific genes lined up one after another. These arrays can encompass 100 or more genes, which are probably transcribed into long polycistronic pre-mRNAs that are processed into monocistronic mRNAs as described above for the polycistronic B-ESs (Fig. 1A) and as described previously for the genes of another trypanosomatid, Leishmania major (Myler et al., 1999). All indications are that most of the other megabase chromosome pairs are organized in a similar manner, i.e., the B-ESs and M-ESs are adjacent to the telomeres, while other genes, including unexpressed VSG s, are at interior sites. Interestingly, the diploid megabase chromosomes appear to harbor haploid ESs at their telomeres, i.e., the ESs at the ends of chromosome Ia are not identical to the ESs at the ends of chromosome Ib. Thus, the 11 megabase chromosome pairs could harbor 44 different telomeres (11 pairs /2 chromo/pair / 2 telomeres/chromo), consistent with the estimate of 40 different telomere-linked B-ESs and M-ESs (see above). The genomic sequences currently available suggest that the boundaries between the telomere-linked haploid B-ESs and M-ESs and the gene-rich, diploid interiors of their chromosomes are long repetitive, non-transcribed sequences including INGI and RIME retrotransposons and PHS (pseudo) genes (Bringaud et al., 2002; LaCount et al., 2001). Thus, it seems likely that the megabase chromosomes of different T. brucei field isolates will have different haploid telomere-linked regions, but similar clusters of chromosome-interior housekeeping genes. The 100 or more linear minichromosomes of 50/150 kb in the T. brucei nucleus possess the same telomere repeats (TTAGGG)n as the megabase chromosomes (Weiden et al., 1991; Sloof et al., 1983). They are comprised predominately of internal tandem arrays of a 177-bp repeat, which consume /90% of some minichromosomes. Many minichromosome telomeres are linked to silent VSG s, but at least some are not. To date none of the minichromosomes have been found to possess an active VSG ES. Therefore, to be expressed, these minichromosomal VSG s must

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undergo either an interchromosomal duplication or be part of a telomere exchange. It has been difficult to determine the ploidy of minichromosomes, although one report hints they may be diploid (Zomerdijk et al., 1992). The reason for the existence of minichromosomes is not known, but if their only function is to serve as repositories for silent, telomere-linked VSG s, then a considerable portion of the nuclear genome, i.e., 10 /20%, is devoted to this purpose. The intermediate-sized chromosomes of 200/ 900 kb in most T. brucei stocks are even more mysterious than minichromosomes. Their numbers and sizes vary among stocks and they contain few, if any, unique markers or housekeeping genes. Their ploidy is uncertain and it is not known if they contain their own unique repeat sequences. They do not hybridize to the minichromosomespecific 177-bp repeat, although they can possess telomere-linked VSG s or VSG -like sequences (Rudenko et al., 1998; Lips et al., 1993). In one case, an expressed B-ES, i.e., the VO2 B-ES shown in Fig. 1A, has been found to be located on a 330kb intermediate chromosome (Rudenko et al., 1998). It is possible the intermediate chromosomes also serve primarily as repositories for telomerelinked VSG s or even VSG ESs, although, similar to minichromosomes, it seems unnecessary for the trypanosome to devote this much DNA to solely that function. In summary, the 120 or more DNA molecules in the T. brucei nucleus include at least 11 pairs of megabase chromosomes, and an indeterminate number of minichromosomes and intermediate chromosomes of uncertain function and ploidy. Unexpressed VSG s are located on most, if not all, of these chromosomes.

5. Regulation of antigenic variation at the level of cell biology The presence of a VSG in a telomere-linked BES or M-ES is necessary, but not sufficient, for that VSG ’s expression. When a VSG is in one of the approximately 40 ESs, it is ‘on deck’ for potential expression, but additional events are required to activate that ES. Thus, T. brucei

