Molecular and Biochemical Parasitology, 9 (1983) 255-269 Elsevier

MBP 00367

255

G E N O M I C O R G A N I Z A T I O N O F T R Y P A N O S O M A B R U C E I V A R I A N T

A N T I G E N G E N E F A M I L I E S IN S E Q U E N T I A L P A R A S I T E M I A S

MARILYN PARSONS 1, RICHARD G. NELSON 1, GEORGE NEWPORT l, MICHAEL MILHAUSEN ~, KENNETH STUART 2 and NINA AGABIAN 1

~Department of Biochemistry, University of Washington, Seattle, WA 98195 and 21ssaquah Health Research Institute, 1595 N.W. Gilman Blvd., Issaquah, WA 98127, U.S.A.

(Received 17 March 1983; accepted 3 May 1983)

cDNA libraries were made from mRNA purified from each of seven sequentially isolated variant antigen types (VATs) of the IsTat 1 serodeme. Plasmids containing variant surface glycoprotein (VSG) sequences corresponding to each of the isolates were used in Southern analyses to examine the genomic organization of VSG nucleotide sequences. In most cases, cells expressing a given VSG were shown to have an extra copy of the corresponding ~SG gene. In one case an expression-linked copy (ELC) was not detectable. VSG gene rearrangements not obviously correlated with the expression of homologous sequences were detected in four of six VSG gene families. Thus, even cDNAs which detected an ELC revealed additional genomic reorganization in regions flanking VSG sequences. The cells used to initiate the chronic infection expressed the same VSG as those isolated from the first parasitemia. The extent of genomic rearrangement observed between these two sequentially derived populations was comparable to that observed between any of the other serially derived VATs. Thus, within a short period of time and in the absence of detectable antigenic variation, the amount of genetic flux in sequences associated with VSG genes can be substantial.

Key words: Trypanosoma brucei; Antigenic variation; Gene rearrangement; cDNA cloning; Variant surface glycoprotein

INTRODUCTION

T h e su r face o f the p a r a s i t e Trypanosoma brucei brucei is c o v e r e d wi th a s ingle

g l y c o p r o t e i n a n t i g e n [ 1]. W h e n these A f r i c a n t r y p a n o s o m e s are s u b j e c t e d to i m m u n o -

logical p r e s s u r e in the m a m m a l i a n h o s t , a p o p u l a t i o n b e a r i n g n e w sur face a n t i g e n

r ap id ly ar ises [2]. Th i s p h e n o m e n o n , k n o w n as an t i gen i c v a r i a t i o n , resu l t s in a

r e l aps ing p a r a s i t e m i a as success ive an t i gen i ca l l y d i s t inc t t r y p a n o s o m e p o p u l a t i o n s

e m e r g e whi le p r e v i o u s p o p u l a t i o n s are r e m o v e d by the h o s t i m m u n e sys t em.

Abbreviations: VAT, variant antigen type; VSG, variant surface glycoprotein; SDS, sodium dodecyl sulfate; SSC, standard saline citrate (150 mM NaCI, 15 mM sodium citrate, pH 7.0); SSPE, (180 mM NaCI, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4); ELC, expression-linked copy; bp, base pair.

0166-6851/83/$03.00 © 1983 Elsevier Science Publishers B.V.

256

The mechanisms underlying ~ntigenic variation have recently come under the scrutiny of molecular biologists. An unknown mechanism operating at the transcrip- tional level specifies which gene from the variant antigen repertoire is expressed [3,4].

In an at tempt to elucidate the mechanism of transcriptional control, the organization of particular VSG sequences in DNA derived from cloned populations of different variant antigen types (VAT) has been compared. Two types of genomic rearrange- ments have been observed. One class o fVSG cDNA probes recognizes gene sequences which show identical genomic organizations in different VAT populations, except those expressing the VSG examined. In the latter VATs, there is an extra hybridizing sequence, presumably resulting from duplication of a basic gene and transposition of the copy to a novel site [5,6]. It appears that the new gene copy is the one transcribed, hence the term expression-linked copy (ELC) [7,8]. It has been postulated that there is a VSG gene expression site(s), and that transposition into this site via a mechanism coupled with gene duplication allows transcription of the VSG gene [7,8]. In similar analyses, however, other VSG cDNA probes do not detect an ELC [9,10]. Gene sequences detected by these cDNA probes show no apparent duplications but reside in different contexts in different VAT populations. None of the latter genomic rearrange- ments, probably resulting from DNA insertions and deletions 3' to the genes [11], have been correlated with VSG gene expression. However, both the genes which show 3' rearrangements and the ELCs appear to be within several kilobases of a double stranded break in the DNA (i.e. a telomere) [12,13].

