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Diversity and Dynamics of the Minichromosomal Karyotype in Trypanosoma Brucei 

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Diversity and Dynamics of the Minichromosomal Karyotype in Trypanosoma Brucei  Molecular & Biochemical Parasitology 113 (2001) 79–88 www.parasitology-online.com. Diversity and dynamics of the minichromosomal karyotype in Trypanosoma brucei Sam Alsford 1, Bill Wickstead1, Klaus Ersfeld *, Keith Gull School of Biological Sciences, Uni6ersity of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK Received 18 August 2000; received in revised form 28 November 2000; accepted 8 December 2000 Abstract The genome of African trypanosomes contains a large number of minichromosomes. Their only proposed role is in the expansion of the parasites’ repertoire of telomeric variant surface glycoprotein (VSG) genes as minichromosomes carry silent VSG gene copies in telomeric locations. Despite their importance as VSG gene donors, little is known about the actual composition of the minichromosomal karyotype and the stability of its inheritance. In this study we show, by using high-resolution pulsed-field electrophoresis, that a non-clonal trypanosome population contains an extremely diverse pattern of minichromosomes, which can be resolved into less complex clone-specific karyotypes by non-selective cloning. We show that the minichromosome patterns of such clones are stable over at least 360 generations. Furthermore, using DNA markers for specific minichromosomes, we demonstrate the mitotic stability of these minichromosomes within the population over a period of more than 5 years. Length variation is observed for an individual minichromosome and is most likely caused by a continuous telomeric growth of approximately 6 bp per telomere per cell division. This steady telomeric growth, counteracted by stochastic large losses of telomeric sequences is the most likely cause of minichromosome karyotype heterogeneity within a population. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Chromosome; Karyotype; Minichromosome; VSG; Telomeres; Trypanosoma brucei; Pulsed-field gel electrophoresis 1. Introduction between 30 and 150 kbp in size, constitute the group of smallest chromosomes. The sequence of a minichromo- The nuclear genome of Trypanosoma brucei consists some mainly consists of a 177-bp tandem repeat, with of three chromosome classes (reviewed in [1,2]). The the remaining sequence consisting of other repeats and megabase chromosomes of this diploid genome contain a silent telomeric VSG gene [5,6]. Chromosome ends the expressed genes and form 11 homologous chromo- consist of typical eukaryotic telomeres. The presence of some pairs [3]. The remainder of the nuclear genome VSG genes on minichromosomes has led to the sugges- consists of the intermediate chromosomes and the tion that they act as a reservoir of telomeric VSG genes, minichromosomes. The ploidy and function of the in- which may be transferred to expression sites by duplica- termediate chromosomes is unclear, though they con- tive transposition [7,8]. tain variant surface glycoprotein (VSG) gene expression The organisation of megabase chromosomes and sites [4]. The 100 or so minichromosomes, which are minichromosomes in the interphase nucleus of the cul- tured procyclic trypanosome and their subsequent be- haviour during mitosis has been extensively Abbre6iations: PFGE, pulsed-field gel electrophoresis; VSG, variant surface glycoprotein. characterised by fluorescence in situ hybridisation Note: Nucleotide sequence data reported in this paper are avail- [9,10]. During mitosis minichromosomes are associated able in the GenBank™, EMBL and DDBJ-databases under the with the mitotic spindle and disruption of the spindle accession numbers AF294806 and AF294807. using the microtubule-depolymerising drug rhizoxin re- * Corresponding author. Tel.: +44-161-2755112; fax: +44-161- 2755763. sults in non-segregation of minichromosomes. The E-mail address: [email protected] (K. Ersfeld). analysis of minichromosome segregation at a popula- 1 These two authors contributed equally to this paper. tion level by FISH revealed the separation of the total 0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0166-6851(00)00388-1 80 S. Alsford et al. / Molecular & Biochemical Parasitology 113 (2001) 79–88 minichromosomal population into two equally sized limiting dilution cloning in conditioned media. Condi- daughter populations, suggesting a high degree of tioned media was prepared as follows. Procyclics were fidelity of inheritance. There remains the problem of grown at 28°C in SDM79 [13] supplemented with 10% envisaging how so many minichromosomes can be seg- (v/v) foetal calf serum (FCS) to a density of approxi- regated on a small spindle that does not exhibit large mately 1×107 cells ml−1. The culture was pelleted and numbers of microtubules or kinetochores. A model has then was supplemented with 10% (v/v) FCS, filter ster- been proposed to explain how a faithful segregation of ilised through a 0.22 mm filter, stored at 4°C and used large numbers of minichromosomes could