ELSEVIER FEMS Microbiology Letters 129 (1995) 27-32 Downloaded from https://academic.oup.com/femsle/article/129/1/27/441076 by guest on 24 September 2021 Assignment of genes to Leishmania infantum chromosomes: karyotype and ploidy

Manuel Soto, Jose M. Requena, David Moreira, Carlos Alonso * Centro de Biologia Molecular “Se~ro Ochoa’ ‘, Unimmrdad Authoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 20 March 1995; accepted 23 March 1995

Abstract

The use of various pulsed-field electrophoresis methodologies under different conditions allowed us to determine the Leishmania infanturn karyotype. A total of 25 chromosomal bands ranging in size from 375 to 3300 kb were resolved amounting to a minimum genomic DNA mass of about 2.6 X IO’ pb. By molecular hybridization and on the basisof the karyotype. specific gene sequences could be assigned to particular chromosomes. A bias in the chromosomal distribution of different markers was found since 9 out of the 12 analysed gene markers hybridize with chromosomal bands XIXa and XIXb. We infer that chromosomal bands XlXa and XIXb, differing in about 30 kb, could be representing a pair of homologous chromosomes and that another pair of homologs may be also defined by chromosomal bands XVII and XVIII.

Keywords: Leishmania; Pulsed-field gel electrophoresis; Ploidy; Homologous chromosomes; Gene location

1. Introduction It seems that the housekeeping genes usually are located in diploid chromosomes[4], whereas part of Since genetic material of trypanosomes never its chromosomal complement could be haploid [5]. condensesduring their life cycle, karyotype analysis Based on the molecular karyotype of Leishmania through conventional cytogenetics is not possible. infantum, determined by the use of different PFGE However, the advent of pulse-field gradient gel elec- methods and conditions, we report the chromosomal trophoresis (PFGE) presentsan opportunity to char- location of several relevant genes. This study pro- acterize the number and organization of the chromo- vides strong evidence for the diploid condition of somes of parasitic protozoa. These studies have chromosomeXIX and showsthat chromosomesXVII demonstratedthe existence of variations in the chro- and XVIII may also represent a pair of homologs. mosome size and chromosomal distribution of se- lected genesamong several speciesof kinetoplastid parasites [l-3]. In spite of the knowledge about the 2. Materials and methods genome structure of these organisms, there is a controversy related with the problem of their ploidy. 2.1. Strains and media

* Corresponding author. Tel.: + 34 (1) 397 84.54; Fax: + 34 (1) Leishmania donor>ani infantum LEM75 397 4799 (MHOM/FR/78/LEM75) was used as reference

037%1097/95/$09.50 % 19YS Federation of European Microbiological Societies. All rights reserved SSDI 0378.1097(YS)OO129-8 organism, grown at 2h” C in RPM1 medium contain- stained in 0.5 X TBE containing 0.5 pg ml-’ of ing 10% of heat-inactivated fetal calf serum. ethidium bromide. Chromosome sizes were determined using the 2.2. Preparation of genotnic DNA following size standards: Pichiu sp. mix (P. scolytii and P. mississlppiensis)and Saccharomyces cere- Cells grown to stationary phase (10’ cells ml- ‘) r.i.siae YN295 as well as phage lambda concatemers (Pharmacia). were collected by centrifugation at low speed, and Downloaded from https://academic.oup.com/femsle/article/129/1/27/441076 by guest on 24 September 2021 DNA in agarose blocks was prepared as described [61. 2.4. DNA probes

The DNA probes used in the present work and their sources were: L. infanturn hsp70 and hsp83 Gels were cast with 1% agaroae in 0.5 X TBE cDNAs, and Trypunosomu cruzi 1% rDNA, cloned buffer (45 mM Tris, 45 mM borate, 1 mM EDTA, in our laboratory (unpublished); T. cruzi (Y- and pH X.0). Both orthogonal field alternating gel elec- P-tubulin cDNAs, clones pTc a3 and pTcp4 [9]; trophoresis (OFAGE) [7] and contour-clamped ho- Drosophila melanogaster ubiquitin, clone pDm63F mogeneouselectric field electrophoresis(CHEF) [8] [lo]; L. infantum histone H2A cDNA, clone cL71 were performed at 15” C in a Pharmacia-LKB appa- [ 111; L. infanturn ribosomal protein PO cDNA [12], ratus. Different chromosomesizes were resolved by cDNAs coding for the acidic ribosomal proteins LIP several pulse times between 60 and 500 s. Gels were and LIP’ [ 131; L. infuntutn histone H3 cDNA [ 141; L.

