Stable Variant-Specific Transcripts of the Variant Cell Surface ...

Vol. 8, No. 2

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1988, p. 854-859

0270-7306/88/020854-06$02.00/0 Copyright © 1988, American Society for Microbiology

Stable Variant-Specific Transcripts of the Variant Cell Surface Glycoprotein Gene 1.8 Expression Site in Trypanosoma brucei CATHY SHEA AND LEX H. T. VAN DER PLOEG* Department of Genetics and Development, Columbia University, New York, New York 10032 Received 10 September 1987/Accepted 24 November 1987

The structure and transcriptional regulation of the 1.8 variant cell surface glycoprotein (VSG) gene expression site located on a 430-kilobase (kb) chromosome was examined in a 430-kb-chromosome-specific library. Using 32P-labeled nascent transcripts generated by nuclear run-on, we selected recombinant clones derived from the 430-kb chromosome which were coordinately activated with the 1.8 VSG gene. The results show that a repetitive region with a minimum size of 27 kb is coordinately activated with the 1.8 VSG gene. As with the 1.8 VSG gene, transcription is by RNA polymerases that are insensitive to the drug alpha-amanitin at concentrations up to 1 mg/ml. Transcription results in the generation of several stable variant-specific mRNAs. These mRNAs most likely belong to a family of repetitive expression-site-associated genes. in variant 118 clone 1 has extensive sequence homology with the rRNA gene promoter of T. brucei, we concluded that VSG genes are most likely transcribed by an RNA polymerase I-like enzyme (pol I*; 26). Pre-mRNAs in trypanosomes mature by the addition, in trans, of a small 35-nucleotide miniexon bearing a 5' cap. pol I*-transcribed mRNAs and RNA polymerase II (pol II)-transcribed mRNAs may thus carry 5' caps and have 5' ends which are indistinguishable due to the addition in trans of a 5' miniexon. To study the regulation of transcription at expression sites, we examined the transcriptional control of the expression site of the 1.8 VSG gene located on a small 430-kilobase (kb) chromosome. Separation of chromosomes by pulsed field gradient (PFG) gel electrophoresis (24) showed that chromosomal rearrangements coincide with the activation and inactivation of the 1.8 VSG gene expression site (29). The telomeric BC 1.8 VSG gene was activated when it was duplicated and transposed on a 90-kb stretch of DNA to a 340-kb chromosome, which thus became 430 kb (Fig. 1). Subsequently, the inactivation of the 1.8 ELC on the 430-kb chromosome was studied in two independent single-relapse experiments. In both instances the 1.8 ELC was retained in inactive form but translocated to a larger chromosome. Since new small (430-kb-chromosome derived) chromosomes were generated (140 kb, variant 118b'; 350 kb, variant MiTat 1.2000), the translocations of the ELC were most likely the result of the reciprocal exchange of chromosome arms (25, 28, 29).

Trypanosoma brucei is a parasitic protozoan which lives in the bloodstream of its mammalian host. In the bloodstream the parasite is covered with a dense protein coat composed of a single type of glycoprotein, the variant cell surface glycoprotein (VSG). By periodically expressing different VSG genes, T. brucei changes the antigenic identity of its cell surface coat and thus escapes the immune response of the host (8, 32, 33). Only a single VSG gene is expressed at a given time, and this gene is invariably located at one of the telomeric VSG gene expression sites (10, 20, 23, 31, 34). Three telomeric expression sites, which are located on different chromosomes, have been identified in T. brucei 427 stock 60 (25, 29, 30), and their transcription is regulated in a mutually exclusive manner. The transcriptional activation of a VSG gene can result from one of two different mechanisms. In the first mechanism, basic copy (BC) VSG genes can be translocated to an active expression site, generating a transcribed expression-linked copy (ELC). Different recombinational events, duplicative transposition (2, 3, 13, 19, 20, 22, 27, 35), telomere conversion (11), and the reciprocal exchange of telomeres (21) can mediate the transposition of BC VSG genes to the expression site. In the second mechanism, VSG genes which are already located at an expression site can become transcribed due to the activation of the expression site (for reviews, see references 6 and 7). The mechanism by which transcription at expression sites is controlled in a mutually exclusive manner remains unclear. However, we have recently shown that the duplicative transposition of the BC of VSG gene 118 in variant 118 clone 1 to its expression site results in the transcriptional activation of a cotransposed VSG gene promoter (26). Positional control is therefore an important mechanism in the regulation of VSG gene transcription. It follows that the chromosomal translocations which coincide with the activation and inactivation of expression sites may, in a similar manner, control expression site transcription through the positional control of the expression site promoter(s) (29). VSG genes, unlike other known trypanosome or eucaryotic protein-coding genes, are transcribed by RNA polymerases that are insensitive to the drug alpha-amanitin at concentrations up to 1 mg/ml (15). Since the VSG gene promoter *