must have mechanisms that facilitate transcription at one telomere-linked ES and silence expression at the other 40 telomere-linked B-ESs and M-ESs. A modified base (b-D-glucosylhydroxymethyluracil or ‘base J’) occurs in the silent ESs and is absent from the expressed ES, but this modification is more likely to stabilize repression of silent ESs than to induce ES activation (van Leeuwen et al., 1998). No other nucleotide differences have been detected in an ES when it is active versus silent although, as expected, the chromatin structure of an actively transcribed ES is in a more open configuration than a silent ES (Horn, 2001; Pays et al., 1981). An early observation that remained a curiosity for years, but has more significance now, is that transcription of both B-ESs and M-ESs is resistant to a-amanitin, a hallmark of genes transcribed by RNA polymerase I, the enzyme that transcribes rRNA genes (Kooter and Borst, 1984). In contrast, transcription of most other protein-encoding genes in T. brucei is sensitive to a-amanitin, consistent with their transcription by RNA polymerase II, the enzyme that conventionally transcribes protein-encoding genes. The RNA polymerase I-like transcription of ESs led to the suggestion that the active ES might be confined to a sub-compartment of the nucleus */perhaps even the nucleolus itself where rRNA genes are transcribed (Lee and Van der Ploeg, 1997). However, subsequent analyses using fluorescence in situ hybridization indicated that ES transcription does not occur in the nucleolus (Chaves et al., 1998). Nevertheless, the hypothesis that one and only one of the 40 ESs is sequestered in a subcompartment of the nucleus for its transcription remained an attractive model. Such a model proposes that ES activation is regulated epigenetically at the level of cell biology, rather than at the level of molecular biology. Recently, strong evidence for this model has emerged (Navarro and Gull, 2001; reviewed by Borst, 2002). Immunofluorescence was used to show that RNA polymerase I is located in the nucleolus of T. brucei , as expected, and also in an ‘extra-nucleolar body’ positioned near, but distinctly separate from, the nucleolus (Fig. 3). Nucleic acid in situ hybridizations demonstrated that this extra-nucleolar body

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Fig. 3. Depiction of the T. brucei nucleus showing the nucleolus and ‘extra-nucleolar body’, both of which contain RNA polymerase I (Navarro and Gull, 2001). Only one of the approximately 40 telomere-linked VSG expression sites (ESs) is situated at a time in the extra-nucleolar body, where it undergoes transcription. The diploid chromosomes are shown as paired to emphasize that homologous chromosomes have different telomeric ESs.

contains the active telomere-linked ES, but does not contain the silent ESs or the rRNA genes. This ES-bearing nuclear body occurs in post-mitotic nuclei before cytokinesis is complete, indicting that each daughter cell inherits the body plus its ES. The integrity of the body is not destroyed by digestion with DNase I, suggesting it is more than simply a loose association of a telomere and the transcription machinery. The telomere-linked MESs were not examined, but since their transcription is also a-amanitin resistant, it is likely they are also transcribed in the body. The biochemical components of the body, other than RNA polymerase I and the ES, remain to be elucidated. Thus, VSG expression appears to be regulated by an unknown epigenetic mechanism that permits one and only one telomere-linked ES to enter, or become a part of, this extra-nucleolar body at a time. A switch in VSG expression is due to either a switch in the B-ES in the body or a switch in the VSG of a B-ES already in the body. Consistent with this mechanism are the findings that a single parasite rarely, if ever, co-expresses two telomerelinked ESs (Mun˜oz-Jorda´n et al., 1996; Esser and Schoenbechler, 1985; Baltz et al., 1986), and the activation of one ES is linked to the silencing of

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the previously expressed ES (Chaves et al., 1999). Somewhat inconsistent with the model are data indicating that silent ESs undergo ‘leaky’ transcription that becomes attenuated as it progresses through the polycistronic ES (Alarcon et al., 1999; Vanhamme and Pays, 1998; Ansorge et al., 1999). Furthermore, how one telomere gets into the body while the others are excluded is only one of the questions raised by this new model. Other questions include the following. What triggers the switch from one B-ES in the body to another? Why is the VSG switch rate much higher in trypanosomes recently isolated from field than in laboratory-adapted laboratory strains? How can a new VSG be duplicatively translocated into an active ES that is in a different compartment than its donor VSG ? Why do only B-ESs enter the extra-nucleolar body during the bloodstream stages and only M-ESs during the metacyclic stage? Are the chromosome-interior genes for EP-procyclin and GPEET-procyclin, whose transcription at the procyclic stage is also resistant to a-amanitin (Rudenko et al., 1989), transcribed in the body?

6. Conclusion African trypanosomes utilize a sophisticated system of antigenic variation to evade the immune response of their hosts that in some respects remains as much of an enigma as it was when first described in the early 1900s. The system requires an enormous reservoir of genetic information consuming at least 10% and maybe 30% or more of the trypanosome’s 35-Mb genome. At a minimum, 1000 VSG s, 40 telomeres and ESs, countless ESAG s, an unknown number of DNA recombination genes, 100 minichromosomes and an ‘extra-nucleolar body’ are involved. The ongoing determination of the trypanosome’s genomic sequence should provide the foundation of the next generation of experiments devoted to elucidating how antigenic variation is generated, regulated and maintained. The technique of RNA interference for diminishing expression of specific genes works well in African trypanosomes (Ngo et al., 1998) and can be used to examine which genes

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or sets of genes are essential for antigenic variation. Similarly, approaches for answering some of the questions about the extra-nucleolar body raised above are also available. It is hoped that these new techniques and experimental approaches will ultimately reveal new ways to control or eliminate this pathogen, but it is equally likely that the parasite itself will continue to provide new surprises.