P A

Fig. I. Molecular karyotypc of I.. infumm. Two elcctrophtrretic techniques were used: OFAGE (lanes A. C) and CHEF (lanes B, B’). Pulse time and voltage conditionb WCTL‘: lane A and marker p, ilKI s and 200 V; lanes B and B’, 65-W s ramping pulse and 170 V; lane C and markers y and Ic, 6OLSOO 5 ramping pulse and 3X) V. All gels wcrc run for Jb’ h at 15” C. The chromosomal bands have been numbered from lower to higher molecular Gzc. The chromnwmal size marker\ used are: Pickia sp. mix (law p), S. crrerGze (lane y) and phage lambda concatemcrs (lane Ic). M. Soto et al. / FEMS Microbiology Letten 129 (1995) 27-32 29 infanturn PSA clone was derived from the nucleotide for more details). In agreement with previously re- sequence of the L. major promastigote surface anti- ported data for the same strain of L. infanturn [16], gen (PSA) gene [151 by PCR amplification. 24 chromosomal bands can be clearly resolved. The only difference with the data reported is that the 2.5. Hybridization conditions chromosomal band XIX was resolved in two sepa- rate bands (lane A) when a 500-s pulse was used. All hybridizations were performed at 42” C in

The size of the chromosomal bands was estimated Downloaded from https://academic.oup.com/femsle/article/129/1/27/441076 by guest on 24 September 2021 6 x SSC, 50% formamide, 1% SDS and 100 Fg from the direct correlation into the resolution win- ml-’ of denatured herring sperm DNA. The final dow for each pulse-time between the logarithm of post-hybridization washes were done in 0.1 X the band size and its relative mobility using three SSC/O.2% SDS at 60” C. standard size markers (Fig. 1). We estimated that the molecular size of the largest chromosome (XXIV) is about 3300 kb, somewhat higher than that reported 3. Results previously [16]. It is likely that the difference be- tween the two estimates is due to the high degree of Fig. 1 shows the molecular karyotype of L. infan- linear resolution which arises from the PFGE condi- turn as it results from the compositional analysis tion used. A calculation of the total DNA mass of all obtained using different pulse times and elec- the chromosomes indicates that the genomic DNA trophoretic conditions (see Materials and methods mass should be about 2.6 X 10’ bp. This amount of

2 5 6 7

- xlxa -XIX - XlXb

-x

Fig. 2. Chromosomal gene location in L. infanturn karyotype. Chromosomes were separated by PFGE and the DNA was blotted to nylon membranes and hybridized with the probes: lane I, P-tubulin; lane 2, histonc H2A (the same pattern was obtained with the probe of the histone H3); lane 3. cu-tubulin; lane 4, PSA (the same pattern was obtained with the probe Lip’); lane 5, hsp70; lane 6, PO (the same pattern was also obtained with the probes ubiquitin. LIP, hsp83 or 18s rDNA). Lane 7 illustrates the labelling of both chromosomal bands XIX by the probe LIP. Only the chromosomal bands that were labelled are numbered. 30 M. Soto et al. / FEMS Microbiology Letters 129 (1995) 27-32

DNA mass must be considered a minimum estimate of the chromosomes associated with band XIX, all of since we cannot exclude the existence in some bands the nine markers hybridized with band XIXa and of more than one chromosome of similar length. In XIXb with similar hybridization intensity (Fig. 2, fact, the intensity of the ethidium bromide staining of lane 7). Moreover, two of these markers, the acidic the chromosomal band XX, significantly higher than ribosomal protein LiP [13] and LiPO [12] are coded it would be expected for its molecular size, probably by two ligated genes, respectively. Since four of the