MATERIALS AND METHODS Trypanosomes. The trypanosomes used in this study belong to T. brucei 427 stock 60. Variant antigen types 118b, 1.8b, and 118b' have been described previously (17, 18). The parasites were grown and isolated as described by Fairlamb et al. (12). PFG gel electrophoresis. PFG gel electrophoresis was carried out as described previously (30). Cloning of the 430-kb chromosome. Variant 1.8b trypanosomes were isolated and prepared for PFG gel electrophoresis as described by Van der Ploeg et al. (30). The gels were stained with ethidium bromide, and the 430-kb chromosome was visualized with long-wave UV light and excised from the gel. The DNA was electroeluted from the agarose in the PFG electrophoresis apparatus. The isolated chromosomal DNA

Corresponding author. 854

TRANSCRIPTS OF T. BRUCEI VSG GENE 1.8 EXPRESSION SITE

VOL. 8, 1988 118b

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FIG. 1. Schematic outline of chromosomal rearrangements which accompany activation and inactivation of thk e telomeric VSG gene 1.8. The triangles represent the 1.8 gene codin g sequence, and the bar represents the 340-kb chromosome in va liant 118b. The duplicative translocation of 90 kb of DNA to the 340-kb chromosome is indicated in the 430-kb chromosome. We id entified a total of nine mRNAs that were derived from clones trans cribed by alphaamanitin-insensitive polymerases. These are to 9.

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was digested to completion with HindlIl and ligated to HindIII-digested pBR322. This ligation mixtuIre was used to transform Escherichia coli HB101. One hundred clones were obtained from the resultant chromosome-spe cific library by hybridization with 32P-labeled 430-kb-chromc some DNA. Analysis of nascent RNA. All procedure s for the 32P labeling of nascent RNA were performed ass described by Kooter and Borst (15). Briefly, nuclei were iisolated immediately from infected rat blood by disruptinig cells with a Stansted cell disrupter and stored at -90°C. In vitro elongation of nascent RNAs was allowed to proceed for 4 to 10 min in the presence of [32P]UTP. The reaction was terminated by heating at 65°C, followed by digestion with DNase I. Nascent RNAs were hybridized to dot blots of c:loned DNA in the presence of 10% dextran sulfate and 1( )0 ,ug of yeast tRNA per ml for 48 h, followed by washing aLnd autoradiography at -90°C. RNA isolation and Northern blot analysis. Total trypanosome RNA was isolated from purified trypano somes by LiCl precipitation (1). Northern blot (RNA blot) arialysis of RNA was performed as described by Boedtker et zal. (5). Plasmid isolation and dot blots. Plasmids wrere isolated as described by Birnboim and Doly (4). Dot blotsi were made by using 2 pLg of plasmid DNA which was denatured by incubation at 60°C in 0.4 M NaOH and subsequenitly neutralized and bound to nitrocellulose filters in a Bioi-Rad dot blot apparatus.

