Proc. Natl. Acad. Sci. USAVol. 80, pp. 4634-4638, August 1983Biochemistry

Tubulin genes of Trypanosoma brucei: A tightly clustered family ofalternating genes

(sleeping sickness/microtubules/hemoflagellates)

THOMAS SEEBECK*, PAUL A. WHITTAKERt, MARTIN A. IMBODEN*, NORMAN HARDMANt, ANDRICHARD BRAUN**Institut fur Allgemeine Mikrobiologie, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland; and tDepartment of Biochemistry, Mariscal College,University of Aberdeen, Aberdeen AB9 lAS, Scotland

Communicated by Ewald R. Weibel, April 15, 1983

ABSTRACT African trypanosomes are the causative agents ofmany medically and economically important diseases of man anddomestic animals. The cell body of these blood-dwelling protozoais enveloped with a dense layer of pellicular microtubules, whichconfer both motility and mechanical stability on these cells; mi-crotubules are also important components of the flagellum. Themajor structural components of the microtubuli are two relatedproteins, a- and fl-tubulin. We have analyzed the genomic or-ganization of a- and f-tubulin genes in Trypanosoma brucei. Inthis organism, the majority of these genes are arranged in a sin-gle, tightly packed cluster of alternating a- and j3-tubulin genes,with a basic repeat length together of 3.6 kilobase pairs. A ge-nomic library of T. brucei was constructed by using the phage vec-tor A 1059, and recombinant phages carrying tubulin genes wereisolated by screening the library with heterologous chicken tu-bulin cDNA probes. The results of restriction endonuclease andhybridization analysis of DNA isolated from recombinant phages,and subcloned fragments thereof, were compatible with the re-striction maps derived from digestion and Southern blot hybrid-ization of genomic DNA.

Several members of the protozoan genus of Trypanosoma arepathogenic parasites of a wide variety of species, including man.African trypanosomes are the causative agents of human sleep-ing sickness (1-3) as well as of various economically disastrousdiseases of domestic animals (4). Animal trypanosomiasis pres-ently restricts cattle production in infested areas of Africa toless than 15% of the carrying capacity of the land. Thus, thevarious forms of trypanosomiasis constitute a major obstacle tothe economic and social development of much of sub-SaharanAfrica.We are currently studying the microtubular system of try-

panosomes in view of its potential as a target structure forchemotherapeutic attack. The cell body of trypanosomes is tightlyenveloped by a regular array of pellicular microtubules (5), whichconfer both motility and mechanical stability on these cells. Inaddition, microtubules are prominently involved in the struc-ture of the flagellum, where they are arranged in the canonical9 + 2 pattern.

The principal components of microtubules are two relatedbut distinct polypeptides, a- and f3-tubulin. Microtubules areformed from these subunits by a reversible polymerization re-action of a/f3-tubulin heterodimers (6). The tubulin proteinshave been strongly conserved throughout the evolution of eu-karyotes. For instance, hybrid microtubules can readily be cre-ated in vitro by copolymerization of tubulins from differentspecies (7); furthermore, sequence analyses of tubulin proteins

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(8, 9) and tubulin genes (10-14) have confirmed a high degreeof evolutionary conservation.

All eukaryotic organisms analyzed so far carry more than onecopy of both a- and ,-tubulin genes (15-19). The individualgenes are unlinked and dispersed throughout the genome (20).In the human genome, several tubulin pseudogenes have beendetected in addition to the families of functional tubulin genes(11, 12).

In this paper, we describe the organization of the tubulingenes in Trypanosoma brucei. In this organism, a- and ,B-tu-bulin genes are also present in multiple copies but, unlike thesituation in most other organisms, the majority of these genesare arranged within a tightly packed cluster of alternating a-and f3tubulin genes.

MATERIALS AND METHODSGrowth of Cells and Preparation of DNA. Procyclic try-

panosomes (Trypanosoma brucei brucei, stock STIB 366) weregrown in SDM-79 medium (21) at 26°C. High molecular weightDNA was prepared by digesting whole cells in 10 vol of 500mM Tris HCI, pH 9.5/200 mM EDTA/4% Sarkosyl/0.5 mg ofproteinase K per ml for 5 hr at 37°C. The viscous lysate wascentrifuged through a sucrose step gradient (50%, 20%, and15% sucrose in 800 mM NaCI/20 mM Tris HCI, pH 8.0/10 mMEDTA) in a Beckman SW 27 rotor at 25,000 rpm and 20°C for3 hr. DNA was collected from the 50% sucrose cushion, con-centrated by dialysis against solid polyethylene glycol 6,000,and further purified by CsCl density gradient centrifugation.

