Identification of a telomeric DNA sequence in Trypanosoma brucei

Cell, Vol. 36, 447-457,

February

1984, Copyright

Q 1984 by MIT

0092-&-!674/94/020447-11

$02.00/O

Identification of a Telomeric DNA Sequence in Trypanosoma brucei Elizabeth H. Blackburn and Peter B. Challoner Department of Molecular Biology University of California Berkeley, California 94720

Summary A simple repetitive DNA sequence in the nuclear genome of Trypanosoma brucei, consisting of tandem repeats of the hexanucleotide 5’ CCCTAA 3’, was identified as being telomeric by several criteria. This sequence was specfically labeled with T. brucei genomic DNA as the template for in vitro nick translation by DNA polymerase I, and was present in Bal 31 nuclease sensitive, genomic restriction fragments of the large sizes expected in this organism for at least some telomeric regions. The same repeated sequence was found in six other flagellates tested. A segment of DNA from T. brucei including this telomeric sequence was cloned in pBR322 and characterized. The cloned segment contained a sequence highly homologous to the 3’ ends of several variant surface glycoprotein mRNAs, upstream of the tandemly repeated hexanucleotide sequence. Introduction Telomeric regions of chromosomes in the hemoflagellate Trypanosoma brucei have several interesting properties, as revealed by studies on variant surface glycoprotein (VSG) genes in both this species (reviewed in Bernards et al., 1983) and the related T. equiperdum (Longacre et al., 1983). Analyses of VSG gene families have shown that many examples of VSG gene copies occur at positions within several kb from chromosome ends, with one such telomerically located gene copy, the expression linked copy (ELC), being in a site from which it is transcribed in cells expressing that particular VSG (Williams et al., 1982; De Lange et al., 1982; Young et al., 1983; Bernards et al., 1983; Laurent et al., 1983). However, the ELC with associated telomeric DNA sequences from T. brucei has proven difficult to clone in recombinant vectors in E. coli (Van Der Ploeg et al., 1982). We decided to attempt to identify and clone telomeric sequences from T. brucei by exploiting properties we and others have found to be common to termini of chromosomal or linear DNAs from several lower eucaryotes. We reasoned that because these generalized properties, described below, are common to phylogenetitally diverse organisms, they were likely to occur in hemoflagellates such as T. brucei as well. The telomeric sequences from several lower eucaryotes have been characterized. In each case, the terminal region of the chromosome or linear DNA analyzed has been shown to consist of tandem repeats of simple sequences (reviewed in Blackburn et al., 1983). These repeats are 5’ CCCCAA 3’ for holotrichous ciliated protozoa including

Tetrahymena and Glaucoma (Blackburn and Gall, 1978; Katzen et al., 1981) 5’ CCCCAAAA 3’ for hypotrichous ciliated protozoa (Oka et al., 1981; Klobutcher et al., 1981) and 5’ CCCTA, 3’ and 5’ CIdT 3’ for the rDNAs of the slime molds Physarum and Dictyostelium respectively (Johnson, 1980; Emery and Weiner, 1981). In addition, very similar types of repeats have been found at the termini of yeast chromosomes (Szostak and Blackburn, 1982; Blackburn et al., 1983; J. Shampay, J. Szostak, and E. Blackburn, unpublished data). A second property of many of these DNA termini is the presence of single-stranded discontinuities near the molecular ends of the DNAs or chromosomes. These were shown to occur within the tandemly repeated terminal sequences, in a sequencespecific array on both strands, in Tetrahymena rDNA, and in Glaucoma (Blackburn and Gall, 1978; Katzen et al., 1981). Similar discontinuities have also been identified in the linear rDNA of Physarum (Johnson, 1980) and in a yeast chromosomal telomere cloned on a linear, selfreplicating DNA vector in yeast (Szostak and Blackburn, 1982; J. Shampay, J. W. Szostak, and E. H. Blackburn, unpublished data). These general properties-that is, the presence of single-stranded discontinuities embedded in a block of tandemly repeated simple sequences-were exploited in the method we have used to identify T. brucei telomeres. Specifically, it has been shown that if a telomeric DNA is used as a template for in vitro nick translation by E. coli DNA polymerase I in the absence of DNAase I, preferential and specific incorporation of radioactive deoxynucleotides occurs by synthesis initiated at 3’ hydroxyl groups of the single-stranded discontinuities. Addition to the in vitro labeling reaction of combinations of deoxynucleoside triphosphates appropriate to the simple repeated telomeric sequence resulted in specific labeling of one or the other strand of the telomeric restriction fragment in the cases tested (Blackburn and Gall, 1978; Johnson, 1980; Szostak and Blackburn, 1982). In order to determine whether these studies could be extended to T. brucei telomeres, we were guided by information that had emerged as a result of extensive analyses of VSG genes. Several VSG genes have been found in chromosomal loci identified as telomeric by molecular criteria (Williams et al., 1982; DeLange et al., 1982; Bernards et al., 1983). These criteria included an apparent “cluster” of restriction sites for over 20 different restriction enzymes (that is, the end of the chromosame) and susceptibility to shortening of this free end by Bal31 nuclease. If these VSG-adjoining telomeres are representative then properties expected for at least some T. brucei telomeres, based on these studies, are, first, long regions of DNA resistant to restriction enzyme digestion, as had been found between the VSG gene and the chromosomal terminus in many cases. These regions ranged from <5 to 35 kb in length (reviewed in Bernards et al., 1983). One possible reason proposed for this absence of restriction enzyme sites was that these “barren” regions consist of

Cell 448

simple repetitive sequences. Second, the presence of at least one minichromosome suggested that there may be a high number of telomeres in a T. brucei nucleus relative to the total amount of genomic DNA (Williams et al., 1982; Sloof et al., 1983). Both of these expected properties would make T. brucei a favorable system for direct analysis of telomeric sequences. In the work described in this paper, we have combined the predictions made on the basis of features expected to be common to telomeres with information specific to telomeric VSG genes in T. brucei in order to identify and clone telomeric sequences from this organism. Our results strongly support the idea that there is a common sequence containing single-stranded discontinuities at or near the ends of chromosomes in T. brucei and related hemoflagellates. This sequence consists predominantly of tandem repeats of the hexanucleotide 5’ CCCTAA 3’. One example of this sequence we have cloned lies near a VSG gene whose sequence matches those of known ELCs.