RESULTS

Alpha-amanitin-insensitive transcription of the 430-kb chromosome. The repetitive nature of expres.sion sites have made it difficult to clone regions upstream of tthe transcribed VSG gene because in chromosome walking ione frequently jumps to homologous regions on other chroiImosomes (16). To bypass this problem we had isolated the 430-kb chromosome with the 1.8 VSG gene expression site from PFG gels and generated a 430-kb-chromosome-specifiic library (25, 29). One hundred different clones with an aveirage insert size of 2.5 kb were obtained; thus, we assume that approximately one-half of the chromosome length was repriesented in this chromosome-specific library. The clones frc)m this library were analyzed for VSG gene 1.8-specific transcription patterns. VSG genes are transcribed by polym erases that are

855

insensitive to alpha-amanitin. We therefore hybridized all 100 clones of the 430-kb-chromosome library to 32P-labeled nascent RNA probes made in the presence of 1 mg of alpha-amanitin per ml. In this way we wa4ted to identify and isolate the clones from the 430-kb-chromosome library that are transcribed by alpha-amanitin-insensitive RNA polymerases in variant 1.8b specifically. Since Cully et al. (9) had previously shown that the expression site of VSG gene 117 contain an expression-site-associated gene (ESAG), we attempted to determine whether putative ESAGs are also located on the 430-kb chromosome. Dot blots containing all of the clones d6rived from the 430-kb chromosome were hybridized with 32P-labeled nascent transcripts synthesized by run-on transcription of variant 1.8 nuclei in the presence of 1 mg of alpha-amanitin per ml. Results obtained with a representative sample consisting of 48 of these clones are shown in Fig. 2 (top left panel). Eleven clones (seven of which are indicated in Fig. 2 with arrowheads), with an average insert size of 2.5 kb, were transcribed in the presence of alpha-amanitin in nuclei from variant 1.8b only (clones hereafter referred to as alphaamanitin insensitively transcribed clones). In contrast, alpha-amanitin-insensitive transcription of these clones could not be detected with nascent RNA as probes from the variants 118b or 118b', which do not transcribe the 1.8b VSG gene (Fig. 2, top right panel, and data not shown). Hybridization of each of the eleven alpha-amanitin insensitively transcribed clones with restriction-enzyme-digested nuclear DNA showed that they all contained different, repetitive inserts. The results thus imply that a region with an overall minimum size of 27 kb (11 x 2.5) of the 430-kb chromosome is transcribed by alpha-amanitin-insensitive RNA polymerases in variant 1.8b but not in variants 118b and 118b'. As a control, nascent RNAs which were made with nuclei from variants 118b, 1.8b, and 118b' in the absence of alpha-amanitin were used as probes on the same dot blots. In each variant we now consistently found the same transcription patterns (Fig. 2, bottom left panel). Run-on transcription signals for pol I- and pol II-transcribed control rRNA, tubulin, hsp7O, and VSG genes with all three variants were as expected (Fig. 2, bottom right panel, and data not shown). Since all the 430-kb chromosome-derived clones are repetitive and some are present at as many as 60 copies per genome, these data indicate that the repetitive inserts are also part of other alpha-amanitin-sensitive transcription units in the genome. This is also true for the alpha-amanitin insensitively transcribed 430-kb-chromosome-derived clones. None of the alpha-amanitin insensitively transcribed clones hybridized with the 70-base-pair repeats that flank the VSG 1.8 gene in its expression site (data not shown). These clones must therefore be located upstream of the 70-basepair-repeat array (about 20 kb in size) which precedes the telomeric 1.8 VSG gene (data not shown; 17, 18). If the alpha-amanitin insensitively transcribed clones are linked in a single transcription unit, then the minimal length of the 1.8 VSG gene transcription unit must be 47 kb. However, because the chromosome-specific library contained only one-half of the chromosome length, the length of the transcription unit may be eveti greater than that calculated based on these experiments. The inactivation of the VSG A1.8 gene expression site coincided with chromosomal rearrangements that moved it to a larger chromosome. We determined whether deletions occurred eliminating 430-kb-chromosome-derived alpha-