Restriction Mapping and Southern Blot Analysis. DNA wasdigested with restriction endonucleases (Boehringer Mann-heim and Bethesda Research Laboratories) under the condi-tions suggested by the suppliers. After electrophoretic sepa-ration on agarose gels, duplicate Southern blots of each gel wereprepared (22) and were analyzed with the appropriate 32P-la-beled DNA probes.

Construction of a Genomic DNA Library from T. brucei.High molecular weight trypanosomal DNA was digested to1/200th, 1/100th, and 1/50th of completion with Sau3A. The12- to 20-kilobase pair (kb) size class of fragments was isolatedand cloned in phage A 1059 (23) by using published procedures(24). A total of 1.2 x 106 recombinants was obtained. The orig-inal phage population was then amplified once. This secondaryphage stock was screened by using chicken cDNA probes fora- and ,B-tubulin (10, 15). All cloning experiments were reg-istered with the committee for recombinant DNA research ofthe Swiss Academy of Medical Sciences and were performedin accordance with the U.S. National Institutes of Healthguidelines.

Abbreviation: kb, kilobase pairs.

4634

Proc. Natl. Acad. Sci. USA 80 (1983) 4635

Hybridization Conditions. DNA-containing filters were in-cubated overnight at 37°C with 100 ,ud/cm2 of 0.6 M NaCl/0.06M sodium citrate, pH 7.2/0.1% bovine serum albumin/0.01%Ficoll/0.01% polyvinylpyrrolidone/50% (vol/vol) formamide/0.2% NaDodSO4/0.01% sodium-pyrophosphate/100 ,ug of de-purinated calf thymus DNA per ml. Subsequent hybridizationwas carried out with 106-107 cpm of 32P-labeled DNA per filterin 20 Al/cm2 of the above buffer.

After hybridization, the filters were washed successively oncein 0.5 M sodium phosphate, pH 7.0, and then three times in0.3 M NaCl/0.03 M sodium citrate, pH 7.2/0.1% NaDodSO4.Filters containing DNA hybridized with homologous probes werewashed at 60WC, whereas filters containing DNA hybridized withheterologous probes were washed at room temperature.

RESULTSRestriction Analysis of Genomic DNA. Previous experi-

ments (25) had indicated that chicken tubulin cDNA probes canbe used to detect trypanosomal tubulin genes under appro-priate hybridization conditions. In order to confirm and extendthese findings, high molecular weight DNA of T. brucei, stockSTIB 366, was digested with a variety of restriction enzymesand fractionated on agarose gels. Duplicate blots of each gelwere hybridized with the chicken cDNA probes pTl (a-tu-bulin) and pT2 ((3tubulin) (10, 15), probing for the distributionof tubulin genes in the trypanosome genome. The results ofsuch a blotting experiment are presented in Fig. 1. A strikingfeature of this autoradiogram is the near-complete congruenceof the hybridization patterns obtained with the two probes. Cross-hybridization between the two was ruled out by appropriatecontrol hybridizations. Several restriction enzymes (BamHI,

1 2 3 4 5 6 7 8 9

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Bgl I, HindIII, Kpn I, and EcoRI) each produce a single stronglyhybridizing band 3.6 kb long, which hybridizes equally wellwith pT1 and with pT2. Upon longer exposure, a few faint butreproducible bands become visible in some of the digests.BamHI-digested DNA, for example, produces two faint bandsof DNA 6.0 and about 15 kb long. Because these bands mostprobably represent single-copy fragments, the much strongersignal obtained from the 3.6-kb bands suggests that these frag-ments are present in a larger copy number. Digestion of ge-nomic DNA with Pst I results in two major bands of 2.7 and 0.8kb, the former hybridizing to both pT1 and pT2 probes, whereasthe latter hybridizes exclusively to pT2, indicating that this lat-ter fragment contains exclusively f3-tubulin DNA sequences(marked by an arrow in Fig. 1). On the other hand, a numberof enzymes were found (e.g., Xho I, Hpa I, Xba I, and Sma I)that did not generate hybridizeable fragments of less than 20kb, though these enzymes cleave bulk trypanosomal DNA per-fectly well as visualized by ethidium bromide staining.