In light of the complexity of the molecular data which is emerging, a well-charac- terized biological framework in which to interpret such results seems essential. We have developed a T. brucei serodeme, the IsTat 1 serodeme, which was derived from relapsing parasitemias in a single deermouse [14]. In a preceding paper [15] we have demonstrated that they are distinct antigenic variants by classical immunological and biochemical criteria. Expression of each VSG was regulated at the level of transcrip- tion. In this comtnunication, we describe the generation of cDNA clones correspon- ding to each of the early VATs and present an initial Southern analysis of the sequences which they detect. Each VSG cDNA is used to examine the genomic organization of VSG sequences in each of the early VATs of the IsTat 1 serodeme. Taken together, these analyses describe the nature of genomic rearrangement of VSG sequences expressed in the early phases of a syringe-passed infection, and demonstrate both the generation of an ELC and rearrangement of 3' flanking sequences can occur within a single VSG gene family.

MATERIALS AND METHODS

Trypanosomes. The IsTat serodeme of T. b. brucei is fully described by Stuart et al. [14]. Trypanosome populations 1.A and 1.D were isolated from a rat infected with a single cell of the IsTat 1.0 clone and represent the first parasitemia and the first relapse respectively. 1.D cells were injected into a deermouse, Peromyscus leucopus, and

257

popula t ions 1.1 through 1.48 were isolated as sequential b lood samples start ing with

the first parasi temia. F r o m the early isolates, clones were obta ined, as designated by a

subscript to the popula t ion number . The antigens expressed were characterized by

polyclonal and monoc lona l ant ibodies, as well as biochemical criteria [151. Cells were

expanded in lethally irradiated rats as described and VAT homogenei ty (~99%)

assayed by immunof luorescence on live cells using VAT specific antisera [ 14]. Table I

fully describes the t rypanosome popula t ions used in this study.

Preparation of VSG cDNA clones. Prepara t ion of RNA is described in the preceding

paper [15]. cDNAs were synthesized by the procedure of Buell et al. [16] with minor

modificat ions. The cDNAs were tailed with dCTP [17] and annealed with pBR322

that had been cleaved with Pst 1 and tailed with dGTP. We then used these annealed

plasmids to t ransform Escherichia coli HB101, and selected for t ransformants on

media conta in ing tetracycline (20 lag ml-~). The resultant colonies were screened for

hybr id iza t ion with c D N A probes made from randomly pr imed po ly (A+) RNA

isolated from homologous and heterologous VATs. Those exhibit ing differential

hybr id iza t ion were presumed to conta in the t ranscr ipt ional ly control led VSG se-

quences. This was verified by Nor thern analysis [15] and hybr idizat ion selection

t rans la t ion (see below). Clones were characterized by restriction mapping. The direc-

t ion of t ranscr ipt ion was determined by either the expression of cloned sequences

(Parsons, unpubl i shed results), hybr idizat ion of cDNAs to a synthetic probe contai-

ning a por t ion of the 35-mer found to date at the ul t imate 5' end of all VSG sequences

[19], or by Nor thern hybr id iza t ion with single s t randed probes made from c D N A

fragments cloned asymmetrical ly into M 13 vectors MP 8 and MP 9 [20].

TABLE I

Early VATs of the IsTat 1 serodeme

Nomenclature Stabilate Clone Antigen

Complete This paper Day a Designation

1.A - A A 8 A - A 1.,%- A Af A f A I.D - 1 D 15 D - 1 I.D a - 1 D a D a 1 1. I a - 1 1 19 1 a 1 1.3~ - 3 3 27 3 a 3 1.5~ - 5 5 35 5 a 5 1.7 a - 7 7 43 7 a 7 1.11 a - 11 11 59 11 a 11

a Number of days is from the single injected IsTat 1.0 cell to isolation of the stabilate. Cloning and amplification required another l0 days. There are approximately 4.5 generations per clay.

258

cDNA probes, cDNA probes were made from poly(A+) RNA by fhe random priming procedure [18] using calf thymus oligo(DNA) as primer for DNA synthesis by avian myeloblastosis virus reverse transcriptase (Life Sciences, Inc., St. Petersburg, FL). Conditions were as described for first strand cDNA synthesis, except that dCTP was replaced with its 32p-labeled homologue.