References Alarcon, C.M., Pedram, M., Donelson, J.E., 1999. Leaky transcription from telomere-linked metacyclic VSG genes in African trypanosomes. J. Biol. Chem. 274, 16884 /16893. Alarcon, C.M., Son, H.J., Hall, T., Donelson, J.E., 1994. A monocistronic transcript for a trypanosome VSG. Mol. Cell Biol. 14, 5579 /5591. Ansorge, I., Dietmar, S., Melville, S., Hartmann, C., Clayton, C., 1999. Transcription of ‘inactive’ expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms. Mol. Biochem. Parasitol. 101, 81 /94. Baltz, T., Giroud, C., Baltz, D., Roth, C., Raibaud, A., Eisen, H., 1986. Stable expression of two variable surface glycoproteins by cloned Trypanosoma equiperdum . Nature 319, 602 /604. Barry, J.D., Crowe, J.S., Vickerman, K., 1983. Instability of the Trypanosoma brucei rhodesiense metacyclic variable antigen repertoire. Nature 306, 699 /701. Barry, J.D., Graham, S.V., Fortheringham, M., Graham, V.S., Kobryn, K., Wymer, B., 1998. VSG gene control and infectivity strategy of metacyclic stage Trypanosoma brucei . Mol. Biochem. Parasitol. 91, 93 /105. Berriman, M., Hall, N., Sheader, K., Bringaud, F., Tiwari, B., Isobe, T., Bowman, S., Corton, C., Clark, L., Cross, G.A., Hoek, M., Zanders, T., Berberof, M., Borst, P., Rudenko, G., 2002. The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei . Mol. Biochem. Parasitol. 122, 131 /140. Bingle, L.E., Eastlake, J.L., Bailey, M., Gibson, W.C., 2001. A novel GFP approach for the analysis of genetic exchange in trypanosomes allowing the in situ detection of mating events. Microbiology 147, 3231 /3240. Bitter, W., Gerrits, H., Kieft, R., Borst, P., 1998. The role of transferrin-receptor variation in the host range of Trypanosoma brucei . Nature 391, 499 /502. Blum, M.L., Down, J.A., Gurnett, A.M., Carrington, M., Turner, M.J., Wiley, D.C., 1993. A structural motif in the variant surface glycoproteins of Trypanosoma brucei . Nature 362, 603 /609. Borst, P., 2002. Antigenic variation and allelic exclusion. Cell 109, 5 /8.