Very similar hybridization patterns were obtained in all caseswhen blots were analyzed with a homologous trypanosomal a-and ,3tubulin probe (pTBtu9A, see below). These observationsstrongly suggest that the chicken tubulin probes pT1 and pT2are able to detect all tubulin genes present in the trypanosomalgenome. Furthermore, they support the idea that the weaklyhybridizing bands-observed, e.g., in BamHI-digested DNA-are true low copy number genes, rather than variant genes thathybridize poorly to the heterologous chicken cDNA probes.The results obtained so far strongly suggest that the majority

of the tubulin genes of T. brucei are located on a stretch of re-iterated DNA consisting of regular head-to-tail repeats, eachwith a unit length of 3.6 kb. This possibility was further ex-plored by a series of double digestions of genomic DNA withrestriction enzymes that should cleave each repeat once (EcoRI,BamHI, HindIII, and Kpn I) or not at all (Xho I), as judged onthe basis of the results from Fig. 1. Fig. 2 represents a South-ern blot hybridization of double-digested genomic DNA hy-bridized to the a-tubulin probe pTl. The autoradiogram dem-onstrates a band pattern that agrees well with the concept ofthe tubulin genes being located on a segment of DNA con-sisting of head-to-tail repeats. Furthermore, the bands ob-tained, and their respective hybridization with pTl, as well aswith pT2 (not shown), are in complete agreement with the re-striction map subsequently derived from a cloned repeat unit(see below).

Thus, this experiment confirms our earlier contention thatthe strongly hybridizing band in Fig. 1 represents multiple copiesof the basic repeat unit. The more weakly hybridizing bandsobserved are currently thought to represent the junction frag-ments between the tubulin repeat and adjacent chromosomal

R R R RR + + H

B H X K

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B X K X K

FIG. 1. Southern blot analysis of restriction digests of genomic DNAfrom T. brucei with an a-tubulin-specific probe. DNA (2 jgper slot) wasdigestedwith restriction enzymes and fractionated on a 1% agarose gel.HindI-digested ADNA was included as a size marker, fiagment lengthsare given in kb. The Southern blot of the gel was analyzed by hybrid-ization withpT1 DNA. Lane 1, control (no enzyme); lane 2,BamHI; lane3, Bgl I; lane 4, HindmI; lane 5, Hpa I; lane 6, Kpn I; lane 7, Pst I; lane8, EcoRI; lane 9, Sal I. The arrow in lane 7 indicates a DNA fragmentthat hybridizes with the (-tubulin-specific probe pT2 exclusively (seetext).

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aIG. 2. Southern blot analysis of double-digested genomic DNA withpTl. DNA (2 tg per slot) was digested and was fractionated on a 1%agarose gel. A blot of the gel was then analyzed by hybridization. R,EcoRI; B, BamHI; X, Xho I; K, Kpn I; H, HindI.

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4636 Biochemistry: Seebeck et al.

DNA. This idea is supported by the isolation of a recombinantphage that carries a 6-kb BamHI fragment immediately adja-cent to a number of 3.6-kb tubulin repeats (see below).

The number of tubulin genes in the trypanosomal genomewas estimated by dot-blot hybridization and by quantitation bySouthern blotting (Fig. 3). The results from both types of ex-periments were in agreement and indicate that about eight cop-ies of the tubulin repeat are present per haploid trypanosomegenome of 5-104 kb (26).

Analysis of Recombinant Phages. From an amplified ge-nomic library of trypanosomal DNA, 60,000 individual plaqueswere screened with the chicken cDNA probes. Eighty-fourpositive plaques were identified. All but two of them were pos-itive for both pT1 and pT2. This observation confirmed the aboveresult, indicating a very close localization of a- and (3-tubulingenes within the genome, and it agrees well with the conclu-sions described above.