Hybridization selection translation. We employed the procedure ofCochet et al. [21]. 8 lag of plasmid DNA was bound to 0.75 cm nitrocellulose circles and hybridized to total (50 lag) or poly(A+) RNA (10 lag). Following thorough washing to remove non-speci- fically bound material, RNA was eluted by boiling in sodium dodecyl sulfate (SDS). It was then precipitated with ethanol, redissolved, and translated using the rabbit reticulocyte lysate system plus [35S]methionine to label the synthesized peptides [ 15]. Translation products were analyzed on SDS polyacrylamide gels and compared with homologous VSG immunoprecipitated with monospecific or monoclonal anti-VSG antibodies from the mixture of peptides synthesized in vitro.

Southern analysis. DNA was isolated as previously described [ 15]. Restriction enzyme digests of genomic DNA were prepared by incubating DNA with a two- to four-fold excess of restriction enzyme for 2 h at 37°C. A further two-fold excess of enzyme was then added and following another hour of incubation purified k phage DNA was added. After 1 h the mixture was removed to 4°C. An aliquot was subjected to Southern analysis using a 32p-labeled ~. phage DNA probe to insure that restriction proceeded to completion. Alternatively, complete digestion was confirmed by South- ern analysis of the t rypanosome a- and [3-tubulin genes.

Restricted genomic DNA (2-3 tag/lane) was fractionated on 0.8% agarose gels in 40 mM Tris acetate buffer, pH 8. Each gel contained k and ~pX phage DNAs restricted with Hind III and Hae III, respectively, as molecular weight markers. After electro- phoresis the gel .was treated with 0.25 N HC1 for 15 rain to ensure efficient transfer of high molecular weight DNA, and then prepared for blotting [22]. Duplicate nitrocel- lulose blots were made from each gel [23]. Blots were prehybridized for several hours in 5 )< SSPE, 200 tag m1-1) heat denatured salmon testis DNA, and 0.25% sarkosyl. After a 16-40 h hybridization at 65°C with 106 cpm of nick-translated plasmid [24] (specific activity ~> 5 X 108 cpm lag -t) per ml of solution, the blots were washed twice for 45 min at 65°C in 3 >( SSC plus 20 mM sodium phosphate, pH 6.5, 0.1% sarkosyl and 1 mM EDTA. Finally, they were washed twice for 45 rain at 65°C in a 1/30

dilution of the buffer above.

RESULTS

The IsTat VATs A and D were isolated sequentially from a rat, and 1,3, 5, 7 and 11 VATs were isolated serially from a single deermouse [14]. cDNA libraries generated from RNA isolated from each of these early VATs were screened for differential

259

hybridization with cDNA probes made from homologous and heterologous ran- domly primed poly (A-k) RNA. The putative VSG sequences thus identified were then checked for hybridization with RNA from the early VATs by Northern analysis [15]. Each cDNA detects an RNA species in homologous RNA only, with the exceptions of D and 1 cDNA clones which both hybridize to D and 1 VSG mRNA. The RNAs encoding the VSGs studied range from 1.5 to 1.9 kb in size. On average, 4 percent of the cDNA clones in each library contain VSG sequences.

Further confirmation that the clones contained VSG sequences was provided by hybridization selection translation. In these experiments a plasmid containing VSG sequences was used to isolate homologous mRNA, which was subsequently translated in vitro. An example is shown in Fig. 1. Here a cloned 7 cDNA was used to enrich sequences from poly(A+) RNA isolated from VAT 7. The enriched mRNA was translated (lane 3) and the resultant peptides were compared to VSG 7 isolated by immunoprecipitation from total 7 RNA translation products synthesized in vitro from VAT 7 RNA (lane 5). This comparison shows that the mRNA selected by hybridization with the 7 cDNA encodes a protein of the appropriate molecular weight. Furthermore, the major protein obtained by hybridization selection transla- tion is immunoprecipitated by anti-VSG 7 antiserum (lane 4), and thus bears determi- nants which reside on VSG 7. Control translations using 11 RNA 'enriched' by selection with 7 cDNA yielded no specific translation product (lane 6) proving that the 7 cDNA sequence is VAT-specific. Similar studies using clones corresponding to each of the VATs demonstrated that each cDNA enriched for sequences from homologous mRNA only, and that the enriched mRNA encoded a protein of the appropriate molecular weight bearing VAT specific VSG determinants [13]. We have applied these criteria to cDNA clones for each VAT used in this analysis.

