Chromosome Organization of the Protozoan Trypanosoma brucei - NCBI

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1990, p. 6079-6083 0270-7306/90/116079-05$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 11

Chromosome Organization of the Protozoan Trypanosoma brucei KEITH GOTTESDIENER,1 JAIME GARCIA-ANOVEROS,l MARY GWO-SHU LEE,2 AND LEX H. T. VAN DER PLOEGl* Department of Genetics and Development, College of Physicians and Surgeons,' and Division of Tropical Medicine, School of Public Health,2 Columbia University, New York, New York 10032 Received 14 May 1990/Accepted 30 July 1990

The genome of the protozoan Trypanosoma brucei is known to be diploid. Karyotype analysis has, however, failed to identify homologous chromosomes. Having refined the technique for separating trypanosome chromosomes (L. H. T. Van der Ploeg, C. L. Smith, R. I. Polvere, and K. Gottesdiener, Nucleic Acids Res. 17:3217-3227, 1989), we can now provide evidence for the presence of homologous chromosomes. By determniing the chromosomal location of different genetic markers, most of the chromosomes (14, excluding the minichromosomes), could be organized into seven chromosome pairs. In most instances, the putative homologs of a pair differed in size by about 20%. Restriction enzyme analysis of chromosome-sized DNA showed that these chromosome pairs contained large stretches of homologous DNA sequences. From these data, we infer that the chromosome pairs represent homologs. The identification of homologous chromosomes gives valuable insight into the organization of the trypanosome genome, will facilitate the genetic analysis of T. brucei, and suggests the presence of haploid gametes.

we show the hybridization of a probe for the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes to chromosome-sized DNA (Fig. 1). In Fig. 1A, bands 1 (the minichromosomes) through 6 are well resolved, leaving the gGAPDH genes in the compression zone (C). In Fig. 1B, Bands 1 to 6 are compressed in the low-molecular-weight range and bands 7 through 12 are separated, still leaving the gGAPDH genes in the compression zone in the high-molecular-weight range. Figure 1C shows the location of gGAPDH genes on bands 14 and 15, as does Fig. 1D, where the location of gGAPDH genes on band 15 is obvious while bands 13 and 14 now appear compressed. Representative examples of PFG gels showing the chromosomal locations of additional markers are shown in Fig. 2. These panels illustrate the fact that two chromosomes hybridize in all cases. Most of the panels in Fig. 2 were not used to accurately assign the chromosomal locations of particular markers. Chromosome assignments were performed in a series of PFG gels (Fig. 1). Figure 2 shows the separation of bands 13 through 15 (panel A), bands 15 through 17 (panel B), and bands 17 through 19 (panel C). The hybridizations of different genetic markers to bands in the PFG gels are shown below the ethidium bromide-stained gels in panels A and B and to the right of the ethidium bromidestained gel in panel C and as follows: gGAPDH hybridized to bands 14 and 15, glucosephosphate isomerase (PGI) hybridized to bands 11 and 16, a gene encoding a protein located in the flagellum of T. brucei hybridized to bands 15 and 16 (TBS 17), a gene encoding a protein of unknown function hybridized to bands 7 and 8 (TBS 4), the procyclic acidic repetitive protein (PARP) A loci hybridized to bands 14 and 15, and the PARP B loci hybridized to bands 18 and 19 (please note that an apparent inconsistency in the hybridization pattern in Fig. 2B, where band 16 in the panel labeled PGI did not comigrate with band 16 in the panel labeled TBS 17, resulted from the fact that panels were from different regions of the same PFG gel; also, DNA concentrations between lanes were different, to assure that the DNA concentration would not affect separation quality). The panels also show hybridization in