Four individual phages were repurified through three cyclesof plaque purification, and their DNA was analyzed by restric-tion enzyme digestion and hybridization. Three of these phagescontained various numbers of the basic tubulin DNA repeatunit, whereas one phage (ATBtu9) also carried a 6-kb BamHIfragment adjacent to a number of 3.6-kb repeat units. This 6-kb fragment migrates with the weakly hybridizing fragment de-tected in BamHI-digested genomic DNA (see above). Prelim-inary restriction mapping indicated that this fragment containsan incomplete tubulin repeat plus an additional 3 kb of non-tubulin DNA. At present, this fragment is most easily inter-preted as representing a linker between the tubulin gene clus-ter and flanking chromosomal DNA.The basic repeat units from two phages (ATBtu3 and ATBtu9)

were subcloned in pBR322, using EcoRI, BamHI, or HindIIIto cleave the DNA. The restriction maps of these various sub-clones were compatible with each other. For a more detailedanalysis, a BamHI subclone from ATBtu9 was selected. The lo-calization of the a- and (3-tubulin genes on the cloned fragmentwas established by Southern blot hybridization. A map ofpTBtu9A, representing the basic tubulin repeat unit in the try-panosomal genome, is given in Fig. 4. This map correlates wellwith the structure of the repeat as derived from Soutern blot-ting experiments of genomic DNA.

In order to identify unambiguously the tubulin genes, thesequences of the fragments of the coding regions of pTBtu9Awere partially determined. The comparison of the correspond-ing amino acid sequences with their chicken counterparts con-firmed the identity of the trypanosomal tubulin genes.

Divergence of Trypanosome Tubulin Genes from Those inthe Chicken. Previous studies (25) as well as our own resultshave indicated that the chicken tubulin cDNA probes pT1 andpT2 are fairly divergent from trypanosomal tubulin sequences.

genomic DNA

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FIG. 3. Hybridization titration of tubulin genes. Genomic DNA (5,ug) was digested to completion with BamHI and fractionated on a 1%agarose gel. Increasing amounts of linearized plasmid pTBtu3A (seelegend to Fig. 4) were electrophoresed in parallel. The Southern blot ofthe gel was analyzed by hybridization with nick-translated DNA ofATBtul.

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FIG. 4. Restriction map of plasmid pTBtu9A. The trypanosomalDNA is inserted into pBR322 at the BamHI site. As presented in thisfigure, the EcoRI site of the vector would lie 0.275 kb to the left of theinsert. pTBtu3A is an analogous clone, carrying the insert in the op-posite orientation. o, BamHI; o, HindE; v, Sal I; *, Pst I; *, Bgl I; *,Kpn I; *, EcoRI. The bars above the map indicate the approximate lo-cation of the a- and the 3-tubulin genes.

We have quantified this divergence by hybrid melting exper-iments (27). Nitrocellulose filters were loaded with pT1, pT2,and pTBtu9A DNA, and the DNA on the filters was then hy-bridized with recombinant phage ATBtu1 DNA containing ex-clusively trypanosomal a- and /-tubulin genes. Control ex-periments were done to establish that the phage DNA does notcross-hybridize with the plasmid vector. Hybrids were thenwashed extensively, first at room temperature and then at con-stantly higher temperatures, in 30 mM NaCl/8 mM Tris HCl,pH 8.0/1 mM EDTA. The results given in Fig. 5 demonstratethat tm values (the temperatures at which the DNAs were 50%melted) of the heterologous sequences pT1 and pT2 are 10 and13°C lower, respectively, than the tm of the homologous genes.Because, under the conditions employed, a 1T difference intm corresponds to approximately a 1% difference in sequence(28, 29), the a-tubulin sequence from chicken has diverged byabout 10% from the corresponding trypanosomal sequence, andthe chicken -tubulin sequence has diverged by about 13% fromits trypanosomal counterpart.