These cloned VSG cDNAs were used to examine the genomic organization of their homologous sequences in the IsTat VATs. Southern analyses of at least three restric- tion digests (Sal 1, Bam HI, and Pvu II) of DNA isolated from each VAT, using cDNA probes corresponding to each VSG, were performed. Each VSG cDNA probe hybridizes to one or more restriction fragments under the stringent conditions em- ployed (see Methods) and thus operationally defines a VSG sequence family. Three categories of VSG sequence families were observed: gene families which show gene duplications associated with expression, families which show no ELC but show variation not associated with expression, and finally families which show both pheno- mena.

The first group is typified by the 11 and 7 VSG gene families. The 1050 base pair (bp) 11 cDNA does not contain a Pvu II site. Fig. 2 shows the hybridization pattern obtained when this 11 VSG cDNA was hybridized with Pvu II digested genomic DNA from each of the VATs. The 11 VSG probe hybridizes with two fragments in each DNA, except that expressing 11 VSG: here an additional fragment is observed, at 11.5 kb. Restriction of genomic DNAs with Barn HI or Sal 1 similarly reveal an additional fragment containing 11 VSG nucleotide sequences in VAT 11 DNA. These digests

260

1 2 3 4 5 6 7

6 7 - -

41 . -

31--

----58

14- -

Fig. 1. Verification that plasmid pTbl.7-cl contains sequences encoding VSG 7 by hybridizaton selected translation. Translation products synthesized in the rabbit reticulocyte lysate system were analyzed by SDS polyacrylamide gel electrophoresis. Lane 1 shows the profile generated by the lysate translation system with no added RNA. Lane 2 depicts total translation products from VAT 7 poly (A+) RNA. Lane 3 contains peptides synthesized by the VAT 7 RNA species enriched by hybridization with 7 cDNA and Lane 4 shows the immunoprecipitation of these products with anti-VSG 7 antiserum. The migration of the labeled product is disturbed by comigration of the immunoglobulin heavy chains. Lane 5 contains the peptides precipitated by anti-VSG 7 from total translation products. Control Lanes 6 and 7 show the translation products of VAT 11 RNA species selected by hybridization with 7 cDNA, and the immunoprecipitation of these with anti-VSG 11 antiserum.

261

Fig. 2. Hybridization of the 1050 bp clone pTb 1.11-c2 to Pvu II digested genomic DNA from the IsTat 1 VATs. The VAT from which each DNA was derived is indicated above the lanes, and the mobility and size (in kb) of molecular weight markers is shown on the left. The probe has no Pvu II site.

also show two to three f ragments which did not vary in size a m o n g the D N A s

examined. Thus the s i tua t ion is ana logous to the appea rance of an ELC, as has been

descr ibed by others [5,6].

The express ion of the 7 gene is also a c c o m p a n i e d by the appea rance of an extra copy

of the 7 gene, as shown in Fig. 3. Here a 7 c D N A is hybr id ized with an Eco R l / B a m H1

doub le digest o f I sTa t V A T D N A s (the c D N A probe conta ins no Eco RI o r Bam H1

sites). Aga in no o ther a l te ra t ions in genomic o rgan iza t ion are observed o ther than the

appea rance o f the ELC.

The second ca tegory of V S G gene families was revealed by the D and 1 c D N A

probes . These c D N A s were made f rom sequent ia l ly isola ted t r y p a n o s o m e popu la -

t ions expressing the same V S G [15]. The sequences con ta ined in the c D N A s conta in

no Sal 1 sites. Fig. 4 shows a Sal 1 digest of serodeme D N A s hybr id ized with the 1

c D N A (an ident ical picture was ob ta ined with the D c D N A ) . It appears in this and

several o ther digests that there is a single copy of the 1 sequence in expressing and

non-express ing cells. Thus, in clones D a and 1 a, both of which express the 1 ant igen,

there is no appa ren t ELC. The context of the 1 gene clearly varies, as evidenced by the

different sized res t r ic t ion f ragments on which it resides in different VATs. In fact the

gene appears to be hypervar iab le , in that two different clones expressing the A ant igen

and recently der ived f rom a single cell ( IsTat 1.0) show different a r rangements of the 1

262

A D 1 3 5 7 1 1

9 ° 6 --

6 . 6 -

Fig. 3. Hybridization of the 850 bp clone pTb 1.7-c 1 to DNA restricted with Eco R 1 and Bam H 1. The VAT

from which each DNA was isolated is indicated above each lane, and the size (in kb) and mobility of

molecular weight standards is shown on the left. The probe has no sites for Eco RI or Barn HI.