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SHEA AND VAN DER PLOEG

amanitin insensitively transcribed DNA sequences, thus explaining the differences in the transcription patterns as due to loss of the genes. We tested nine alpha-amanitin insensitively transcribed clones and 25 of the alpha-amanitin sensitively transcribed clones. Polymorphic restriction fragments or copy-number alterations of restriction fragments indicative of deletions (resulting from chromosomal rearrangements) could not however be detected (data not shown). Variant-specific transcripts detected with clones transcribed by alpha-amanitin-insensitive polymerases. To ascertain whether the alpha-amanitin insensitively transcribed 430-kbchromosome-derived clones from variant 1.8b represented additional ESAGs, we determined whether variant-specific steady-state mRNAs could be detected with these clones as probes. Each of the clones detected common transcripts in both variant 118b and 1.8b. However, they also hybridized to one or more variant 1.8b-specific transcripts (Fig. 3, bands indicated with stars). We do not know whether these mRNAs have miniexons (which are common to all mRNAs of trypanosomes) spliced onto their 5' ends. These variantspecific transcripts were presumably derived from the 1.8b VSG gene expression site, since the cloned probes were isolated from the 430-kb chromosome and were transcribed only by alpha-amanitin-insensitive RNA polymerases in

variant 1.8b. None of these clones hybridized to the repetitive ESAG1 described by Cully et al. (9; data not shown). It is possible, therefore, that the variant-specific mRNAs represent additional ESAGs of the VSG gene 1.8 expression site. Because the clones contain repetitive inserts, they also hybridized to common RNA molecules which were presumably derived from other transcription units. Since these common transcripts were found in variants 118b, 1.8b, and 118b', they must have resulted from -transcription by the RNA polymerases which are sensitive to alpha-amanitin. In addition, some minor transcripts unique to either variant 118b or 1.8b were detected with one of the alphaamanitin insensitively transcribed clones (Fig. 3, probe A7). This probe also hybridized to a common transcript of variants 118b and 1.8b. The most likely explanation for this is that divergent copies of this clone can also be found in the expression site of variant 118b. Since this clone did not hybridize with nascent RNA probes from variant 118b synthesized in the presence of 1 mg of alpha-amanitin per ml, (Fig. 2, top right panel, dot A7), we conclude that the stringency difference between the Northern hybridization and the nuclear run-on hybridization and the difference in abundance of accumulated steady-state RNA in the North-

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FIG. 2. Hybridization of labeled nascent transcripts to 430-kb-chromosome-specific clones. The results for a representative sample consisting of 48 of the 100 clones tested for alpha-amanitin-insensitive transcription are shown. Run-on transcription was carried out using nuclei from variant antigen type (VAT) 1.8b (left panels) and variant 118b' (top right panel) in the presence or absence of alpha-amanitin as indicated above each panel. Dots indicated by arrowheads contained clones which were transcribed in the presence of alpha-amanitin in variant 1.8b only. Most clones were transcribed in the absence of alpha-amanitin (bottom left panel), although some clones were never transcribed (for example, bottom left panel, row D, dots 3 to 11). Control run-on transcription was carried out in the presence or absence of alpha-amanitin as indicated (bottom right panel). In the control panel, hybridization was with labeled nascent transcripts synthesized in nuclei from variant 118b'. The rRNA (dot r) control was cut off the panel without alpha-amanitin because of its great signal intensity. Only VSG gene 118 (dot v) and rRNA (dot r) transcription was unaffected by the addition of 1 mg of alpha-amanitin per ml to the nuclear run-on. Transcription of genes transcribed by pol II such as the tubulin gene (dot t) and hsp7O (dot h) was undetectable under these conditions. pBR322 (dot p), the plasmid vector into which the 430-kb-chromosome-specific library was cloned, showed no hybridization under any condition. Hybridizations were carried out at 65°C for 48 h. Posthybrizational washes were at 65°C with 3 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate).

TRANSCRIPTS OF T. BRUCEI VSG GENE 1.8 EXPRESSION SITE

VOL. 8, 1988

em blots compared with low levels of 32P-labeled run-on RNA allowed the detection of these steady-state RNAs in Northern blots only. As a control, several of the clones which were transcribed only by alpha-amanitin-sensitive polymerases (a total of eight were tested, and results for four are shown in Fig. 4) were used as probes on total RNA from variants 118b and 1.8b. None of these probes detected variant-specific transcripts, and they hybridized only to RNAs common to both variants.