occur along within a week of preparation. the central mitotic spindle [11]. In this model, minichro- For cloning, the culture was diluted to a final density mosomes are assumed to form lateral associations with of about one cell ml−1 in 20 ml conditioned media and the spindle microtubules instead of the terminal associ- transferred to a 96-well plate at 200 ml per well. After 7 ations seen in classical kinetochore-mediated chromo- days at 28°C, populated wells were inoculated into 10 some segregation. ml SDM79 supplemented with 10% (v/v) FCS and Although the available evidence on a population placed at 28°C. Primary clones were identified by the level suggests that the minichromosomes are segregated co-ordinate of their source well (letter, row; number, with a certain degree of fidelity, these observations are column) and secondary clones were identified by the not sufficient to make exact statements on segregation source primary clone and their source well. Therefore, accuracy [10,12]. In order to achieve this it is essential primary clone C10 was isolated from well C10 of a to analyse the fate of individual minichromosomes over 96-well plate, while secondary clone C10D4 was iso- time in a defined population of cells. lated from well D4 following the re-cloning of primary In this study, we used high-resolution pulsed-field gel clone C10. electrophoresis (PFGE) to analyse the karyotype pat- tern of minichromosomes in clonal cell lines. In addi- 2.2. Pulsed-field gel electrophoresis tion to employing probes detecting the entire minichromosomal complement of a cell, we also devel- Chromosome blocks were prepared as earlier de- oped markers for specific minichromosomes in this scribed [3]. PFGE was carried out in a contour-clamped population. We demonstrate that minichromosomes are homogeneous electric field apparatus (CHEF DRII or accurately segregated over time. We also describe a CHEF Mapper, BioRad). Chromosome separation was high degree of minichromosomal diversity in non-clonal performed in 1 or 1.5% agarose (PFGE grade, Amer- populations and provide evidence that this heterogene- sham-Pharmacia Biotech) in 90 mM Tris–borate pH ity is unlikely to be due to non-disjunction or chromo- 8.2, 0.2 mM EDTA (1×TB[0.1]E) at constant tempera- some loss but rather is a consequence of size variations ture with buffer circulation. Specific conditions are of individual chromosomes within the cells of the described in the figure legends. population. Concurrent electrophoresis of l-concatamers (Promega) and EcoRI/HindIII digested l-phage (Appligene) allowed T.brucei chromosome sizes to be 2. Materials and methods estimated. Following separation, chromosomes were stained in 2.5 mgml−1 ethidium bromide for 30 min 2.1. Limiting dilution cloning of procyclic T. brucei and destained in distilled water for 30 min. Images were digitally captured under 300 nm UV illumination. Our laboratory stock of T. brucei strain 427 has been maintained in culture over a period of 10 years, during 2.3. Southern blot analysis which time it has not been cloned. Therefore, it was thought that the cloning of this population might reveal Separated chromosomes were depurinated in 0.25 M the degree of fidelity with which the minichromosomes HCl for 20 min, denatured and transferred onto nylon are segregated by analysing the stability of the kary- membranes by standard procedures. DNA probes for otype pattern. Sets of primary clones were produced by the 177-bp minichromosome repeat and the telomere non-selective limiting dilution cloning from a contem- repeat oligonucleotide was labelled with digoxigenin as porary laboratory stock of EATRO 427 (1998 and described [10,14]. The 177 bp repeat-specific probe was 2000) and from a sample that had been frozen in liquid hybridised in EasyHyb–buffer (Roche Diagnostics) at nitrogen in our laboratory in 1993. All primary clones 40°C overnight and the final washes were carried out in were subsequently recloned to produce secondary clone 15 mM NaCl, 1.5 mM sodium citrate (0.1×SSC)/0.1% pairs, with each pair corresponding to a specific pri- (w/v) SDS at 60°C. The telomere-specific probe was mary clone. hybridised at 37°C for 3 h and the final washes were Procyclic stage cells of the culture-adapted 427 strain carried out at 50°C in 0.5×SSC/0.1% (w/v) SDS. To were cloned in the absence of selective pressure by optimise detection of the less abundant probes, we used S. Alsford et al. / Molecular & Biochemical Parasitology 113 (2001) 79–88 81 UV-nicking (80 mJ at 250 nm) instead of depurination We subjected this trypanosome population, which and transfer under alkaline conditions in 0.4 M NaOH, has been grown virtually constantly and has not been 1.5 M NaCl. Blots were developed with an anti-digoxi- cloned within a 10-year window, to non-selective genin antibody (Fab-fragment) conjugated to alkaline cloning by limiting dilution. Analysis of the resulting phosphatase and CSPD as a chemiluminescent sub- clones presented essentially the same pattern for all strate (all from Roche Diagnostics). chromosomes at this level of resolution.
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