Af D a 1 3 5 7 11

- 2 3

- 9 . 6

- 6 . 6

Fig. 4. Hybridization of the 1060 bp clone pTb 1.1 -c 1 to Sal 1 restricted DNA. The VAT derivation of each

DNA is noted above the lanes and migration and size (in kb) of molecular weight markers is indicated on the

left. The hybridization pattern obtained using the 600 bp pTb l.D-cl cDNA is identical. Neither probe has a

Sal 1 site.

263

gene [15]. Restriction digests which cleave the 1 cDNA have allowed us to map the

genomic rearrangement 3' to the gene, proximal to a cluster of restriction enzyme sites (i.e. a telomere) (Fig. 5). Thus the 1 gene family appears similar to the VSG gene families described by Young et al. [11]: telomeric genes which are not duplicated in conjunction with expression and which show insertions or deletions of DNA in 3' flanking regions. However, it differs in that the IsTat 1 gene family has only one member.

The last category of VSG genes is that where both an ELC and non-expression associated variation occur. These gene families are shown in Figs. 6, 7 and 8. Fig. 6 shows Pvu II digested genomic DNAs hybridized with the 5 eDNA probe, which contains two Pvu II sites. The largest hybridizing restriction fragment varies from VAT to VAT. In VAT A the fragment is 12 kb, VAT D 12.5 kb (not shown), VAT 1 4.3 kb, and so on. There is an extra fragment in VAT 5 at 6.5 kb, and the 1.3 kb fragment is increased in intensity. The internal 0.4 kb fragment, not seen on this blot, is also duplicated in VAT 5. That the 5 ELC is composed of the 6.5, 1.3, and 0.4 kb fragments is confirmed by hybridization of similar blots with probes corresponding to the 5' and 3' halves of the cDNA (see Fig. 6).

The 3 gene family also shows non-expression associated variation (Fig. 7A) and an ELC (Fig. 7B). In Fig. 7A, a Pst digest of serodeme DNAs was hybridized to the 3' Pst fragment of a 3 cDNA, revealing a large multigene family. One copy of the gene varies throughout the serodeme. This fragment did not hybridize to the 5' Pst fragment of the eDNA, indicating that the variation is occurring 3' to the VSG gene, a finding corroborated by several other restriction digests. Fig. 7B shows the presence of the ELC, as revealed by Ava 1 digestion.

The last gene family of this group is the A family. Sal 1 restricted serodeme DNAs are hybridized with the A probe in Fig. 8 (there is one Sai 1 site in the cDNA). In non-expressing DNAs, 3 fragments are observed: one at 14.5 kb, one at 1.3 kb and one which ranges from 6 to 8 kb depending on the VAT examined. The variable fragment and the 14.5 kb fragment correspond to 3' portions of the genes, while the 1.3 kb

VAT I s~,orB H 7 ................ !

11 III ............... '~4

51 I I I I I ~ I 3 ' 2 0 2 4 6 1 0

Fig. 5. G e n o m i c m a p of the 1 gene in VATs 7 and l l . The shaded area indicates the loca t ion of the 1 800 bp

s t ruc tura l gene. The a r row marks the locat ion of a c luster of seven restr ic t ion sites (Pst 1, Pvu If, Sal 1, H ind

III , Bg 1 II, Kpn 1, and Barn H 1). Other enzyme sites are marked as follows: S, Sal 1 ; D, Hind III , B, Barn H 1;

H, Hinc II. Res t r ic t ion sites within the cod ing sequence are not shown. Aside f rom the Hinc I] site

a p p r o x i m a t e l y 100 bp 3' to the gene, no restr ic t ion sites were found between the gene and the c lus ter of sites.

F rom the 5' end of the gene, the cluster lies 7.8 kb 3' in VAT 7 and 8.2 kb 3' in VAT 11. Other VATs show

s imi la r var ia t ion.

264

1 3 5 7 1 1 A

A A D 1 3 5 7 1 1

4 . - .3 °

"--5'

Fig. 6.

B A 3

• m I

Fig. 7.