Results In Vitro Labeling of T. brucei DNA by DNA Polymerase I T. brucei DNA was labeled in vitro using nick translation conditions for E. coli DNA polymerase I holoenzyme. Since the aim of the experiments was to allow only incorporation of label by nick translation initiated at any specific endog-

enous single-stranded break of the type identified previously at the termini of Tetrahymena rDNA molecules, DNAase I was not included in the reactions (Blackburn and Gall, 1978). a=P-deoxyribonucleoside triphosphates (dNTPs) were included in the nick translation reactions in all possible combinations of one, two, three, or all four dNTPs at a time. After a short labeling reaction (1.5 minutes at 25°C) the reactions were stopped, and unincorporated dNTPs removed. The labeled DNA samples were digested with a frequently cutting restriction enzyme, and electrophoresed on an agarose gel, which was then dried and autoradiographed directly. Figure la shows the results of one such experiment. It was found that with two of the combinations of three dNTPs, several Dde I restriction fragments, from about 4-22 kb in length, were specifically labeled. No such labeling was seen with the other combinations of three or fewer dNTPs. This contrasted with the majority of Dde I restriction fragments, as visualized by ethidium bromide staining, where over 90% migrated as a broad distribution of bands less than 5 kb in length. Similar results were found using Mbo I or Hae III (Figure 1b), Hinf I, Rsa I, or Hpa II (data not shown), instead of Dde I. When all four dNTPs were included in the labeling reaction, the amount of incorporation was greatly increased, and the distribution of label in the restriction fragments followed more closely the total DNA as seen by staining with ethidium bromide (Figure 1 b). These results showed that using two out of the four

b

_

3.8

-

2.7

Dde I Figure 1. Specificity

of Labeling of T. brucei DNA by Nick Translation

(a) Equal amounts of T. brucei DNA were labeled for 15 min at 25°C by nick translation with E. coli DNA polymerase I holoenzyme, in the presence of different combinations of a32P-deoxyribonucleoside trtphosphates (dNTPs). as described previously (Blackburn et al., 1979). The labeled DNA from each reaction was then digested with the restriction enzyme Dde I, and fractionated by electrophoresis in a 9.7% agarose gel at 2 volts/cm for 15 hr. An autoradicgram of the dried gel is shown. The dNTP(s) present in the reaction shown in each lane are indicated at the top of the lane. (b) In labeling reactions similar to those shown rn (a), T. brucei DNA was labeled with E. coli DNA polymerase I using either dCTP + dATP + dTTF’, dGTP + dATP + dTTP or all four dNTPs. The single a32P-labeled dNTP present in each reaction is indicated at the tops of the lanes by an asterisk. a--dCTP was the labeled dNTP used in the reaction with all four dNTPs. The labeled DNA resulting from each reaction was digested with either Mbo I, Dde I or Hae Ill, and fractionated by electrophoresis on a 1% agarose gel. An autoradiogram of the dried gel is shown.

Telomeric 449

DNA Sequence

in T. brucei

possible combinations of three dNTPs resulted in the specific incorporation of label into genomic regions that were generally much more resistant than the total T. brucei DNA to digestion into small fragments. This is consistent with the result expected from in vitro labeling of the “barren” regions previously identified between the VSG genes and the molecular ends of the chromosomes.

Analysis of Pyrimidine Tracts Labeled In Vitro by DNA Polymerase I The strong specificity of in vitro labeling described above suggested that simple repetitive DNA sequences were being synthesized in the nick translation reactions. In particular, the increase in specific labeling upon addition of three, as opposed to two, dNTPs in the nick translation reaction was very similar to that previously observed for the Tetrahymena rDNA telomeres. In Tetrahymena rDNA, incorporation of aZP-dCTP at single-stranded breaks was greatly increased by the addition of dATP, which allowed in vitro synthesis of tandem 5’ CCCCAA 3’ repeats (Blackburn and Gall, 1978). Similarly, using the T. brucei DNA as a template, the addition of the third dNTP to make up a combination consisting of dCTP + dATP + dTTP or dGTP + dlTP + dATP resulted in greatly increased incorporation relative to any combination of two dNTPs at a time. This indicated that the appropriate combination of three dNTPs made possible nick translation through long tracts of DNA, made up of only those three bases, for each of the two combinations of three dNTPs. The simplest interpretation of three dNTPs giving increased for two combinations incorporation is that, as for Tetrahymena rDNA and yeast chromosomal telomeres, one combination allowed synthesis by copying one strand, and the other by copying the complementary strand (Blackburn and Gall, 1978; Szostak and Blackburn, 1982). Pyrimidine tract analysis of the labeled DNA confirmed these expectations. Figure 2 shows pyrimidine tract analysis performed after depurination of the labeled DNAs seen in Figure 1b. A pyrimidine tract with the composition (C,, T) was the predominant product of depurination of T. brucei DNA labeled with a3’P-dTTP plus dCTP and dATP. This pyrimidine tract was also a prominent product after labeling with all four dNTPs; a very similar result has been seen in the comparable labeling regimes used on Tetrahymena and Physarum rDNAs (Blackburn and Gall, 1978; Johnson, 1980) in which tracts of composition C4 or C3T respectively are the prominent labeled product. Similarly, labeling with dGTP + dTTP + dATP resulted in a simple pyrimidine tract pattern (Figure 2) again indicating that a simple, most probably repetitive, DNA sequence was being synthesized in this nick translation reaction. Confirming results were obtained in turn by alternating the single a=P-dNTP within the two combinations of three dNTPs. These pyrimidine tract analyses are summarized in Table 1. For this table, the predominant (which in every case was a single) product of depurination is shown for the two combinations of three dNTPs that gave specific labeling of large restriction fragments. If it is assumed that each of

the two combinations of three dNTPs label the complementary strands of the same simple repetitive sequence, 5’ CCCTAA 3’ then the unambiguous sequence 3’ GGGATT 5’ can be deduced to be the predominant sequence synthesized in vitro. This interpretation is confirmed by results described below, and is also strengthened by the similarity to previous findings for Tetrahymena rDNA, in which telomeric 5’ CCCCAA 3’ 3’ GGGGTT 5’ repeats were labeled in vitro by incorporation of only dCTP + dATP, or the complementary dGTP + dTTP (Blackburn and Gall, 1978).

ol

t

lFigure 2. Pyrimidine Translation Reactions

Tract Analysis

of T. brucei

DNA Labeled

in Nick

The four labeled DNA samples shown in Figure 1 b were each depurinated. The pyrimrdine tracts produced were fractionated by two-dimensional fingerprinting and autoradiographed. First dimension fractionation was on cellulose acetate strips at pH 3.5; second dimension fractionation was homochromatography on DEAE-cellulose thin layers as described previously (Blackburn and Gall, 1978). The major depurination product is indicated on each fingerprint. The assignment of pyrimidine tracts was made as described by Ling (1974). Table 1. Analysis of Trypanosoma brucei DNA Sequences Preferentially In Vitro by DNA Polymerase I Holoenzyme dNTPs Present in Reaction with DNA Polvmerase: Labeled a-“P-dNTP: Depurination Product(s) Labeled:

Labeled

G+T+A

C+A+T C

T

G

T

CCCTf A)

CCCT(A)

Pi

l-T(A)

Major repeat deduced: 5’ CCCTAA 3’ 3’ GGGATT 5’