Borst, P., van der Ploeg, L.H., van Hoek, J.F., Tas, J., James, J., 1982. On the DNA content and ploidy of trypanosomes. Mol. Biochem. Parasitol. 6, 13 /23. Borst, P., Ulbert, S., 2001. Control of VSG gene expression sites. Mol. Biochem. Parasitol. 114, 17 /27. Bringaud, F., Biteau, N., Melville, S.E., Hez, S., El-Sayed, N.M., Berrima, M., Hall, N., Donelson, J.E., Baltz, T., 2002. A new, expressed multigene family containing a hot spot of insertion for retroelements is associated with polymorphic subtelomeric regions of Trypanosoma brucei . Eukaryotic Cell 1, 137 /151. Chaves, I., Zomerdijk, J., Dirks-Mulder, A., Dirks, R.W., Raap, A.K., Borst, P., 1998. Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei . Proc. Natl. Acad. Sci. USA 95, 12328 /12333. Chaves, I., Rudenko, G., Dircks-Mulder, A., Cross, M., Borst, P., 1999. Control of variant surface glycoprotein geneexpression sites in Trypanosoma brucei . EMBO J. 18, 4846 / 4855. Cross, G.A.M., Wirtz, L.E., Navarro, M., 1998. Regulation of vsg expression site transcription and switching in Trypanosoma brucei . Mol. Biochem. Parasitol. 91, 77 /91. Davies, K.P., Carruthers, V.B., Cross, G.A., 1997. Manipulation of the vsg co-transposed region increases expressionsite switching in Trypanosoma brucei . Mol. Biochem. Parasitol. 86, 163 /177. Donelson, J.E., Hill, K.L., El-Sayed, N.M.A., 1998. Multiple mechanisms of immune evasion by African trypanosomes. Mol. Biochem. Parasitol. 91, 51 /66. Esser, K.M., Schoenbechler, M.J., 1985. Expression of two variant surface glycoproteins on individual African trypanosomes during antigen switching. Science 229, 190 /193. Esser, K.M., Schoenbechler, M.J., Gingrich, J.B., 1982. Trypanosoma rhodesiense blood forms express all antigen specificities relevant to protection against metacyclic (insect form) challenge. J. Immunol. 129, 1715 /1718. Gerrits, H., Mußmann, R., Bitter, W., Kieft, R., Borst, P., 2002. The physiological significance of transferrin receptor variations in Trypanosoma brucei . Mol. Biochem. Parasitol. 119, 237 /247. Gibson, W., Garside, L., Bailey, M., 1992. Trisomy and chromosome size changes in hybrid trypanosomes from a genetic cross between Trypanosoma brucei rhodesiense and T . b . brucei . Mol. Biochem. Parasitol. 51, 189 /199. Hide, G., 1999. History of sleeping sickness in East Africa. Clin. Micro. Rev. 12, 112 /125. Hoek, M., Cross, G.A.M., 2001. Expression-site-associatedgene-8 (ESAG8) is not required for regulation of the VSG expression site in Trypanosoma brucei . Mol. Biochem. Parasitol. 117, 211 /215. Hoek, M., Zanders, T., Cross, G.A.M., 2002. Trypanosoma brucei expression-site-associated-gene-8 protein interacts with a Pumilio family protein. Mol. Biochem. Parasitol. 120, 269 /283. Hope, M., MacLeod, A., Leech, V., Melville, S.E., Sasse, J., Tait, A., Turner, C.M.R., 1999. Analysis of ploidy (in

J.E. Donelson / Acta Tropica 85 (2003) 391 /404 megabase chromosomes) in Trypanosoma brucei after genetic exchange. Mol. Biochem. Parasitol. 104, 1 /10. Horn, D., 2001. Nuclear gene transcription and chromatin in Trypanosoma brucei . Int. J. Parasitol. 31, 1157 /1165. Hotz, H.R., Biebinger, S., Flaspohler, J., Clayton, C., 1998. PARP gene expression: control at many levels. Mol. Biochem. Parasitol. 91, 131 /143. Kanmogne, G.D., Bailey, M., Gibson, W.C., 1997. Wide variation in DNA content among isolates of Trypanosoma brucei ssp. Acta Trop. 63, 75 /87. Kim, K.S., Donelson, J.E., 1997. Co-duplication of a VSG gene and its promoter to an expression site in African trypanosomes. J. Biol. Chem. 272, 24637 /24645. Kooter, J.M., Borst, P., 1984. Alpha-amanitin-insensitive transcription of variant surface glycoprotein genes provides further evidence for discontinuous transcription in trypanosomes. Nucleic Acids Res. 12, 9457 /9472. LaCount, D.J., El-Sayed, N.M., Kaul, S., Wanless, D., Turner, C.M.R., Donelson, J.E., 2001. Analysis of a donor gene region for a variant surface glycoprotein and its expression site in African trypanosomes. Nucleic Acids Res. 29, 2012 / 2019. Lee, M.G., Van der Ploeg, L.H., 1997. Transcription of proteincoding genes in trypanosomes by RNA polymerase I. Annu. Rev. Microbiol. 51, 463 /489. Lenardo, M.J., Esser, K.M., Moon, A.M., Van der Ploeg, L.H., Donelson, J.E., 1986. Metacyclic variant surface glycoprotein genes of Trypanosoma brucei subsp. rhodesiense are activated in situ, and their expression is transcriptionally regulated. Mol. Cell Biol. 6, 1991 /1997. Lenardo, M.J., Rice-Ficht, A.C., Kelly, G., Esser, K.M., Donelson, J.E., 1984. Characterization of the genes specifying two metacyclic variable antigen types in Trypanosoma brucei rhodesiense . Proc. Natl. Acad. Sci. USA 81, 6642 / 6646. Lips, S., Revelard, P., Pays, E., 1993. Identification of a new expression site-associated gene in the complete 30.5 kb sequence from the AnTat 1.3A variant surface protein gene expression site of Trypanosoma brucei . Mol. Biochem. Parasitol. 162, 135 /137. Lu, Y., Hall, T., Gay, L.S., Donelson, J.E., 1993. Point mutations are associated with a gene duplication leading to the bloodstream re-expression of a trypanosome metacyclic VSG. Cell 72, 397 /406. Mair, G., Shi, H., Li, H., Djikeng, A., Aviles, H.O., Bishop, J.R., Falcone, F.H., Gavrilescu, C., Montgomery, J.L., Santori, M.I., Stern, L.S., Wang, Z., Ullu, E., Tschudi, C., 2000. A new twist in trypanosome RNA metabolism: cissplicing of pre-mRNA. RNA 6, 163 /169. McCulloch, R., Rudenko, G., Borst, P., 1997. Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences. Mol. Cell Biol. 17, 833 /843. McCullock, R., Barry, J.D., 1999. A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation. Genes Dev. 13, 2875 /2888.