DISCUSSIONThe tubulin genes in the genome of Trypanosoma brucei arearranged in a tightly packed cluster of alternating a- and ,B-tu-bulin genes. This cluster contains most of the genes detectedin the genome under our experimental conditions. The basicrepeat unit, which is generated by digestion with a variety ofrestriction enzymes, is 3.6 kb long. This mode of genomic or-ganization of tubulin genes differs significantly from the sit-uation in other organisms analyzed so far (15-20). At present,it is not quite clear if other species of trypanosomes share thisgene arrangement. However, preliminary observations withdifferent stocks of T. brucei (25), with T. rhodesiense (30), andwith T. equiperdum (U. Hibner, personal communication) sug-gest that this arrangement might be a general feature of thisgenus. A very recent study of the arrangement of tubulin genesin the genome of T. brucei grown in vivo (31) fully agrees withthe results presented here. This study, in conjunction with ourown results, confirms the stability of the tubulin gene arrange-ment irrespective of the physiological state [procyclic forms (thispaper) vs. bloodstream forms (31)] of the organisms.

Upon digestion with certain restriction enzymes, one or twoweakly hybridizing DNA bands were detected in addition tothe major bands. These fragments are detected equally wellwith heterologous and homologous tubulin DNA probes. Thisobservation indicates that these bands represent single-copyfragments carrying normal tubulin sequences, rather thanpseudogenes with strongly diverged sequences. In the latter

Proc. Natl. Acad. Sci. USA 80 (1983)

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FIG. 5. Melting profiles of chicken and trypanosome tubulin DNA hybrids. Nitrocellulose filters were loaded with pTl, pT2, or pTBtu9A DNA(1 jig per filter, five filters per DNA species) and the DNAs were hybridized with ATBtul DNA. Hybrids were washed extensively in 30 mM NaCl/8mM Tris;HCl, pH 8.0/1 mM EDTA at room temperature and then at increasing temperatures. Each temperature step was maintained for 10 min.The cpm ofDNA washed off at each temperature was measured. Each point of the graphs represents the mean ofthe five filters processed in parallel.The 100% values correspond to 10,000-14,000 cpm in the three hybridizations. (Inset) Derivatives of the melting curves. Curve 1, pTBtu9A ATBtul(homologous hybridization); curve 2, pTlPATBtul (chicken a-tubulin gene hybridized with trypanosome a-tubulin gene); curve 3, pT2'ATBtul (chicken,3tubulin gene hybridized with trypanosome ,B-tubulin gene).

case, the heterologous probes would have been expected to hy-bridize far less well than homologous probes. Therefore, weinterpret these findings to indicate that these fragments rep-resent the linker fragments between the tubulin gene clusterand adjacent chromosomal DNA. This assumption has beensupported by the finding that one of the recombinant phages,ATBtu9, carries a 6.0-kb BamHI fragment of predominantlynontubulin DNA adjacent to basic repeat fragments.

The results presented above can most readily be interpretedby a concept of one (or a very few) tight cluster of alternatinga- and 13-tubulin genes in the genome of T. brucei. These clus-ters consist of repeated DNA fragments, in a head-to-tail ar-rangement, 3.6 kb long.

At present, our data do not allow a close estimate of the de-gree of similarity between the individual tubulin genes of thecluster. However, we can rule out the presence of strongly di-verged genes within the cluster-e.g., as a consequence of thegeneration of tubulin pseudogenes of the type that has recentlybeen discovered in different species (11, 14).

Judging from present knowledge about the size of a typicaleukaroytic gene, a 3.6-kb fragment of DNA appears rather shortfor harboring two genes, each of which codes for a polypeptideof about 45 kilodaltons. Without even taking account of anypossible introns, no more than a few hundred nucleotides areavailable for providing transcriptional signals and mRNA leadersequences. Therefore, such a tight cluster of homologous genesmight be an interesting phenomenon from the point of view oftranscriptional regulation.

A detailed analysis of this gene cluster and a full sequenceanalysis of the basic repeat unit are expected to provide a muchmore complete understanding of the functional organization ofthis unusually close-knit gene family.

We thank T. Ackermann-Dick for her competent and cheerful assis-tance, L. Jenni and R. Brun of the Swiss Tropical Institute in Basel forcontinuous encouragement and support, and G. Ryffel for his valuablecomments on the manuscript. We are indebted to D. W. Cleveland ofthe Johns Hopkins Medical School for providing us his chicken cDNAclones pTl and pT2. This work was supported from the United NationsDevelopment Program/World Bank/World Health Organization Spe-cial Programme for Research and Training in Tropical Diseases.

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