Cell 450

Bal31 Nuclease Sensitivity of the DNA Regions Specifically Labeled In Vitro To test whether the specific labeling we observed was the result of incorporation into sequences near chromosome ends, unlabeled T. brucei DNA was digested with Bal 31 nuclease for increasing times. This enzyme progressively shortens chromosomal DNA molecules from their ends. Therefore, when Bal 314reated DNA is subsequently digested with a restriction enzyme, those fragments that included the original chromosome termini at one end are progressively shortened with increasing Bal 31 digestion, while internal fragments are unaffected. Accordingly, after digesting T. brucei DNA with Bal 31 for increasing times, the DNA was digested with either Dde I, Hae Ill or Hpa II, electrcphoresed on an agarose gel, and blotted by the Southern method onto duplicate nitrocellulose filters. As a hybridization probe, T. brucei DNA was labeled with a3*P-labeled dGTP + dATP + dTTP as described above, and digested with Mbo I. The digestion products were fractionated by agarose gel electrophoresis and fragments between 5 and 15 kb were eluted as a group. As shown in Figure 1 b, this size class includes virtually all the specifically labeled DNA, with the incorporated label predominantly in simple sequence DNA. We will refer to such a preparation of labeled DNA as T. brucei G+A+T probe. Figures 3a, 3b, and 3c show the result of hybridizing radioactive T. brucei G+A+T probe to the Bal 31-treated T. brucei DNA subsequently digested with Dde I, Hae Ill or Hpa II. Before Bal 31 digestion (0 min lanes), a large number of fragments hybridized to this probe. Many of these Dde I or Hae III fragments could be matched by measured sizes to those fragments specifically labeled by in vitro nick translation shown in Figure 1a or 1 b. However, the relative intensities within the banding patterns differed in many cases between the directly labeled DNA (Figure 1) and the hybridizing bands Figure 3). This result suggested that fragments that contained relatively large amounts of hybridizing DNA sequences did not necessarily have all these sequences accessible to labeling in vitro. After 5 min of Bal 31 digestion, a number of prominent hybridizing bands were lost, This finding indicated that the hybridizing sequences in these fragments were confined to regions within -1 kb from the original free ends from which Bal 31 digestion was initiated. A band initially -15 kb in length, seen most clearly in the Dde I and Hpa tl digested samples, could be seen to migrate progressively faster with increasing times of Bal31 digestion, suggesting that shortening occurred but that hybridizing sequences in this band occur several kb in from the chromosomal tern-tint in these fragments. In addition, for each restriction enzyme used, hybridizing material at limit mobility position (>30 kb) decreased in intensity as Bal 31 digestion proceeded, While this could be due to progressive shortening of these fragments from one end, in the case of such high molecular weight fragments migrating at limit mobility, the possibility cannot be excluded that they are degraded by Bal31 from internal nicks in these long regions. In control experiments,

a

0 1 5 10204060

Ddel

d 0

1

5 10 20

Ode1

b 0

C 1510204060

0

1

Haem

e 0 1

5

10204060

HpaII

f 51020

H.SeUl

Figure 3. Sensitivity to Bal 31 Nwclease of T. brucei Sequences Specifically Labeled by Nick Translation

0151020

Hpatl

DNA Hybridizing

to

T. brucei DNA was digested for the times shown in minutes above each lane with Bal31 nuclease under the conditions described by Williams et al. (1962). Aliquots of the DNA from each time point were then digested to completion with either Dde I, Hae Ill or Hpa II as indicated in the figure. After electrophoresis in a 0.7% agarose gel, the DNA was stained with ethidium bromide, photographed, and then transfered to two duplicate nitrocellulose filters by the bidirectional transfer method described by Smith and Summers (1961). DNA from one filter was hybridized to the =P T. brucei G+A+T probe, prepared as described in the text (a, b, and c), or to a T-labeled 209 bp Msp I-Xba I restriction fragment, purified from plasmid pTb17, which hybridized strongly to the T. brucei G+A+T probe (d, e, and f). The duplicate filter from each digest was hybridized to a cloned segment of the Tetrahymena thermophila rFiNA gene, spanning most of the 26s and part of the 17s rRNA coding regions (bottom parts of a, d, e, and f). The horizontal bars mark the positions of restriction fragments of lengths 15.6, 10.1, 5.5, 3.6, 2.3, and 2.1 kb, included as size markers in the gel electrophoresis. Numbers to the left of the rRNA probe hybridizations shown in the lower parts of the panels are the sizes in kb of the hybridizing bands produced in each Bal 3l/restriction digestion set.

in which a ribosomal RNA coding sequence probe was hybridized to the Bal 31 digests, no shortening of hybridizing restriction fragments was observed, although some decrease in the intensity of hybridization occurred in some experiments (Figure 3). These Bal 31 digestion experiments showed that the labeled sequences in the T. brucei G+A+T probe hybridized preferentially to many restriction fragments, which

Telomeric 451

DNA Sequence

in T. brucei

were on the average longer than the sizes of the bulk of the genomic DNA restriction fragments. Furthermore, these hybridizing fragments were preferentially sensitive to digestion or shortening by Bal 31 treatment of chromosomal DNA, as expected for sequences at telomeric loci.

Cloning and Analysis of Telomeric Sequences The strong specificity of labeling observed in preparing the T. brucei G+A+T probe suggested its use for screening putative telomeric sequences inserted into pBR322. These plasmids were constructed by a protocol similar to that previously used to clone Tetrahymena and Dictyostelium rDNA termini (Emery and Weiner, 1982; Blackburn et al., 1983; P. B. Challoner, unpublished data). Specifically, the extreme molecular termini of native T. brucei chromosomal DNA were removed by treatment with Sl nuclease. This Sl digestion, presumably by cutting at the endogenous single-strand breaks within the telomeric repeated sequence, produces a blunt end suitable for ligation to a Pvu II-generated end of the vector. The Sl -treated T. brucei DNA was subsequently digested with Mbo I, and telomeric fragments were enriched by isolating the 5 to 15 kb size class from a preparative agarose gel. These fragments were then ligated to the 2.7 kb Barn HI-Pvu II fragment of pBR322 and used to transform E. coli DHl cells. Of 536 ampicillin resistant colonies recovered in this experiment, 18 hybridized to the T. brucei G+A+T probe. The 18 plasmids with homology to the T. brucei G+A+T probe were digested with Pal I (an isoschizomer of Hae Ill), and analyzed by Southern blotting and hybridization to the same probe. While all 18 plasmids contained Pal I restriction fragments that hybridized to varying degrees, the sizes of those fragments were generally smaller than that predicted by the 5 to 15 kb size fraction used in the cloning and the absence of restriction sites at T. brucei chromosomal termini. Furthermore, several small hybridizing Pal I fragments in submolar amounts were observed in three of the plasmid DNA preparations, suggesting that the inserts in these plasmids suffered deletion during growth in the host cells. Finally, hybridization to the Pal I digested plasmids with the 540 bp Hae Ill fragment of pBR322, which contains the Pvu II site, showed that in all but five weakly hybridizing plasmids the region with homology to the T. brucei G+A+T probe was not adjacent to the Pvu II end of the vector. These results suggested that the cloning experiment did not generate the expected plasmids containing inserts of intact telomeric regions with simple repeats at one end. We therefore selected the plasmid that most strongly hybridized the T. brucei G+A+T probe, pTbl7, and the next most strongly hybridizing plasmid, pTb3, for further analysis to establish whether that hybridization was in fact because of the presence of tandem 5’ CCCTAA 3’ repeats, as deduced from the pyrimidine tract experiments. The region of pTb17 with homology to T. brucei G+A+T probe was mapped more accurately by digestion with several restriction enzymes and hybridization to that probe