403

Mehlert, A., Zitzmann, N., Richardson, J.M., Treumann, A., Ferguson, M.A., 1998. The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei . Mol. Biochem. Parasitol. 91, 145 / 152. Melville, S.E., Gerrard, C.S., Blackwell, J.M., 1999. Multiple causes of size variation in the diploid megabase chromosomes of African trypanosomes. Chromosome Res. 7, 191 / 203. Melville, S.E., Leech, V., Gerrard, C.S., Tait, A., Blackwell, J.M., 1998. The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers. Mol. Biochem. Parasitol. 94, 155 / 173. Mewes, H.W., Albermann, K., Bahr, M., Frishman, D., Gleissner, A., Hani, J., Heumann, K., Kleine, K., Maierl, A., Oliver, S.G., Pfeiffer, F., Zollner, A., 1997. Overview of the yeast genome. Nature 387, 7 /65. Mun˜oz-Jorda´n, J.L., Davies, K.P., Cross, G.A.M., 1996. Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei . Science 272, 1791 /1794. Myler, P.J., Audleman, L., deVos, T., Hixson, G., Kiser, P., Lemley, C., Magness, C., Rickel, E., Sisk, E., Sunkin, S., et al., 1999. Leishmania major Friedlin chromosome 1 has an unusual distribution of protein-coding genes. Proc. Natl. Acad. Sci. USA 96, 2902 /2906. Navarro, M., Cross, G.A., 1996. DNA rearrangements associated with multiple consecutive directed antigenic switches in Trypanosoma brucei . Mol. Cell Biol. 16, 3615 /3625. Navarro, M., Gull, K., 2001. A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei . Nature 414, 759 /763. Ngo, H., Tschudi, C., Ullu, E., 1998. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei . Proc. Natl. Acad. Sci. USA 95, 14687 /14692. Pays, E., Lheureux, M., Steinert, M., 1981. The expressionlinked copy of a surface antigen gene in Trypanosoma is probably the one transcribed. Nature 292, 265 /267. Pays, E., Lips, S., Nolan, D., Vanhamme, L., Perez-Morga, D., 2001. The VSG expression sites of Trypanosoma brucei : multipurpose tools for the adaptation of the parasite to mammalian hosts. Mol. Biochem. Parasitol. 114, 1 /16. Pays, E., Nolan, D.P., 1998. Expression and function of surface proteins in Trypanosoma brucei . Mol. Biochem. Parasitol. 91, 3 /36. Pays, E., Tebabi, P., Pays, A., Coquelet, H., Revelard, P., Salmon, D., Steinert, M., 1989. The genes and transcripts of an antigen gene expression site from T . brucei . Cell 57, 835 /845. Pedram, M., Donelson, J.E., 1999. The anatomy and transcription of a monocistronic expression site for a metacyclic variant surface glycoprotein gene in Trypanosoma brucei . J. Biol. Chem. 274, 16876 /16883. Robinson, N.P., Burman, N., Melville, S.A., Barry, J.D., 1999. Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes. Mol. Cell Biol. 19, 5839 /5846.