suggests that more than a single chromosome exist in genetic markers belonging to the histone H2A, his- Downloaded from https://academic.oup.com/femsle/article/129/1/27/441076 by guest on 24 September 2021 that band. tone H3, P-tubulin and hsp70 genes that hybridized In order to assign the location of genes to chro- with the XIXa and XIXb chromosomal bands also mosomal bands and based in the rationale that if a hybridize with chromosomal bands XIV, XIV, VIII group of genes have alleles in chromosomes of and XIV, and with X, respectively, it is likely that in similar molecular size they could be considered ho- L.infantum a group of gene families might exist that mologous we have determined the chromosomal lo- map to multiple unlinked chromosomal loci, as re- cation of 12 different genetic markers. Fig. 2 (lanes ported for the L. major tubulin gene family [17]. l-6) and Table 1 show examples of the chromoso- The promastigote surface antigen and ribosomal Lip’ ma1 location of some of the markers and a summary protein DNA markers hybridize with chromosomal of the chromosomal positioning of all the markers bands XVII and XVIII. Since in the Leishmania tested, respectively. The experiments also indicated genome there are only two genes tandemly arrayed, that 9 out of the 12 genetic markers locate in the coding for the ribosomal Lip’ proteins [13], it is chromosomal band XIX and this band could be probable that also thesetwo bandscan be considered resolved in two. As expected from a diploid nature as a pair of homologs.

Table I Karyotypc analysis of Leishmania infanturn Chromosomal band Band size (kb) Gene locations XXXIV 3306 XXIII 2703 XXII 2164 XXI I889 xx 1731 XIXb 1316 hsp70, P-tubulin, ubiquitin. histone HZA. XIXa 1286 18s rRNA, LIP, LiPO. hsp83, histone H3 XVIII 1086 PSA. LIP’ XVII 998 XVI 884 xv 822 XIV 7X8 Histone H2A, P-tubulin, histone H3 XIII 749 XII 713 XI 673 X 644 hsp70, cr.tubulin IX 588 VIII 567 P-Tubulin VII 543 VI 519 V 505 IV 479 III 462 11 399 37s M. Soto el al. / FEMS Microbiology Letters 129 (19951 27-32 31