(Figure 4a). The DNA sequence of this region was determined (Figure 4b). It was found to include 92 bp consisting of blocks of four and five perfect tandem repeats of 5’ CCCTAA 3’, as well as eight degenerate tandem repeats such as 5’ GCCTAA 3’ or 5’ CCTA 3’. However, as discussed in more detail below, this repeated DNA sequence did not immediately adjoin the pBR322 F’vu II site. However, the appropriate restriction fragment from pTb17 could be used as a probe for the repeated 5’ CCCTAA 3’ sequence we had inferred from the in vitro labeling experiments described above to be at telomeric loci. Knowledge of the primary sequence allowed the identification of the closest restriction sites to these tandem 5’ CCCTAA 3’ repeats in pTb17: Msp I and Xba I cut at positions 215 and 424 respectively. This 209 bp Msp I-Xba I fragment was isolated, nick translated, and used as the probe to Bal31 treated T. brucei genomic DNA as shown in Figures 3d, 3e, and 3f. The patterns of hybridization at the 0 time points of Bal 31 digestion were very similar to those seen

a

b

Figure 4. Restriction Mapping and Sequence Analysis of a Cloned Segment of T. brucei DNA Containing Telomeric DNA Sequences (a) Partial restriction map of pTb17 region hybridizing T. brucei G+A+T probe (see text). Upper lines indicate restriction fragments hybridizing the G+A+T probe. Expanded portion of map shows sequencing strategy, the location of tandem 5’ CCCTAA 3’ repeats (thick bar) and the Msp I-Xba I fragment used as the hybridization probe for experiments in Figures 3. 6, and 7. Restriction sites: D = Dde I, Hf = Hinf I, Hh = Hha I, M = Msp I, R = Rsa I, X = Xba I. (b) DNA sequence of pTbl7 region shown in (a). The Msp I-Xba I restriction fragment spanning nucleotides 215-424 is underlined. The tandem 5’ CCCTAA 3’ repeats are shown in larger letters.

Cell 452

with the T. brucei G+A+T probe shown in (a), (b), and (c). Using both probes, several hybridizing fragments were sensitive to removal or shortening with up to 5 min of Bal 31 digestion. One prominent hybridizing fragment, seen in all three restriction digests, showed progressive shortening for up to 20 min of Bat 31 treatment (Figures 3d, 3e, and 3f). The 209 bp Msp I-Xba I fragment of pTb17 contained tandem repeats of the canonical sequence 5’ CCCTAA 3’, which is the same as the major repeated sequence deduced from the pyrimidine tract analysis of the in vitro labeling experiments described above. However, because regions 215-247 and 340-424 in this fragment contained sequences other than the basic 5’ CCCTAA 3’ repeat (Figure 4b), we wished to determine whether the 5’ CCCTAA 3’ repeats were in fact responsible for the observed hybridization to Bal 31 -sensitive restriction fragments. Regions 215-247 and 340-424 contain sequences rich in homopolymeric tracts which would produce unusual pyrimidine tracts upon depurination: the tract (T12, C,) would be derived from the 215-247 region, and (C,, Tn), (Tg, C), (Tj6, C,) and (Tr, C) would be derived from the 340-424 region, these tracts together comprising 62 out of the total 117 bp of these non-5’ CCCTAA 3’ repeat regions. Tracts of such unusual composition would have been clearly distinguishable in the pyrimidine tract analyses carried out in the in vitro labeling experiments and were not detected. Furthermore, the Dde I fragments from positions 1 to 274 and 287 to 900 (Figure 4b) each hybridized strongly to the T. brucei G+A+T probe, and the only sequence common to both fragments is the 5’ CCCTAA 3’ repeat. Since the 209 bp Msp I-Xba I fragment also hybridized strongly to the T. brucei G+A+T probe, which as described earlier contains a predominant 5’ CCCTAA 3’ repeat, we conclude that any common hybridization behavior of the pTbl7 Msp I-Xba I fragment and the T. brucei G+A+T probe is attributable to tandem repeats of the sequence 5’ CCCTAA 3’. Further data confirming this conclusion was obtained by analysis of a second recombinant plasmid, pTb3, which was selected by the same screening procedure as pTbl7. This plasmid also hybridized to several of the large (>5 kb) genomic fragments in common with the pTbl7 Msp lXba I fragment and the T. brucei G+A+T probe, as well as to lo-15 lower molecular weight (<5 kb) bands not hybridized by the latter two probes, Bal 31 digestion of T. brucei DNA, followed by restriction with Hpa II or Hae Ill and hybridization to the pTb3 insert, showed that the high molecular weight (>5 kb) fragments were sensitive to Bal 31 digestion, whereas the IO-15 low molecular weight fragments were not digested by Bal 31 (data not shown). Preliminary sequence analysis of a 500 bp fragment from the insert of pTb3 that hybridized to the T. brucei G+A+T probe showed that it contained at least four tandem 5’ CCCTAA 3’ sequences, flanked on one side by se quences different from those flanking the 5’ CCCTAA 3’ repeats in pTb17. Again, as in pTbl7, the 5’ CCCTAA 3’