DISCUSSION We present evidence showing that a large region of the 430-kb chromosome (minimally up to 47 kb upstream of the 1.8 VSG coding exon) is transcribed by alpha-amanitininsensitive RNA polymerases specifically in variant 1.8b.

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The trypanosome variants used in the comparisons differ only in the expression site which is active. We assume, therefore, that the variant-specific transcripts which resulted from transcription that is sensitive to alpha-amanitin were all expression site derived. However, due to the repetitive nature of the expression sites, this cannot be proven. For the same reason, we were unable to determine whether the alpha-amanitin insensitively transcribed clones are linked in a single polycistronic gene or are present as multiple independent genes. This does not alter the interpretation of our data, which show that many variant-specific and alphaamanitin insensitively transcribed genes exist in the genome of T. brucei. Kooter et al. (16) and Johnson et al. (14) recently determined the anatomy of the expression site of VSG gene 221 in variant 221a. Like the VSG gene 1.8 expression site, the VSG gene 221 expression site contains multiple repeats,

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FIG. 3. Northern blot analysis of clones transcribed by alpha-amanitin-insensitive polymerases. Total RNA (20 F.g each) from bloodstream-form variants 118b, 1.8b, and procyclic trypanosomes (lanes pro) was size fractionated in a 1% agarose-formaldehyde gel and transferred to nitrocellulose filters. Three 32P-labeled ESAG clones derived from the 90-kb transposed segment (probes are indicated at the tops of the panels) were hybridized at 65°C; posthybridizational washes were carried out at 65°C with 0.1 x SSC. Stars indicate transcripts that were found only in variant 1.8b. Additional common transcripts that were found in every antigenic variant and which must have been transcribed by pol II were also detected. The panel designated 1.8 cDNA was hybridized with the VSG 1.8 coding sequence. Long (left panel) and short (right panel) exposures are shown for the hybridization with probe A7.

MOL. CELL. BIOL.

SHEA AND VAN DER PLOEG

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ACKNOWLEDGMENTS

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We thank all our colleagues from the laboratory for critical reading of the manuscript. This work was supported by Public Health Service grant Al 21784 from the National Institutes of Health and a Searle Scholarship Award to L.H.T.V.D.P. We are supported by a grant from the John D. and Catherine T. MacArthur Foundation.

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FIG. 4. Northern blot analysis of clones transcribed by alphaamanitin-sensitive polymerases. Total RNA (20 ,ug each) from bloodstream-form variants 118b and 1.8b was size fractionated and transferred to nitrocellulose filters as described in the legend to Fig. 3. The probes used were clones derived from the 430-kb-chromosome-specific library which had failed to hybridize to labeled nascent transcripts synthesized by run-on transcription in variant 1.8b in the presence of 1 mg of alpha-amanitin per ml. All the clones tested were transcribed in the absence of alpha-amanitin in variants 118b, 1.8b, and 118b', however. Hybridization and posthybridizational washing conditions were as described in the legend to Fig. 3.

making it difficult to determine the origin of its variantspecific transcripts. However, the transcribed domain of the 221 expression site was determined by UV inactivation of transcription to be 60 kb. Maturation of the large premRNA, presumably through trans-splicing with the miniexon, generated multiple variant-specific stable mRNAs. A protein-coding gene (ESAG1) has been shown to be part of a third expression site in variant 117a (9) and variant 118 clone 1 (26). In addition, Shea et al. (26) previously showed that the expression site of VSG gene 118 in variant 118 clone 1 contains not one (as in the 221a expression site) but two separate polycistronic genes. We assume, therefore, that the clones encoding variant-specific mRNAs of the 430-kb chromosome are derived from a single gene or a few linked genes of the 1.8 expression site. This assumption is supported by the fact that two of these clones were shown to be cotransposed with the 1.8 VSG gene to the 340-kb chromosome when the 1.8 VSG gene was activated (data not shown). Thus, it is possible that the stable variant 1.8b- and variant 221a-specific mRNAs represent additional ESAGs of the expression sites. The function of the ESAGs in different

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