Trypanosoma brucei is a unicellular, eucaryotic parasite of humans and livestock that is transmitted by the tsetse fly. The genetic organization, karyotype, and nuclear structure of this protozoan have been studied intensively (3, 28). Both the bloodstream form and insect (procyclic) form of T. brucei are diploid, as determined by DNA renaturation experiments and quantitation of the DNA content per nucleus (1, 2, 6). A sexual cycle exists in T. brucei, but the presence of gametes and homologous chromosomes has remained elusive. Different models to explain genetic exchange in trypanosomes have been proposed. Some models propose cell fusion generating a tetraploid heterokaryon which, by random chromosome loss, reestablishes a neardiploid genome content, while other models propose haploid gametes (8, 12, 17, 21, 22, 24-26, 33). The genome of T. brucei is organized into about 100 minichromosomes, ranging in size from 50 to 150 kilobases (kb), and at least 18 individual larger chromosomes up to 5.7 Mbp, as determined by pulsed-field gel electrophoresis (PFG) (29-31). The numerical values of the chromosome sizes add up to more than 60,000 kb, accounting for almost the entire estimated diploid trypanosome genome. Paradoxically, only single copies of several of these chromosomes are present in each cell. The trypanosome genome, therefore either contains homologous chromosomes of different sizes or is not organized into homologous chromosomes. The latter could be explained if frequent chromosome rearrangements had scrambled the genome. To identify whether homologous chromosomes exist, the trypanosome chromosomes were size separated by PFG (20, 30, 31) and the chromosomal locations of 18 different genetic markers were determined. By separating the chromosomesized DNA under different PFG conditions, we determined the precise chromosomal location of each marker gene. A representative example of the methods used to assign markers to specific chromosomes is shown in Fig. 1. In this figure, *

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FIG. 1. Determination of the chromosomal location of the gGAPDH genes. PFG electrophoresis of genomic DNA from T. brucei stock 427-60, variants 221a and 1.208R (left and right lanes, respectively) was performed under separation conditions described previously and as indicated below (31). Bands with chromosomes are numbered, starting with the minichromosomes of 50 to 150 kb (band 1), up to the largest chromosome separated in this series (band 17). Band C indicates the compression zone. Panels A to D, respectively, show ethidium-stained 1% agarose PFG gels run at 10 V/cm at a 50-s pulse frequency for 40 h, 5 V/cm at a 900-s pulse frequency for 5 days, 3 V/cm, at a 2,400-s pulse frequency for 7 days, and 3 V/cm at a pulse frequency of 3,600 s for 7 days. To the left of each ethidium-stained gel, the hybridization of the same gel with a probe for the gGAPDH genes (the 1.3-kb AluI to KpnI genomic restriction enzyme fragment [16]) is shown. Hybridizations were performed as described previously (31), and posthybridization washing conditions were 65°C with 0.1 x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate).

the well, which we have previously shown to result from chromosomes that are nonspecifically trapped at that position (31). These hybridizations clearly reveal that the genetic markers tested each hybridized with a pair of chromosomes. The chromosomal location of the marker genes is identical in the five different antigenic variants tested. A summary of the chromosomal positioning of all markers tested is shown in Table 1. Almost every marker recognized two different bands in the PFG gels, which is indicative of alleles of genes on homologous chromosomes. These putative homologs, however, have markedly different sizes. For instance, bands 18 and 19, which differ in size by several hundred kilobase pairs, hybridized with the same five markers. Six additional chromosome pairs could be identified, and most of these pairs were characterized on the basis of the location of two different marker genes. These chromosome pairs are bracketed at the left side of Table 1 (chromosomes in bands 19 and 18, 17 and 14', 16 and 15', 16' and 11, 15 and 14, 10 and 9, and 8 and 7). Our data confirm the prediction that alleles of the PARP genes are present on homologous chromosomes (19). Some PFG bands (e.g., 14 and 16) include chromosomes belonging to different chromosome pairs. This apparent inconsistency may be accounted for by the presence of more than one type of chromosome per band, as indicated by the nonstoichiometrically stained ethidium bands 19, 16, 15, 14, 10, and 9 (bands marked with asterisks in Table 1). Different chromosomes thus might comigrate in a single band. That this is indeed the case can be seen from a comparison of band 14 in different antigenic variants. This band can be separated into two bands in some variants, presumably due to a DNA rearrangement event which affected the size of one of the chromosomes (Fig. 2A, arrow). As a result, one of the chromosomes of band 14 (Table 1, 14') hybridized with the medRNA gene, while the gGAPDH and a PARP A alleles