repeats did not abut the Pvu II site of the pBR322 vector, suggesting these repeats were not cloned directly from a free chromosomal end. To summarize, each of the three probes described hybridized to a common set of high molecular weight, Bal 31 -sensitive genomic restriction fragments. Because these three probes had as their only common identifiable sequence tandemly repeated 5’ CCCTAA 3’, we conclude that this sequence is enriched in telomeric fragments of T. brucei genomic DNA. However, the results obtained from sequence analysis of pTb17 and pTb3, and the finding that the hybridization signal to at least one genomic restriction fragment persisted through Bal 31 shortening for up to 5 kb, argue that blocks of this repeated sequence are also located some distance in from chromosomal ends, and are flanked by additional DNA sequences. Finally, a necessary series of control experiments we carried out involved eliminating the possibility that the simple repeated sequence we identify as telomeric was derived from kinetoplast DNA (Fairlamb et al., 1978; Chen and Donelson, 1980). Minicircle DNA could be identified as an ethidium bromide staining band of the expected size in agarose gels. This band did not hybridize to pTbl7, and did not hybridize preferentially to T. brucei G+A+T probe. To eliminate the possibility that the hybridizing high molecular weight bands seen after restriction digestion might be arrays of catenated minicircles lacking sites for the restriction enzyme used, these restricted DNAs were treated with T4 topoisomerase II or gyrase under conditions in which an interlocked pair of circular DNA molecules added to the reaction was completely converted to free single circles. No effect on the electrophoretic mobility of telomeric restriction fragments was found with either topoisomerase II or gyrase, consistent with their being linear fragments, Furthermore, where the DNA sequence of minicircles has been determined, no tandem repeats of the sequence 5’ CCCTAA 3’ have been found (Chen and Donelson, 1980). Lastly, the sizes of the genomic restriction fragments hybridizing to telomeric sequences were too large to be derived from maxicircle DNA. pTb17 Includes and Extends a Region Homologous to VSG mRNAs The sequence of pTb17 was compared with those previously determined for several VSG mRNAs and VSG basic copy genes. While none of these VSG genes contained tandemly repeated 5’ CCCTAA 3’ sequences, the pTb17 sequence complementary to that shown in Figure 4b, from positions 469 to 654, was highly homologous to the 3’ portion of several VSG mRNAs (Figure 5a). The sequence in pTbl7 contained an open reading frame encoding 111 residues between positions 900 (the furthest base determined) and 565. The 3’ end of this sequence (bp 579-469) was very similar to both the region preceding and at the stop codon (bp 579-562) and through the entire 3’ untranslated region (bp 561-469), of all characterized VSG mRNAs.

Telomerii 453

DNA Sequence

in T. brucei

Figure 5. VSG Homology

C

From positions 654 to 562, the sequence of pTb17 was also homologous to the sequences of cDNAs encoding Subset 2 VSGs as defined by Borst and Cross (1982). The sequence from bp 615 to 562 encoded a C-terminal hydrophobic extension highly homologous to those seen in Subset 2 VSGs, and the sequence from bp 654 to 616 encoded an amino acid sequence very similar to the Cterminal portion of the mature protein of Subset 2 VSGs (Figure 5b). Inspection of the sequence of pTb17 therefore strongly suggests that it encodes a functional VSG in Subset 2, since all the highly conserved features of these genes are present. Thus the 3’ untranslated region, and coding sequences for the hydrophobic C-terminal extension, the processing site for cleavage to produce the correct C-terminal end of the mature protein, and a similar (though not identical) distribution of four cysteine residues in a region of hydrophilic amino acids (bp 783-631) are all features of pTb17 that are shared with Subset 2 VSG genes. The only exception to the similarity in conserved features is the absence of an asparagine residue (a glycosylation site in Subset 2 VSGs) at the fifth position in from the C-terminus of the mature protein; this is replaced by a serine residue in the pTbl7 sequence (Figure 5b). A map summarizing the organization of the VSG cDNA homologous region and 5’ CCCTAA 3’ repeats in pTb17 is shown in Figure 5c. These findings indicated that the sequence cloned in pTb17 includes a VSG expression linked gene copy, although there is no direct evidence for its being transcribed

in pTb17

(a) The complementary strand of the pTb17 sequence from bp 900 to 465 in Figure 4b is shown below the corresponding amino acids encoded by a 333 bp open reading frame. Homologous regions from all Subset 2 VSG cDNAs (lITat 1 .I from Rice-Ficht et al.. 1961; TXTat 1 from Merritt et at.. 1963; MfTat 1.2 from Majumder et al., 1961) and representative Subset 1 VSG cDNAs (IlTat 1.3 from Rice-Ficht et al., 1961; AnTat 1 .l and AnTat 1.6r from Matthyssens et al., 1961) are given below the pTb17 sequence. The alignment of the 3 nontranslated section from bp 562 to 465 follows that suggested in Borst and Cross (1962). Vertical line denotes C-terminus of mature proteins. Cysteine residues in locations common to VSG proteins are boxed. See text for a detailed discussion of VSG features shared by pTb17. (b) C-terminal ends of mature Subset 2 VSG proteins (left of vertical line) and hydrophobic C-terminal extension (right of vertical line) are afigned relative to the amino acid sequence predicted from pTbl7. MlTat 1.1 and MlTat 1.7 protein data from Holder and Cross (1961). (c) Map of pTb17 sequence. Thick sokd line = 333 bp open reading frame. Thick open line = homology with 3’ nontranslated portion of VSG cDNAs. Alternating thin solid and thin open line = blocks of alternating 64-65 %GC and O-26 %GC sequence respectively. C = cysteine residue. C term = C-terminus of mature protein predicted by analogy to known VSG proteins.

in the T. brucei stock from which it was isolated. The sequence we have determined in pTbl7 extended past the poly (A) addition site (Figure 5~). This neighboring sequence was very rich in runs of T residues on the sense strand downstream of the poly (A) addition site (370-470 in Figure 4b). If the sequence we have cloned is at the VSG expression site, such a sequence may act as a termination signal for RNA polymerase II, in which case the 5’ CCCTAA 3’ repeats downstream of the region homologous to VSG mRNA may not be transcribed. Therefore, because the 209 Msp I-Xba I fragment begins 44 bp away from the closest polyadenylation site, the hybridization results seen using this probe are unlikely to be attributable to VSG-specific sequences.

Presence of the Telomeric Repeat of T. brucei in DNAs of Other Protozoans DNAs from several other protozoans were screened for the presence of repeated 5’ CCCTAA 3’ sequence by hybridization with two probes. The T. brucei G+A+T probe and the 209 bp Msp I-Xba I fragment from pTb17 were hybridized to DNAs of Trypanosoma brucei, Tetrahymena thermophila, Giardia lamblia, Trichomonas vaginalis, Euglena, Crithidia fasciculata, Leishmania tropica, Leishmania tarantolae, Trypanosoma cruzi, and Leptomonas. Strong hybridization under stringent conditions was observed with DNAs of all the flagellates except Giardia and Trichomonas, and no hybridization to Tetrahymena DNA was detected (Figure 6). C. fasciculata, L. tropica, L. tarantolae, T. cruzi,

Cell 454

a

b

BglII 123456789lO

123456789x)

EtBr Figure 6. Hybridization

T. brucei of T. brucer Telomeric

123456789x)