Af D a 1 3 5 7 11

265

Fig. 8. Hybridization of the 650 bp clone pTbl.A-cl with Sal 1 fragments of IsTat VAT DNA. The VAT

from which each DNA was derived is indicated above the lanes and the mobility in size (in kb) of molecular

weight markers is shown on the right. The probe has one Sal site.

Fig. 6. Hybridization of the 1.2 kb clone pTbl.5-cl to Pvu 11 digested DNA from each of the IsTat 1 early

VATs. The VAT from which each DNA was derived is indicated above the lanes, and the size (in kb) and

mobility of molecular weight markers is shown on the left. The probe has two Pvu II sites, which create a 400

bp internal Pvu II fragment not seen on this gel. Restriction fragments which contain the 5' and the 3'

portions of the ELC are marked.

Fig. 7A. Hybridization of the 3' 450 bp Pst fragment of pTb 1.3-c I to Pst 1 digested IsTat VAT DNA. The

VAT from which each DNA was isolated is indicated above each lane and the size (in kb) and migration of

molecular weight markers is shown in the center: 23, 9.6, 6.6 and 4.3 kb.

Fig. 7B. Hybridization of the 3' Ava 1 fragment of pTb 1.3-c2 to Ava 1 digested genomic DNA from VATs 1

and 3.

266

fragment encodes the 5' portion(s). DNA isolated from cells expressing the A antigen

show an extra fragment (which hybridizes to the 3' portion of the probe) and a

moderate, but reproducible increase in intensity of the 1.3 kb fragment. To insure that this reflected the true presence of an ELC and not clonal heterogeneity, we examined

several A clones (see ref. 15). All showed the presence of an extra hybridizing

fragment.

DISCUSSION

We have used cloned VSG cDNAs to analyze the organization of VSG sequence

families in the initial isolates of the IsTat 1 serodeme. The extent of DNA rearrange-

ment we have observed is striking. Majiwa et al. have shown that genes which show an

ELC and those which do not can be expressed by a single trypanosome stock [9]. Our

data indicates that early in a single infection both types of genes can be expressed.

Furthermore, within a sequence family, we have presented evidence that an ELC can

occur in conjunction with other genomic rearrangements. VSG sequences detected by the A, 3, 5, 7 and 11 VSG cDNAs all show some features

of the ELC model, but differ in other respects. The 11 and 7 VSG genes follow the ELC model most closely. They show no rearrangements when not expressed and expression

is accompanied by the appearance of an extra copy of the gene. Preliminary data

however suggest the duplication and transposition events generating the 11 ELC

involve a larger segment of DNA than has been described for other ELCs (1~8,25,26]

and Nelson, unpublished results). The appearance of an ELC in a VSG gene family

which also shows other genomic rearrangements has not yet been described. Three of the six cDNAs used in this study reveal this combination. It appears that in each of

these families, the rearrangements occur by insertions or deletions of DNA 3' to the

gene (unpublished data) and thus are analogous to the rearrangements observed by

Young et al. [15]. VAT 1 was isolated from the first parasitemia in P. leucopus, four days (18

generations) after infection with D cells [4]. Although the antigens expressed on VAT

D and 1 trypanosomes appear identical [ 15], the genomes of the D and 1 VATs differ with respect to the organization of non-expressed VSG gene families. In particular,

the putatively telomeric members of the A (Fig. 8), D/1 (Fig. 4), 3 (Fig. 7A) and 5 (not

shown) gene families all reside in different contexts in the D and 1 populations. The number of differences between D and 1 VAT DNAs is not small considering the number of sequences surveyed, and suggests that the amount of genomic rearrange-

ment in a relatively short period of time is substantial. Since no known antigen expression switch occurred between D and 1, these data suggest that there is conti- nuous genetic flux in telomeric regions which is not associated with a specific variation event. In fact, if one examines Figs. 7A and 8 the rearrangement of VSG flanking sequences seems to progress in a relatively regular fashion over time without regard for the antigenic variation event. However, we have shown that clones expressing the

267

same VSG, isolated at the same point in time, can show distinct patterns in VSG Southern analyses [15], belying the apparent regularity observed in these figures.

