FEMS Microbiology Letters 129 (1995) 27-32 ELSEVIER

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 basis of 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

Since genetic material of trypanosomes never condenses during their life cycle, karyotype analysis through conventional cytogenetics is not possible. However, the advent of pulse-field gradient gel elec- trophoresis (PFGE) presents an opportunity to char- acterize the number and organization of the chromo- somes of parasitic protozoa. These studies have demonstrated the existence of variations in the chro- mosome size and chromosomal distribution of se- lected genes among several species of kinetoplastid parasites [l-3]. In spite of the knowledge about the genome structure of these organisms, there is a controversy related with the problem of their ploidy.

* Corresponding author. Tel.: + 34 (1) 397 84.54; Fax: + 34 (1) 397 4799

It seems that the housekeeping genes usually are located in diploid chromosomes [4], whereas part of its chromosomal complement could be haploid [5].

Based on the molecular karyotype of Leishmania infantum, determined by the use of different PFGE methods and conditions, we report the chromosomal location of several relevant genes. This study pro- vides strong evidence for the diploid condition of chromosome XIX and shows that chromosomes XVII and XVIII may also represent a pair of homologs.

2. Materials and methods

2.1. Strains and media

Leishmania donor>ani infantum LEM75 (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- ing 10% of heat-inactivated fetal calf serum.

2.2. Preparation of genotnic DNA

Cells grown to stationary phase (10’ cells ml- ‘) were collected by centrifugation at low speed, and DNA in agarose blocks was prepared as described

[61.

Gels were cast with 1% agaroae in 0.5 X TBE buffer (45 mM Tris, 45 mM borate, 1 mM EDTA, pH X.0). Both orthogonal field alternating gel elec- trophoresis (OFAGE) [7] and contour-clamped ho- mogeneous electric field electrophoresis (CHEF) [8] were performed at 15” C in a Pharmacia-LKB appa- ratus. Different chromosome sizes were resolved by several pulse times between 60 and 500 s. Gels were

P A

stained in 0.5 X TBE containing 0.5 pg ml-’ of ethidium bromide.

Chromosome sizes were determined using the following size standards: Pichiu sp. mix (P. scolytii and P. mississlppiensis) and Saccharomyces cere- r.i.siae YN295 as well as phage lambda concatemers (Pharmacia).

2.4. DNA probes

The DNA probes used in the present work and their sources were: L. infanturn hsp70 and hsp83 cDNAs, and Trypunosomu cruzi 1% rDNA, cloned in our laboratory (unpublished); T. cruzi (Y- and P-tubulin cDNAs, clones pTc a3 and pTcp4 [9]; Drosophila melanogaster ubiquitin, clone pDm63F [lo]; L. infantum histone H2A cDNA, clone cL71 [ 111; L. infanturn ribosomal protein PO cDNA [12], cDNAs coding for the acidic ribosomal proteins LIP and LIP’ [ 131; L. infuntutn histone H3 cDNA [ 141; L.

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 sequence of the L. major promastigote surface anti- gen (PSA) gene [151 by PCR amplification.

2.5. Hybridization conditions

All hybridizations were performed at 42” C in 6 x SSC, 50% formamide, 1% SDS and 100 Fg ml-’ of denatured herring sperm DNA. The final post-hybridization washes were done in 0.1 X

SSC/O.2% SDS at 60” C.

3. Results

Fig. 1 shows the molecular karyotype of L. infan- turn as it results from the compositional analysis obtained using different pulse times and elec- trophoretic conditions (see Materials and methods

2

-XIX

for more details). In agreement with previously re- ported data for the same strain of L. infanturn [16], 24 chromosomal bands can be clearly resolved. The only difference with the data reported is that the chromosomal band XIX was resolved in two sepa- rate bands (lane A) when a 500-s pulse was used.

The size of the chromosomal bands was estimated from the direct correlation into the resolution win- dow for each pulse-time between the logarithm of the band size and its relative mobility using three 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 previously [16]. It is likely that the difference be- tween the two estimates is due to the high degree of linear resolution which arises from the PFGE condi- tion used. A calculation of the total DNA mass of all the chromosomes indicates that the genomic DNA mass should be about 2.6 X 10’ bp. This amount of

5 6 7

- xlxa - 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 since we cannot exclude the existence in some bands of more than one chromosome of similar length. In fact, the intensity of the ethidium bromide staining of the chromosomal band XX, significantly higher than it would be expected for its molecular size, probably suggests that more than a single chromosome exist in that band.