12345678910

G+A+T

pTb17

Rsal

pTb17

Mspl-Xbal

Probes to DNAs of Several Flagellated Protozoans

and Tetrahymena

Mspl-Xbal

thermophila

DNA purified from T. brucei (lane I), T. thermophila (lane 2) Giardia lamblia (lane 3) Trichomonas vaginalis (lane 4) Euglena (lane 5) Crithidia fasciculata (lane 6) Leishmania tropkx (lane 7) Leishmania tarantolae (lane 8) T. cruzi (lane 9) and Leptomonas (lane 10) was diisted with Bgl II (a) or Rsa I (b). The DNA was fractionated by electrophoresis in a 0.7% agarose gel, stained with ethidium bromide and photographed with 366 nm illumination (EtBr panel). The digested DNA was then blotted to duplicate nitrocellulose filters and hybridized to either YP-labeled T. brucei G+A+T probe (indicated as T. brucei G+A+T in the figure) or the Msp I-Xba I fragment of pTb17 shown in Figure 4 (indicated as pTb17 Msp I-Xba I in the figure). Horizontal bars show the positions of the size markers as described in the legend to Figure 3.

and Leptomonas are in the same taxonomic order, Kinetoplastida, as T. brucei; Euglena belongs to a different flagellate class. Since, as described above, the pTb17 Msp I-Xba I fragment and the T. brucei G+A+T probe have the tandemly repeated sequence 5’ CCCTAA 3’ in common, we conclude that this repeated sequence is highly conserved in all the Kinetoplastidae tested, and in Euglena and therefore possibly other flagellates as well. To determine whether this hybridization to DNAs of other flagellates was occurring to sequences at telomeric loci, we tested the effect of digestion with Bal 31 on DNAs of T. cruzi and C. fasciculata. Following Bal 31 digestion for 0, 1, 5, and 20 min under the same conditions as those used for T. brucei DNA (see Experimental Procedures), the DNAs were digested with Bgl II and hybridized to the Msp I-Xba I fragment of pTb17. All hybridization was lost after 1 min of Bal 31 digestion of T. cruzi DNA, and the hybridizing fragments of C. fasciculata and T. brucei DNA were shortened and decreased in intensity in the 20 min of digestion (Figure 7). In control experiments, a cloned DNA containing an evolutionarily conserved region of the 26s rRNA gene of T. thermophila was hybridized to duplicate Southern blots of these Bal 31 treated DNAs; no decrease in size and little loss of the hybridizing restriction fragments occurred after 20 min of Bal31 digestion (Figure 7). It therefore can be concluded that repeated 5’ CCCTAA 3’ sequences are found in telomeric regions in the genomes of these two flagellates. Consistent with this interpretation, in hybridizations to the smaller (4.5 kb) frag-

ments of L. tropica, L. tarantolae, T. cruzi, and Leptomonas (Figures 6a and 6b, lanes 7-l 0), the hybridizing fragments were in many cases seen to migrate as heterodisperse bands in gel electrophoresis. This was especially evident in the Rsa I digested samples seen in Figure 6b, lanes 710. The rDNA telomeres of Tetrahymena (Blackburn and Gall, 1978) Physarum and Dictyostelium (Emery and Weiner, 1981) and yeast chromosomal telomeres (Szostak and Blackburn, 1982) are also found as heterodisperse fragments following restriction endonuclease digestion. A strikingly higher total amount of hybridization was observed when T. brucei DNA was probed with telomeric DNA sequences, compared to similar amounts of other protozoan DNAs (Figures 6 and 7). Most of the hybridization to T. brucei DNA was to very high molecular weight (>25 kb) Bgl II fragments. A similar result was found when the protozoan DNAs were digested with the frequently cutting enzyme Rsa I and hybridized to the T. brucei telomeric probes (Figure 6b). Because T. brucei has minichromosomes containing VSG genes flanked by long “barren” regions (Williams et al., 1982; Sloof et al., 1983) this result suggests that at least some of these high molecular weight hybridizing restriction fragments may be contributed by minichromosomes. Discussion The experiments described in this paper were designed to exploit known properties of the telomeres of several lower

Telomeric 455

DNA Sequence

in T. brucei

Figure 7. Sensitivity to Bal 31 Nuclease of DNAs from T. brucei, T. cruzi, and C. fasciculata Hybridizing to T. brucei Telomeric Sequence Equal amounts of DNAs from Trypanosoma brucei (left four lanes), Trypanosoma cruzi (middle four lanes), or Crithidia fasciculata (right four lanes) were digested with Bal 31 nuclease for the times shown in minutes at the top of each lane. The Bal 31 treated DNA samples were then digested to completion with Bgl U, blotted to duplicate nitrocellulose filters after electrophoresis through a 0.7% agarose gel, and hybridized to the Msp I-Xba I fragment of pTbl7. Duplicate filters were then hybridized to the ‘P-rRNA coding region probe from Tetrahymena described in the legend to Figure 3. The bands hybridizing to this probe are shown in the lower pan of the panel and their sizes in kb are indicated on the lower left of the figure. Horizontal bars indicate the positions of size markers as described in the legend to Figure 3.

eucaryotes in order to identify the telomeric sequences of the hemoflagellate Trypanosoma brucei. The similarity between some of the results described here and those found for telomeric DNAs of other lower eucaryotes support the idea that we have identified a repeated sequence found at T. brucei telomeres. Furthermore, these results are in agreement with the previously determined structure of telomeric loci carrying VSG genes in T. brucei. In these cases, a region several kb in length between the VSG gene and the terminus of the chromosome was found to be resistant to digestion by many restriction enzymes. The sequence we identified by in vitro labeling also hybridized to similarly resistant genomic fragments. The degree of Bal 31 sensitivity we observed for restriction fragments containing the telomeric sequence was very similar to that previously observed for telomeric regions near VSG genes. Measuring over the first two 20 min intervals in out Bal 31 digestions, the rate of shortening observed was loo-150 bp/min. This rate is not significantly different from the rates seen by Williams et al. (1982) and De Lange et al. (1982) for Bal 31 digestion, under the