4. Discussion port: Plan Regional de la Comunidad de Madrid (160/9), CICYT SAF93-0146, LET1 S.A. and Insti- A combination of two methodsof pulsed-field gel tutional grant of the Fundacion Ramon Areces. electrophoresis (PFGEI using different pulse times have been used to accurately resolve the molecular karyotype of L. infanlum. The number of well re- References solved chromosomal bands for this strain was 25 Downloaded from https://academic.oup.com/femsle/article/129/1/27/441076 by guest on 24 September 2021 ranging in size from 375 to 3300 kb. The pattern of [1] Van der Ploeg. L.H.T., Schwartz, D.C.. Cantor. CR. and the molecular karyotype is similar to that previously Borst. P. (19841 Antigenic variation in Trypanosom~a hrucei reported by Pageset al. [16] with two variations: (i) analyzed by clectrophoretic separation of chromosome-sized DNA molecules. Cell 37, 77-84. the chromosomal band XIX has been resolved in [2] Gibson, W.C., Osinga, K., Michels, P.A.M. and Borst, P. two; and (ii) the estimatedsize of the larger chromo- (1985) Trypanosomcs of the subgenus trypanozoon arc somal band is higher than that previously reported. diploid for housekeeping genes. Mol. Biochem. Parasitol. 16, We have found that 75% of the genetics markers 23 l-242. used map in chromosomeXIX. Since there is no bias [3] Gibson. W.C. and Miles, M.A. (19861 The karyotype and in the election of the genetic markers and most of ploidy of Trypanotama cruzi. EMBO J. 5, 1299-1305. [4] Lighthall, G.K. and Gianini. S.H. (19921 The chromosomes them hybridize with equal intensity to chromosomal of L&hmania. Parasitol. Today 8, lY2-199. bands XIXa and XIXb, which differ in only 30 kb, [5] Bastien, P.. Blaineau. C. and Pages. M. (19921 Lrishmania: based in the rationale previously indicated, we think sex. lies and karyotypes. Parasitol. Today 8, 174-177. that they can be consideredto be homologs. The fact [h] Galindo, I. and Ramirez-Ochoa, J.L. (lYS91 Study of Lrish- that the chromosomal bands XIXa and XIXb hold maniu mexicana electrokaryotypc by clamped homogeneous electric field elcctrophoresis. Mol. Biochcm. Parasitol. 34, homologous chromosomes is in addition suggested 245-251. by the genomic restriction analysis of the DNA [7] Carlc, G.F. and Olson. M.V. (lY841 Separation of chromoso- regions containing the LiP and LiPO genes [12,13], ma1 DNA molecules from yeast by orthogonal-ficld-alterna- which indicates that these genes are located in ho- tion gel clcctrophorcsis. Nucleic Acids Res. 12. 5647-5664. mologous chromosomal loci. Since the genescoding [Xl Chu. G.. Vollrath, D. and Davies, R.W. (19861 Separation of large DNA molecules by contour-clamped homogcnous elec- for the promastigote surface antigen and the LIP’ tric fields. Science 234, 15X2-1585. protein locate in chromosomal bands XVII and [Y] Soarcs, C.M.A., De Carvalho, E.F., Urmcnti, T.P., Carvalho, XVIII, it also may well be that these bands, differing J.F.O., DC Castro. F.T. and Rondinelli, E. (19891 Alpha- and in 90 kb, may hold another pair of homologs. beta-tubulin mRNAs of Trypanmoma cruzi originated from a single multicistronic transcript. FEBS Lett. 250, 497-502. As indicated by Bastien [S], the existence of [IO] Izquicrdo, M., Arribas, C., Galceran, J., Burke, .J. and Cabr- diploidy in Leishmania is currently a controversial era. V.M. (19841 Characterization of a Drosophila repeat question because supporting evidence is not abun- mapping at the early-ecdysonc puff 63F and present in many dant. Either the parasitecan be consideredas haploid eucaryotic gcnomes. Biochim. Biophys. Acta 783, 114-121. being disomic or polysomic for one or more chromo- [ll] Soto, M.. Requcna, J.M.. G6me.z. L.C., Navarretc, I. and somesor as diploid having two sets of almost identi- Alonxo, C. (19921 Molecular characterization of a Leishma- nia donmani irlfantum antigen identified as histone H2A. cal chromosomes.Although the data presented can- Eur. J. Biochem. 205, 211-216. not discriminate between these two alternatives as [12] Soto, M., Requena, J.M. and Alonso. C. (1993) Isolation, far as the entire genome, they favor the hypothesis of characterization and analysis of the expression of the I.&h- the diploid condition of L. infantum, since there are mrrnicr rihosomal PO protein genes. Mol. Biol. Parasitol. 6 1, 265-274. at least two pairs of similar sized homologous chro- [13] Soto. M.. Requena, J.M., Garcia. M.. Gomcz, L.C., Navar- mosomes. rcte. 1. and Alonso. C. (19931 Genomic organization and expression of two independent gene arrays coding for two antigcnic acidic ribosomal proteins of Leishmania. J. Biol. Acknowledgements Chcm. 268, 21835-21843. [14] Soto, M., Requena, J.M.. Morales, G. and Alonso, C. (1994) The Lrishmania infanturn histone H3 possesses an extremely We thank Drs. E. Rondinelli and M. Izquierdo for divergent N-terminal domain. Biochim. Biophys. Acta 1219, the supply of some of the DNA probes. Grant sup- 533-535. 32 M. Soto et al. / FEMS Microbiology Letters 129 (19951 27-32

[15] Murray, P.J. and Spithill, T.W. (1991) Variants of a L&t- suggest amplification/deletion and possible genetic ex- mania surface antigen derived from a multigenic family. J. change. Mol. Biochem. Parasitol. 36, 161-168. Biol. Chem. 266, 24477-24484. [17] Spithill, T.W. and Samaras, N. (1985) The molecular kary- [16] Pages, M., Bastien, P., Veas, F., Rossi, V., Bellis, M., otype of Leishmania major and mapping of (Y and p tubulin Wincker, P., Rioux, J.A. and Roizes, G. (1989) Chromosome gene families to multiple unlinked chromosomal loci. Nu- size and number polymorphisms in Leishmania infantum cleic Acids Res. 13, 4155-4169. Downloaded from https://academic.oup.com/femsle/article/129/1/27/441076 by guest on 24 September 2021