were traced to a second chromosome in band 14. Also, band 9, which always appeared as a nonstoichiometrically staining band, can be separated into two bands, depending on the PFG conditions (data not shown). The relative ethidium bromide staining intensity also suggests that band 19 contains at least three different chromosomes, the largest of which have not been separated. The unique locations of the triosephosphate isomerase (TIM) and heat shock protein 70 (hsp70)-coding genes and one set of rRNA genes (underlined in Table 1) on band 19 suggests that band 19 includes both homologs carrying these genes. The identification of a large number of chromosome pairs suggests that homologous chromosomes differ in size in trypanosomes. We reasoned that if these chromosome pairs represented homologs, their long-range physical maps should be identical in the regions flanking the marker genes. Thus, one would expect to see only one hybridizing band in restriction enzyme-digested genomic DNA, and not two bands, as would be expected if the marker genes were located on nonhomologous chromosomes. Figure 3 shows a representative example of restriction enzyme digestion of trypanosome DNA, followed by separation of the DNA by PFG and hybridization with marker genes that are present on bands 18 and 19. In almost all restriction enzyme digestions, a single hybridizing band was found, showing that the markers, which recognized two different chromosomes in PFG gels, are located on DNA segments with almost identical physical maps. Only a few restriction fragment length polymorphisms could be detected for some markers (in the SmaI digestion with cGAPDH and in the SfiI digestion hybridized with pyruvate kinase). Since none of the markers showed linkage (for any particular restriction enzyme digest, each marker recognized a different size restriction enzyme fragment) we can predict that at least 1 Mb of DNA of these chromosomes has an almost identical physical map. Similar

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FIG. 2. PFG electrophoresis of genomic DNA from T. brucei variants 221a (221) 1.208R, 118 clone 1 bloodstream form (118B), 118 clone 1 insect form (118P), and variant 117a (117) (5, 31) from T. brucei stock 427-60 as described previously (31). Only some bands with chromosomes are numbered, starting with the minichromosomes of 50 to 150 kb (band 1) and the largest chromosomes of 5.7 Mb (band 19). Band C indicates the compression zone of each PFG gel. Panels A to C, respectively, show ethidium bromide-stained 1% agarose PFG gels run for 7 days at 3 V/cm with a pulse frequency of 2,400 s, for 7 days at 3 V/cm with a pulse frequency of 3,600 s, and for 12 days at 2.5 V/cm with a pulse frequency of 5,500 s. Hybridizations were performed as described previously (31), and posthybridization washing conditions were 0.1x SSC and 65°C. Probes used in the hybridizations were the gGAPDH gene (see Fig. 1 for probe identification), a 1.8-kb HindIII-BamHI genomic fragment from the gene for glucosephosphate isomerase (PGI) (13), a cDNA encoding a putative flagellar Ca2"-binding protein of T. brucei (TBS 17) (M. G.-S. Lee, A. Ho, A. D'Alesandro, and L. H. T. Van der Ploeg, Nucleic Acids Res., in press), a cDNA encoding a protein of unknown function (TBS 4 [M. G.-S. Lee and L. H. T. Van der Ploeg, unpublished results]), a 1.6-kb HindIII-PstI genomic fragment specific for the region upstream of the PARP A locus (Parp A) and a 0.47-kb Hinfl genomic fragment specific for the region upstream of the PARP B locus (Parp B) (19).