same conditions, of regions 3’ to the VSG genes they examined. Studies on VSG genes of T. brucei and T. equiperdum at telomeric loci (Bernards et al., 1983; Longacre et al., 1983) have suggested that the DNA between the VSG genes and the chromosome ends not only lacks restriction enzyme cutting sites, but is also resistant to cloning using cosmid or X vector systems. We have observed deletions of T. brucei DNA containing 5’ CCCTAA 3’ repeats inserted in the plasmid vector pBR322. Such deletions would be consistent with selection against this sequence in either cosmid or X vectors, where a minimum insert size is required for packaging in the X phage capsid. However, we do not know whether long blocks consisting of the predominant 5’ CCCTAA 3’ repeat are found in the T. brucei genomic DNA, nor whether the “barrenness” of the telomeric sequences adjacent to VSG genes is attributable solely to the presence of simple tandemly repeated sequences Our results would suggest that this is not necessarily the case, since the two cloned examples of repeated 5’ CCCTAA 3’ we identified had different flanking sequences (one being the 3’ end of a VSG gene). While the sequence from bp l-467 in pTb17, which would be distal to the VSG gene in the chromosome, is notable for its high proportion of homopolymeric tracts, it does contain several restriction sites (see Figure 4b). Therefore, such a sequence cannot alone account for the observed resistance to restriction enzyme digestion of regions 3’ to VSG genes at telomeric loci. It has been suggested that T. equiperdum sequences between many VSG genes and chromosome ends contain an as yet unidentified modified base (Raibaud et al., 1983); our data would be consistent with this if such a base prevents cutting by restriction enzymes. Despite the finding that the 5’ CCCTAA 3’ repeats in pTbl7 are not next to the vector sequence, in contrast to the simple repeated telomeric sequences from Tetrahymena and Dictyostelium cloned by similar procedures, it is likely that the construction of pTb17 did result in a telomeric region being cloned, since pTb17 contains a VSG gene sequence closely resembling that of the ELC. Such VSG sequences have previously been’ mapped to telomeric regions, although up to several kb of DNA have been found between the telomeric VSG gene copy and the end of the chromosome in the cases analyzed. In contrast, in some of the genomic fragments hybridizing to 5’ CCCTAA 3’ repeats, the hybridization signal was lost after Bal 31 digestion of cl kb of sequence, suggesting that in these fragments the 5’ CCCTAA 3’ repeats occurred much closer to the chromosomal ends than VSG gene copies. However, hybridization to at least one restriction fragment, and over a broad size range in the Southern blot pattern in general, did not disappear until after 20 min of Bal 31 digestion. Therefore, two types of hybridization behavior could be distinguished, suggesting that 5’ CCCTAA 3’ repeats occur both within 1 kb of chromosome termini, and at positions several kb in from the chromosome ends. Consistent with previous findings on ELCs, pTb17 may include

an example

of the

latter

situation.

Cell 456

The conservation of the repeated telomeric sequence 5’ CCCTAA 3’ in all but two of the flagellate species examined strongly argues that it is important for proper functioning of telomeres in these organisms, rather than being involved specifically in the structure or expression of VSG genes. Where telomeric sequences have been identified in other lower eucaryotic systems, all chromosomal molecules have been found to share the same telomeric sequence at their extreme molecular termini. Therefore, if T. brucei is similar in this respect to the other species analyzed, the 5’ CCCTAA 3’ repeat sequence we identify may be common to all telomeres in this organism. Recently, it has been found that during growth of the infectious form of T. brucei, the lengths of the telomeric regions of two examples of chromosomes bearing a VSG gene increased in a regular manner (Bernards et al., 1983). These increases were estimated at 10 and 7 bp per generation for the two chromosome ends. Since these estimates are an average increase per cell cycle in a population of cells, it is possible that they reflect incremental increases in the number of 5’ CCCTAA 3’ basic repeats, perhaps of one or two repeat units per generation. The results described in this paper provide further evidence for the very similar structure of all telomeric or terminal sequences of linear or chromosomal DNAs thus far examined in a wide variety of lower eucaryotes, implying that variations in telomere sequences may occur only infrequently during evolution. Hence all of the telomeric sequences analyzed thus far can be written in the general form!Y’C,F, 3’, where n is always >I for at least r 01 some of the tandem repeats and m = 1-4 (Blackburn et al., 1983; J. Shampay, J. W. Szostak, and E. H. Blackburn, unpublished data). These simple repeats all contain short clusters of contiguous cytosine residues on the strand with 5’ + 3’ polarity towards the interior of the chromosome (Blackbum et al., 1983). This generality of the type of sequence at telomeres strongly suggests that there is a recognizable DNA structure conferred by this type of repetitive sequence which is required for telomere function. Furthermore, the results presented here indicate that the properties of telomeric DNAs may be general enough to make the telomeric sequences of other eucaryotes amenable to identification and isolation by the same experimental approach we have used for T. brucei telomeres. While the main technical difficulty that can be envisioned is the extent of background labeling compared to specific labeling, the use of Bal 31 sensitivity as a diagnostic property of sequences near free chromosome ends should make this combined approach feasible for identifying the telomeric sequences of other eucaryotes. Experimental

Procedures

Preparation of DNA from Trypanosoma brucei and Other Protozoans Whole cell DNAs from Trypanosoma brucei, stock EATRO 110, Leishmania tropica strain 252, Iran, Leishmania tarantolae, Trypanosoma cruzi (Peru), Leptomonas. Grardia lamblia (Portland I), Trichomonas vaginalis ATCC

3001, Euglena and Crithidia fasciculata, were all generously provided by Steven Beverley. T. brucet DNA was further purified to remove kinetoplast DNA by equilibrium density gradient centrifugation in a Hoechst 33258. CsCl gradient. Gradients were prepared to the same final concentrations of components and centrifuged, as described by Wild and Gall (1979). Kinetoplast DNA (G+C content -28% [Chen and Donelson, 19801) banded separately from nuclear DNA and the latter was removed using a syringe. Macronuclear DNA from Tetrahymena thermophila was purified as described by Yao and Gall (1977). Digestions with Bal31 and Restriction Enzymes T. brucei DNAs were digested with Bat 31 from Bethesda Research Laboratories using 3.75 U enzyme/l0 rg DNA at 30°C. Conditions were essentially as described by Williams et al. (1982). Restriction enzyme digestions were carried out according to the manufacturer’s instructions. In Vitro Labeling with DNA Polymerase I, Pyrimidine Tract Analysis, and DNA Sequence. Analysis These were carried out as described by Blackburn and Gall (1978) and Blackburn and Chiou (1981) for Tetrahymena rDNA. Pyrimidine tract anafysis was performed as described by Ling (1974) and Blackburn and Gall (1978). DNA sequence determination was performed according to Maxam and Gilbert (1980). Recombinant DNA Plasmids containing T. brucei telomeric sequences were constructed as described in the text and previously in Emery and Weiner (1982) and Blackburn et al. (1983). Si nuclease from Bethesda Research Laboratories and T4 DNA ligase from New England Biolabs were used in the construction according to the manufacturers’ conditions. Standard procedures, described in Maniatis et al. (1982) were used in the screening and manipulation of these plasmids. Southern Blotting and Hybridizations After size fractionation by agarose gel electrophoresis, DNA was transferred to duplicate nitrocellulose filters as described by Smith and Summers (1981). Hybridizations were performed with probes nick translated as described by Maniatis et al. (1975) in 4X SSC (SSC = 0.15 M NaCI. 0.015 M sodium citrate [pH 7.01) and 1X Denharcft’s solution (Denhardt, 1972) at 65OC, for 17 to 20 hr. Post-hybridization washes to remove unspecifically bound probe were in 4X SSC, 0.5% sodium dodecyl sulfate, at 65’C for 3-5 hr, followed by l-2 hr at 42°C in 0.1X SSC, 0.1% sodium dodecyl sulfate. Acknowledgments We are indebted to Steven Beverley for very generous gifts of DNAs purified from Trypanosoma brucei and other protozoans. This research was supported by grant no. GM25926 from the National Institutes of Health to E. H. 8. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to Indicate this fact. Received

September

30, 1983; revised

December

1, 1983

Bernards, A., Michels, P. A. M., Ltncke, C. R., and Borst. P. (1983). Growth of chromosome ends in multiplying trypanosomes. Nature 303. 592-597. Blackburn, E. H., and Gall, J. G. (1978). A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 1.20, 33-53. Blackburn. E. H.. and Chiou, S. S. (1981). Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena. Proc. Nat. Acad. Sci. USA 78. 22632267. Blackburn,

E. H., Budarf, M. L., Challoner,

P. B., Cherry

J. M., Howard,

E.