In order to assign the location of genes to chro- mosomal bands and based in the rationale that if a group of genes have alleles in chromosomes of similar molecular size they could be considered ho- mologous we have determined the chromosomal lo- cation of 12 different genetic markers. Fig. 2 (lanes l-6) and Table 1 show examples of the chromoso- ma1 location of some of the markers and a summary of the chromosomal positioning of all the markers tested, respectively. The experiments also indicated that 9 out of the 12 genetic markers locate in the chromosomal band XIX and this band could be resolved in two. As expected from a diploid nature

of the chromosomes associated with band XIX, all of the nine markers hybridized with band XIXa and XIXb with similar hybridization intensity (Fig. 2, lane 7). Moreover, two of these markers, the acidic ribosomal protein LiP [13] and LiPO [12] are coded by two ligated genes, respectively. Since four of the genetic markers belonging to the histone H2A, his- tone H3, P-tubulin and hsp70 genes that hybridized with the XIXa and XIXb chromosomal bands also hybridize with chromosomal bands XIV, XIV, VIII and XIV, and with X, respectively, it is likely that in L.infantum a group of gene families might exist that map to multiple unlinked chromosomal loci, as re- ported for the L. major tubulin gene family [17]. The promastigote surface antigen and ribosomal Lip’ protein DNA markers hybridize with chromosomal bands XVII and XVIII. Since in the Leishmania

genome there are only two genes tandemly arrayed, coding for the ribosomal Lip’ proteins [13], it is probable that also these two bands can be considered as a pair of homologs.

Table I Karyotypc analysis of Leishmania infanturn

Chromosomal band Band size (kb) Gene locations

XXXIV XXIII XXII XXI xx XIXb XIXa XVIII XVII XVI xv

XIV XIII XII

XI X IX VIII VII

VI V IV III 11

3306 2703

2164 I889 1731 1316

1286 1086 998 884 822

7X8 749 713 673 644

588 567 543

519 505 479 462 399

37s

hsp70, P-tubulin, ubiquitin. histone HZA.

18s rRNA, LIP, LiPO. hsp83, histone H3 PSA. LIP’

Histone H2A, P-tubulin, histone H3

hsp70, cr.tubulin

P-Tubulin

M. Soto el al. / FEMS Microbiology Letters 129 (19951 27-32 31

4. Discussion

A combination of two methods of pulsed-field gel electrophoresis (PFGEI using different pulse times have been used to accurately resolve the molecular karyotype of L. infanlum. The number of well re- solved chromosomal bands for this strain was 25 ranging in size from 375 to 3300 kb. The pattern of the molecular karyotype is similar to that previously reported by Pages et al. [16] with two variations: (i) the chromosomal band XIX has been resolved in two; and (ii) the estimated size of the larger chromo- somal band is higher than that previously reported.

We have found that 75% of the genetics markers used map in chromosome XIX. Since there is no bias in the election of the genetic markers and most of them hybridize with equal intensity to chromosomal bands XIXa and XIXb, which differ in only 30 kb, based in the rationale previously indicated, we think that they can be considered to be homologs. The fact that the chromosomal bands XIXa and XIXb hold homologous chromosomes is in addition suggested by the genomic restriction analysis of the DNA regions containing the LiP and LiPO genes [12,13], which indicates that these genes are located in ho- mologous chromosomal loci. Since the genes coding for the promastigote surface antigen and the LIP’ protein locate in chromosomal bands XVII and

XVIII, it also may well be that these bands, differing in 90 kb, may hold another pair of homologs.

As indicated by Bastien [S], the existence of diploidy in Leishmania is currently a controversial question because supporting evidence is not abun- dant. Either the parasite can be considered as haploid being disomic or polysomic for one or more chromo- somes or as diploid having two sets of almost identi- cal chromosomes. Although the data presented can- not discriminate between these two alternatives as far as the entire genome, they favor the hypothesis of the diploid condition of L. infantum, since there are at least two pairs of similar sized homologous chro- mosomes.

Acknowledgements

We thank Drs. E. Rondinelli and M. Izquierdo for the supply of some of the DNA probes. Grant sup-

port: Plan Regional de la Comunidad de Madrid (160/9), CICYT SAF93-0146, LET1 S.A. and Insti- tutional grant of the Fundacion Ramon Areces.

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