Our findings indicate a greater extent of genomic variation in VSG flanking sequences than might be suspected from previous studies, probably reflecting the unusual propensity for VSG genes to reside adjacent to a telomere. In fact 4 out of 17 VSG genes detected with our cDNA probes seem to lie in telomeric regions. Telomeric regions in other protozoa have been shown to contain sequence repeats and, when cloned in yeast, show heterogeneity which appears to be an intrinsic result of their propagation [27]. This would suggest that the sequence rearrangements 3' to VSG genes reflect the properties of the telomere rather than the VSG gene per se. The dissociation of these rearrangements from the variation event as shown in this study, in ref. 15, and by others [28] strengthens this hypothesis.

Gene duplication, normally a rare event, is the first step in the generation of a multigene family. In the generation of the ELC, the African trypanosome has evolved a specialized mechanism for gene duplication. Telomeric VSG genes have been hypothesized to be the result of the occasional stable maintenance of an ELC after expression [29]. Our findings that approximately 24% of VSG genes fall into this category suggests that such a mechanism could be important in expanding the T. brucei VSG gene repertoire. Furthermore, since at least some telomeric VSG genes can be activated without expression-linked gene duplication, relaxed selection on regions of the DNA important for this function could allow for even more mutational drift.

In order to establish a working hypothesis for examining the molecular basis of variant antigen expression, it is reasonable to study those gene families most amenable to analysis. However, this could bias our understanding of the extent and nature of genomic flux in the VSG gene repertoire. The fact that the genes we examined were selected by the biological relationship of host and parasite suggests we are studying a representative sample of T. brucei VSG gene families expressed early in a syringe-pas- saged infection. In this study, 5 of 6 VATs showed the duplicative mode of expression and one showed non-duplicative VSG gene activation. Approximately one-fourth of the genes surveyed show 3' rearrangements and appear to fall in the class oftelomeric genes. Analyses in progress of VSG gene organization in late VATs, in procyclic culture from trypanosomes derived from homogenous VATs, and in cyclically trans- mitted IsTat VATs, should contribute to our further understanding of the mechanisms of antigenic variation.

ACKNOWLEDGEMENTS

We would like to thank Steve Kosowski, Elke Gobright, and Lyle Rudensey for excellent technical assistance, Dr. Elizabeth Brown for critical reading of the manus- cript, and Pam Horbet t for secretarial assistance. Dr. William Rutter provided helpful advice. This work was supported in part by funds from the Rockefeller Foundation,

268

G e n e t i c S y s t e m s C o r p o r a t i o n , a n d the W o r l d H e a l t h O r g a n i z a t i o n i nc lud ing the

U N D P / W o r l d B a n k / W H O Specia l P r o g r a m fo r R e s e a r c h a n d T r a i n i n g in T r o p i c a l

Disease . O t h e r s u p p o r t i nc ludes g r a n t s to N . A . ( N I H AI 17309, N S F P C M 8 0 2 1 9 9 5 )

a n d K.S. ( N I H A:I17375, U S A M R D C c o n t r a c t D A M D I 7 - 9 2 - C - 2 0 1 6 ) .

REFERENCES

1 Cross, G.A.M. (1975) Identification, purification and properties of clone-specific glycoprotein anti- gens constituting the surface coat of Trypanosoma brucei, Parasitology 71,393-417.

2 Vickerman, K. (1978) Antigenic variation in trypanosomes. Nature 273,613-617. 3 Hoeijmakers, J.H.J., Borst, P., Van den Burg, J., Weissman, C. and Cross, G.A.M. (1980) The

isolation of plasmids containing DNA complementary to messenger RNA for variant surface glyco- proteins of Trypanosoma brucei. Gene 8, 391-417.

4 Pays, E., Delronche, M., Lheureux, M., Vervoort, T., Bloch, J., Gannon, F. and Steinert, M. (1980) Cloning and characterization of DNA sequences complementary to messenger ribonucleic acids coding for the synthesis of two surface antigens of Trypanosoma brucei. Nucl. Acid Res. 8, 5965-5981.

5 Hoeijmakers, J.H.J., Frasch, A.C.C., Bernards, A., Borst, P. and Cross, G.A.M. (1980) Novel expression-linked copies of the genes for variant surface antigens in trypanosomes. Nature 284, 78-80.

6 Pays, E., Van Meirvenne, N., LeRay, D. and Steinert, M. (1981) Gene duplication and transposition linked to antigenic variation in Trypanosoma brucei. Proc. Natl. Acad. Sci. U.S.A. 78, 2673-2677.