results for the other chromosome pairs were obtained with additional enzymes and the genetic markers listed in Table 1. These data show that some of the chromosome pairs contain large stretches of DNA that must be highly homologous. We conclude that the genome of T. brucei contains homologous chromosomes that differ in size. The trypanosome minichromosomes are likely to constitute a separate repertoire, since they do not appear to follow Mendelian inheritance (33), and we therefore assume that the trypanosome genome is aneuploid for these minichromosomes which might represent amplified DNA (M. Weiden and L. H. T. Van der Ploeg, unpublished data). For most of the larger chromosomes, the size differences among the proposed homologs are less than 20%. The size

variability could result from recombinational events affecting the telomeric regions, as has been observed in trypanosomes and the protozoan Plasmodiumfalciparum (4, 18, 29). However, for chromosome pairs 17 and 14' and 16' and 11, this size difference is more drastic, showing a roughly twofold size difference (1.5 Mb). This larger chromosome size variability might reflect translocation events or chromosome breakage and deletions. We thank Hui-min Chung, Jin Huang, Sylvia Le Blancq, and Gloria Rudenko for critical reading of the manuscript; Paul Michels for donation of the marker genes encoding glycolytic enzymes; and Gloria Rudenko for a gift of the PARP locus-specific probes. This work was supported by Public Health Service grant Al 21784 from the National Institutes of Health and by a grant from the

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MOL. CELL. BIOL. TABLE 1. Chromosomal location of genetic markers in T. brucei stock 427-60a

CHROMOSOME SIZE

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TBS17, rRNA cI 16* 15'* TBS17, rRNA * r15 medRNAPARP A 141* gGAPDH,

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a Asterisks identify nonstoichiometric ethidium-staining bands; ' refers to the presumed second chromosome of a band; brackets designate chromosome pairs. Gene probe abbreviations: TIM, 1.2-kb AccI fragment for the triosephosphate isomerase gene (23); HSP70, 1.8-kb HindlIl fragment from the heat shock 70 locus (7); rRNA, RNA repeat clone Pr4 (9, 32); medRNA, miniexon repeat in plasmid T3/T7 (10; J. Huang, unpublished data); TuB, alpha-beta tubulin clone, pTb alpha-beta T-1 (27); ES 118, 3.0-kb PstI-KpnI fragment from a chromosome 10 specific library (K. Gottesdiener and L. H. T. Van der Ploeg, unpublished data); PLC, HindII-NdeI fragment from the phospholipase C gene (15); TBS18, cDNA encoding a T. brucei protein of unknown function (M. G. S. Lee and L. H. T. Van der Ploeg, unpublished data); ALDO, 2.1-kb AccI-EcoRV fragment from the fructose-biphosphate aldolase gene (13); CRAM, cDNA of a gene encoding a putative cell surface receptor of the flagellar pocket (11); CGAPDH, 2.9-kb BamHI genomic fragment encoding the cytosolic glyceraldehyde phosphate dehydrogenase gene (P. A. M. Michels, unpublished data). MB, Megabase pairs.

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FIG. 3. PFG electrophoresis of restriction enzyme-digested genomic DNA from variant 118 clone 1, followed by hybridization with marker located on chromosomes 18 and 19. PFG gels were run for 36 h at 10 V/cm with a pulse frequency of 25 s. Shown are hybridizations of the same filter with each probe consecutively. Probe was removed by boiling the filter for 30 min in 0.1 x SSC and 1% sodium dodecyl sulfate. Size markers in kilobases are indicated to the left and right. Posthybridization washing conditions were at 65°C with 0.1 x SSC. Probes were a 2.9-kb BamHI genomic fragment encoding the cytosolic glyceraldehyde phosphate dehydrogenase gene (cGAPDH) (P. A. M. Michels, unpublished data), a 1.0-kb AccI-PstI genomic restriction enzyme fragment from the gene for pyruvate kinase (PYK) (Michels et al., unpublished data), a 2.1-kb AccI-EcoRV fragment from the gene for fructose-biphosphate aldolase (ALDO) (13), and a cDNA for a gene encoding a putative cell surface receptor of the flagellar pocket (CRAM) (11).

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Catherine T. and John D. MacArthur Foundation to L.H.T.V.D.P. L.H.T.V.D.P. is a Burroughs Wellcome Scholar in Molecular

Parasitology.

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