404

J.E. Donelson / Acta Tropica 85 (2003) 391 /404

Roditi, I., Clayton, C., 1999. An unambiguous nomenclature for the major surface glycoproteins of the procyclic form of Trypanosoma brucei . Mol. Biochem. Parasitol. 103, 99 /100. Roditi, I., Furger, A., Ruepp, S., Schurch, N., Butikofer, P., 1998. Unraveling the procyclin coat of Trypanosoma brucei . Mol. Biochem. Parasitol. 91, 117 /130. Rudenko, G., Bishop, D., Gottesdiener, K., Van der Ploeg, L., 1989. Alpha-amanitin resistant transcription of protein coding genes in insect and bloodstream form Trypanosoma brucei . EMBO J. 8, 4259 /4263. Rudenko, G., Chaves, I., Dirks-Mulder, A., Borst, P., 1998. Selection for activation of a new variant surface glycoprotein gene expression site in Trypanosoma brucei can result in deletion of the old one. Mol. Biochem. Parasitol. 95, 97 / 109. Schneider, A., Martin, J., Agabian, N., 1994. A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria. Mol. Cell Biol. 14, 2317 /2322. Sloof, P., Menke, H.H., Caspers, M.P., Borst, P., 1983. Size fractionation of Trypanosoma brucei DNA: localization of the 177- bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction. Nucleic Acids Res. 11, 3889 /3901. Smith, D.H., Pepin, J., Stich, A.H.R., 1998. Human African trypanosomiasis: an emerging public health crisis. Br. Med. Bull. 54, 341 /351. Sternberg, J., Tait, A., 1990. Genetic exchange in African trypanosomes. Trends Genet. 6, 317 /322. The C. elegans Sequencing Consortium, 1998. Genome sequence of the nematode C . elegans : a platform for investigating biology. Science 282, 2012 /2018. Tschudi, C., Ullu, E., 2002. Unconventional rules of small nuclear RNA transcription and cap modification in trypanosomatids. Gene Expr. 10, 3 /16. Turner, C.M., 1997. The rate of antigenic variation in flytransmitted and syringe-passaged infections of Trypanosoma brucei . FEMS Microbiol. Lett. 153, 227 /231. Turner, C.M., Barry, J.D., 1989. High frequency of antigenic variation in Trypanosoma brucei rhodesiense infections. Parasitology 99, 67 /75. Turner, C.M.R., Barry, J.D., Maudlin, I., Vickerman, K., 1998. An estimate of the size of the metacyclic variable antigen repertoire of Trypanosoma brucei rhodesiense . Parasitology 97, 269 /276. Turner, C.M.R., Melville, S.E., Tait, A., 1997. A proposal for karyotype nomenclature in T. brucei . Parasitol. Today 13, 5 /6.

Turner, C.M.R., Sternberg, J., Buchanan, N., Smith, E., Hide, G., Tait, A., 1990. Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meiosis and syngamy. Parasitology 101, 377 /386. Van der Ploeg, L.H., Schwartz, D.C., Cantor, C.R., Borst, P., 1984. Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell 37, 77 /84. Van der Ploeg, L.H., Smith, C.L., Polvere, R.I., Gottesdiener, K.M., 1989. Improved separation of chromosome-sized DNA from Trypanosoma brucei , stock 427 /60. Nucleic Acids Res. 17, 3217 /3227. Van der Ploeg, L.H., Valerio, D., De Lange, T., Bernards, A., Borst, P., Grosveld, F.G., 1982. An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome. Nucleic Acids Res. 10, 5905 /5923. Vanhamme, L., Pays, E., 1998. Controls of the expression of the VSG in Trypanosoma brucei . Mol. Biochem. Parasitol. 91, 107 /116. van Leeuwen, F., Kieft, R., Cross, M., Borst, P., 1998. Biosynthesis and function of the modified DNA base (bD-glucosyl-hydroxymethyluracil in Trypanosoma brucei . Mol. Cell Biol. 18, 5643 /5651. Weiden, M., Osheim, Y.N., Beyer, A.L., Van der Ploeg, L.H., 1991. Chromosome structure: DNA nucleotide sequence elements of a subset of the minichromosomes of the protozoan Trypanosoma brucei . Mol. Cell Biol. 11, 3823 / 3834. World Health Organization; 1996. Thirteenth Programme Report of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. pp. 124 /133. Xong, H.V., Vanhamme, L., Chamekh, M., Chimfwembe, C.E., Van Den Abbeele, J., Pays, A., Van Meirvenne, N., Hamers, R., De Baetselier, P., Pays, E., 1998. A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense . Cell 95, 839 /846. Zomerdijk, J.C., Kieft, R., Borst, P., 1992. A ribosomal RNA gene promoter at the telomere of a mini-chromosome in Trypanosoma brucei . Nucleic Acids Res. 20, 2725 /2734. Zomerdijk, J.C., Ouellette, M., ten Asbroek, A.L., Kieft, R., Bommer, A.M., Clayton, C.E., Borst, P., 1990. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei . EMBO J. 9, 2791 /2801.