7 Pays, E., Lheureux, M. and Steinert, M. (1981 ) The expression-linked copy of surface antigen gene in Trypanosoma is probably the one transcribed. Nature 292, 265-267.

8 Bernards, A., Van der Ploeg, L.H.T., Frasch, A.C.C. and Borst, P. (1981) Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3' end. Cell 27, 497-505.

9 Majiwa, P.A.O., Young, J.R., Englund, P.T., Shapiro, S.Z. and Williams, R.O. (1982) Two distinct forms of surface antigen gene rearrangement in Trypanosoma brucei. Nature 297, 514-516.

10 Williams, R.W, Young, J.R. and Majiwa, P.A.O. (1981) Contextural genomic rearrangements of variable-antigen genes in Trypanosoma brucei. Cold Spring Harb. Symp. Quant. Biol., Vol. 45, 945-949.

11 Young, J.R., Donelson, J.E., Majiwa, P.A.O., Shapiro, S.Z. and Williams, R.O. (1982) Analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei. Nucl. Acid Res. 10, 803-819.

12 De Lange, T. and Borst, P. (1982) Genomic environment of the expression linked extra copies resembles the end of a chromosome. Nature 299, 451-453.

13 Williams, R.O., Young, J.R. and Majiwa, P.A.O. (1982) Genomic environment of T. brucei VSG

genes: presence of a minichromosome. Nature 299, 417-421. 14 Stuart, K., Goldbright, E., Jenni, L., Milhausen, M., Thomashow, L., and Agabian, N. (1982) The

IsTat 1 serodeme of Trypanosoma brucei: Development of a new serodeme. Parasitology (in press). 15 Milhausen, M., Nelson, R., Parsons, M., Newport, G., Stuart, K. and Agabian, N. (1983) Molecular

characterization of initial variants from the IsTat 1 serodeme of Trypanosoma brucei. Mol. Biochem.

Parasitol. 9, 241-254. 16 Buell, G.N., Wickens, M.P., Payvar, F. and Schimke, R. (1978) Synthesis of full length cDNAs from

four partially purified oviduct mRNAs. J. Biol. Chem. 253, 2471-2482. 17 Roychoudhury, R., Jay, E. and Wu, R. (1976) Terminal labeling and addition of homopolymer tracts

to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucl. Acid Res. 3, 101-116. 18 Taylor, J.M., Illmensee, R. and Sumners, J. (1976) Efficient transcription of RNA into DNA by avian

sarcoma virus polymerase. Biochim. Biophys. Acta 442, 324-330.

269

19 Boothroyd, J.C. and Cross, G.A.M. (1982) Transcripts coding for variant surface glycoproteins have a short, identical exon at their 5' end. Gene 20, 281-289.

20 Messing, J. and Vieira, J. (1982) A new pair of MI3 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19,269-276.

21 Cochet, M., Perrin, R., Gannon, F., Krust, A., Chambon, P., McKnight, G.S., Lee, D.C., Mayo, K.E. and Palmiter, R. (1978) Cloning of an almost full-length chicken conalbumin double-stranded DNA. Nucl. Acid Res. 6, 2435-2452.

22 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 93,503-517.

23 Smith, Gale E. and Summers, Max D. (1980) The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal. Biochem. 109, 123-129.

24 Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113,237-251.

25 Pays, E., Lheureux, M. and Steinert, M. (1981) Analysis of the DNA and RNA changes associated with isotypic variant specific antigens of trypanosomes. Nucl. Acid Res. 9, 4225-4238.

26 Pays, E., Lheureux, M. and Steinert, M. (1982) Structure and expression of a Trypanosoma brucei gambiense variant specific antigen gene. Nucl. Acid Res. 10, 3149-3163.

27 Szostak, J.W. and Blackburn, E.H. (1982) Cloning yeast telomeres on linear plasmid vectors. Cell 29, 249-255.

28 Borst, P., Bernards, A., VanderPloeg, L., Michels, P., Lin, A., de Lange, T,, Veineman, G., Tromp, M. and Van Boom, J. DNA rearrangements controlling the expression of genes for variant surface antigens in trypanosomes. In: 5th John Innes Syrup. on Biological Consequences of DNA Structure and CJenome Rearrangement. In press.

29 Donelson, J.E., Young, J.R., Dorfman, D., Majiwa, P.A.O. and Williams, R.O. (1982) The IsTat 1.4 surface antigen gene family of Trypanosoma brucei. Nucl. Acid Res. 10, 6581-6595.