MOLECULAR AND CELLULAR BIOLOGY, Aug. 1991, p. 3823-3834 Vol. 11, No. 8 0270-7306/91/083823-12$02.00/0 Copyright C) 1991, American Society for Microbiology Chromosome Structure: DNA Nucleotide Sequence Elements of a Subset of the Minichromosomes of the Protozoan Trypanosoma brucei MICHAEL WEIDEN,1 YVONNE N. OSHEIM,2 ANN L. BEYER,2 AND LEX H. T. VAN DER PLOEG3* Department of Medicine' and Department of Genetics and Development,3 College ofPhysicians and Surgeons, Columbia University, 701 West 168 Street, New York, New York 10032, and Department of Microbiology, University of Virginia, Charlottesville,. Virginia 229082 Received 18 December 1990/Accepted 30 April 1991 The genome of the protozoan Trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. The small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. We show that a subset of the minichromosomes is composed predominantly of simple-sequence DNA, with over 90% of the length of the minichromosome consisting of a tandem array of 177-bp repeats, indicating that these molecules have limited protein-coding capacity. Proceeding from the tip of the telomere to a chromosome internal position, a subset of the minichromosomes contained the GGGTTA telomere repeat, a 29-bp telomere-derived repeat, a region containing 74-bp G+C-rich direct repeats separated by approxi- mately 155 bp of A+T-rich DNA that has a bent character, and 50 to 150 kb of the 177-bp repeat. Several of the minichromosome-derived telomeres did not encode protein-coding genes, indicating that the repertoire of telomeric variant cell surface glycoprotein genes is restricted to some telomeres only. The telomere organization in trypanosomes shares striking similarities to the organization of telomeres and subtelomeres in humans, yeasts, and plasmodia. An electron microscopic analysis of the minichromosomes showed that they are linear molecules without abnormal structures in the main body of the chromosome. The structure of replicating molecules indicated that minichromosomes probably have a single bidirectional origin of replication located in the body of the chromosome. We propose a model for the structure of the trypanosome minichromosomes. Trypanosoma brucei is a protozoan parasite that lives in comprise about 10% of the nuclear DNA but represent over the bloodstream of its mammalian host. Trypanosomes can 80% of the chromosomes of T. brucei. The MC are mitoti- escape destruction by the humoral immune response be- cally stable (44); however, their number may vary during cause they are capable of periodically switching to the genetic exchange (49). expression of an entirely new cell surface protein coat made The MC are believed to harbor a large number of telomeric of a single type of protein, the variant cell surface glycopro- VSG genes. The only protozoa identified thus far that tein (VSG). This process, called antigenic variation, relies on contain MC are trypanosomes that undergo antigenic varia- a large repertoire of over 1,000 different VSG genes, only tion; hence, their only function may be to provide a set of one of which is expressed in a trypanosome at any one time. telomerically located VSG genes (42). However, MC do not The expressed VSG gene is invariably located at the very appear to be able to express VSG genes, and the MC-derived end of a chromosome, also referred to as the telomere, and VSG genes need to be translocated to an expression site for telomeres containing expressed VSG genes have been iden- transcription (15, 41, 42). The highly recombinogenic nature tified on a few specific chromosomes only. These telomeres of trypanosome telomeres (1, 43) may lead to a more rapid are referred to as VSG gene expression sites. Switching to accumulation of changes in the telomeric VSG genes com- the expression of a new VSG gene can be accomplished by pared with chromosome internally located VSG genes. A different types of DNA recombinational events which can rapidly evolving group of VSG genes will have epidemiolog- translocate a VSG gene to an expression site. Alternatively, ical implications and intuitively appears to be advantageous differential transcriptional control of the expression sites can to the parasite. The small size ofthese molecules provides an result in an antigenic switch (for reviews, see references 5, opportunity to define the structure of a naturally occurring 13, 32, 33, and 41). chromosome at the nucleotide level, thereby giving a de- The genome of T. brucei contains roughly 20 large chro- tailed overview of the sequences required for chromosome mosomes, ranging in size from 200 kb to 5.7 Mb, and about functioning