Telomeric 457

DNA Sequence

in T. brucei

A., Katzen, A. L., Pan, W.-C., and Ryan, T. (1983). DNA termini in ciliate macronuclei. Cold Spring Harbor Symp. Quant. Biol. 47, 1195-1207.

homologres near the C-termini of the variable Trypanosoma brucei. Nature 294, 53-57.

Borst, P., and Cross, G. A. M. (1982). antigenic variation. Cell 29, 291-303.

Sloof, P., Menke, H. H., Caspers, M. P. M., and 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 minichromosomal DNA fraction. Nucl. Acids Res. 7 7, 3889-3901.

Molecular

basis for trypanosome

Chen, K. K., and Donelson, J. E. (1980). Sequences of two kinetoplast DNA minicircles of Trypanosome brucei. Proc. Nat. Acad. Sci. USA 77, 2445-2449. DeLange, T., and Borst, P. (1982). Genomic environment linked extra copies for surface antigens of Trypanosoma the end of a chromosome. Nature 299, 451-453.

of the expressionbrucei resembles

Emery, H. S., and Weiner, A. M. (1981). An irregular satellite sequence is found at the termini of the linear extrachromosomal rDNA in Dictyostelium discoideum. Cell 26. 41 l-41 9. Fairlamb, A. H., Weislogel, P. O., Hoeijmakers, J. H. J., and Borst, P. (1978). Isolation and characterization of kinetoplast DNA from blood stream form of Trypanosoma brucei. J Cell Biol. 76, 293-309. Holder, A. A., and Cross, G. A. M. (1981). Glycopeptides from variant surface glycoproteins of Jrypanosoma brucei. C-terminal location of antigenitally cross-reacting carbohydrate moieties. Mol. Biochem. Parasit. 2, 135-150. Johnson, E. M. (1980). A family of inverted repeat sequences and specific srngle-strand gaps at the termini of the Physarum rDNA palindrome. Cell 22,075-806. Katzen, A. L., Cann, G. M., and Blackburn, E. H. (1981). Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate. Cell 24, 313320. Klobutcher, L. A., Swanton, M. A., Donini, P., and Prescott, D. M. (1981). All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3’ terminus. Prcc. Nat. Acad. Sci. USA 78.30153019. Laurent, M., Pays, E., Magnus, E., Van Meirvenne, N., Matthyssens, G., Williams, R. O., and Steinert, M. (1983). DNA rearrangements linked to expression of a predominant surface antigen gene of trypanosomes. Nature 302,263-266. Longacre, S., Raibaud, A., Hibner. U., Buck, G., Eisen, f-l., Baltz, T., Giroud, C., and Baltz, D. (1983). DNA rearrangements and antigenic variation in Trypanosoma equiperdum. Expression independent DNA rearrangements in the basic copy of a variant surface glycoprotein gene. Mol. Cell Biol. 3, 410-414. Majumder, H. K., Boothroyd, J. C., and Weber, H. (1981). Homologous 3’. terminal regions of mRNAs for surface antigens of different antigenic variants of Trypanosoma brucei. Nucl. Acids. Res. 9. 4745-4753. Maniatis, T., Jeffrey, A., and Kleid, D. G. (1975). Nucleotide sequence of the rightward operator of phage X. Proc. Nat. Acad. Sci. USA 72, 11841188. Maniatis, T., Fritsch, E. F.. and Sambrook, Laboratory Manual. (Cold Spring Harbor, Laboratory).

J. (1982). Molecular Cloning: A New York: Cold Spring Harbor

Matthyssens, G., Michiels, F., Hamers. R., Pays, E., and Steinert, M. (1981). Two vanant surface glycoproteins of Tfypenosoma brucei have a conserved C-terminus. Nature 293, 230-233. Maxam, A., and Gilbert, W. (1980). Sequencing end-labeled specific chemical cleavages. Meth. Enzymol. 65, 499560.

DNA with base-

Merritt, S. C., Tschudl, C., Konigsberg, W. H., and Richards, F. F. (1983). Reverse transcription of trypanosome variable antigen mRNAs initiated by a specific oligonucleotide primer. Proc. Nat. Acad. Sci. USA 80, 15361540. Oka, Y., Shiota, S., Nakai, S.. Nishida, V., and Okubo, S. (1980). Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata. Gene 10, 301-306. Raibaud, A.. Gaillard, C., Longacre, S., Hibner, U., Buck, G.. Bernardi, G., and Eisen, H. (1983). Genomic environment of variant surface antigen genes of Trypanosoma equip&urn. Proc. Nat. Acad. Sci. USA 80,43064310. Rice-Ficht,

A. C., Chen,

K. K., and Donelson,

J. E. (1981).

Sequence

surface

glycoprotetns

of

Smith, G. E., and Summers, M. D. (1980). The bidrrectronal transfer of DNA and RNA to nitrocellulose of diazobenzyloxymethyl-paper. Analyt. Biochem. 109,123-129. Szostak, J. W., and Blackburn, E. H. (1982). Cloning yeast telomeres linear plasmid vectors, Cell 29, 245-255.

on

Van Der Ploeg, L. H. T., Valerio, D., Delange, T., Bemards, A., Borst, P., and Grosveld. F. G.. (1982). An analysis of cosmtd clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glyco: proteins are clustered In the genome. Nucl. Acids Res. 70, 5905-5923. Wild, M. A., and Gall, J. G. (1979). An intervening sequence in the gene codtng for 25s ribosomal RNA of Tetrahymena pigmentosa. Cell 16, 566 573. Williams, R. O., Young, J. R., and Majiwa, P. A. 0. (1982). Genomic environment of T. brucei VSG genes: presence of a minichromosome. Nature 299, 417-421. Yao, M.-C., and Gall, J. G. (977). A simple integrated gene for ribosomal RNA in a eucaryote, Tetrahymena pyriformis. Cell 12, 121-l 32. Young, J. R., Shah, J. S., Matthyssens, G., and Williams, R. 0. (1983). Relationship between multiple copies of a T. brucei variable surface glycoprotein gene whose expression is not controlled by duplication. Cell 32, 1149-1159.