in trypanosomes. Our goal was to improve our 100 50- to 150-kb minichromosomes (MC) (21, 44, 45). Seven understanding of the genetic elements that are necessary and pairs of homologous chromosomes have been identified sufficient for eukaryotic chromosome function. among the 20 larger chromosomes, while the genome has been proposed to be aneuploid for the MC (21). The chro- mosomes all share terminally located telomere repeats with MATERIALS AND METHODS the sequence (GGGTTA)n (4, 43), recently also identified in humans (9a, 28; for a review see reference 3). The MC DNA isolation, enzyme digestion, and Southern blotting. T. brucei clone 118d (for Fig. 6B and C, 118 clone 1 was used) was grown in rats and purified from blood elements as described by Fairlamb et al. (20). Nuclear DNA was isolated * Corresponding author. from trypanosomes as described by Van der Ploeg et al. (46) 3823 3824 WEIDEN ET AL. MOL. CELL. BIOL. and digested by adding a fourfold excess of restriction nation method (36), using either Sequenase at 25°C with endonuclease for complete digestion; 10% of the recom- [35S]dATP or Taq polymerase at 65°C with dye-labeled mended amount of enzyme was added for partial restriction primers. Subclones of PT4 were inserted into the replicative enzyme digestions. For the partial restriction enzyme diges- forms of bacteriophage M13 or the Bluescript vector (Strat- tions, aliquots were taken at different times and were imme- agene); both single-stranded and double-stranded sequenc- diately phenol-chloroform-isoamyl alcohol extracted. Bal 31 ing were performed. digestions were similarly performed in a time series. The EM. For electron microscopy (EM), the DNA molecules actual reduction in size of the fragments was calculated from were visualized with the drop diffusion method of Lang and rehybridization of the same Southern blots containing the Mitani (25). Forty-microliter drops of the following mixture Bal 31-treated DNA with a 177-bp repeat and dividing the were deposited on a clean plastic petri dish and allowed to observed reduction in size of small bands by 2 to give base diffuse for 20 to 60 min: 20 ,lI of DNA at 1 ,ug/ml; 15 [LI of pairs digested per end. Counts per minute on the filter were 0.01 M Tris-0.001 M EDTA (pH 8.5); 160 RI of 0.25 M measured directly on a Betascope 603 (Betagen, Waltham, ammonium acetate; and 5 ,ul of cytochrome c (1 mg/ml; Mass.) Plasmids were isolated as described by Birnboim and Sigma type V, filtered) diluted 1:20 in distilled H20. Carbon- Doly (2). Agarose gels were treated with 0.25% HCI prior to coated grids (300 mesh) were touched to the surface of each blotting. DNA was transfered to Hybond-N and hybridized drop, washed in distilled H20, and stained for 1 min in with random-primed 32P-labeled DNA. Plasmid DNA and uranyl acetate solution made by diluting a stock of 0.05 M size markers (1-kb ladder or phage lambda DNA digested uranyl acetate in 0.05 N HCl, 1:100 in 90% ethanol. Grids with HindlIl) were end labeled with [32P]ATP and polynu- were then rinsed in 95% ethanol, air dried, and rotary cleotide kinase. Final stringencies of posthybridization shadow cast with platinum. The replicating molecule shown washing were 0.1x SSC (lx SSC is 0.15 M NaCl plus 0.015 in Fig. 8 was prepared as described above, with the addition M sodium citrate), for A2, 0.3 x SSC for the 177-bp repeat, of NaCl to give a final concentration of 6 mM. Grids were and 1.0x SSC for A4. viewed in a JEOL 100 CS transmission electron microscope Sucrose gradient isolation of MC. A modification of the at 80 kV. method published by Sloof et al. (39) was used. A 35-ml linear S to 20% sucrose gradient in 100 mM EDTA-25 mM Tris-HCl (pH 7.5)-1% sodium lauryl sarcosinate (SLS)-50 RESULTS ,ug of proteinase K per ml was overlayered with 2 ml of a Sequence analysis of the telomere domain. In previous solution containing 200 mM EDTA, 1% SLS, and 1 mg of studies, we isolated two telomere-derived clones, PT1 and proteinase K per ml. On top of this solution we layered 4 x PT4 (43). The physical maps of both clones are presented in 108 trypanosomes in 1 ml of phosphate-buffered saline. The Fig. 1A. One of these telomere clones (PT4) contained a trypanosomes were allowed to lyse by incubating the gradi- tandem array of the GGGTTA telomere repeat (44). To ent at 25°C for 3 h, after which the DNA was size separated identify the genetic elements that comprised a telomeric and by spinning at 10,000 rpm in an SW28 rotor for 16 h at 25°C; subtelomeric domain, we determined the DNA nucleotide 1-ml fractions were removed from the top of the gradient sequence of clone PT4 (Fig.