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Assignment of genes to Leishmania infantum chromosomes: karyotype and ploidy
ELSEVIER
FEMS
Microbiology
Letters
129 (1995)
27-32
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’
Received
‘, Unimmrdad
20 March
Authoma
de Madrid,
1995; accepted 23 March
Cantoblanco,
28049 Madrid,
Spain
1995
Abstract The use of various
pulsed-field electrophoresis methodologies under different conditions allowed us to determine the 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.
Leishmania
infanturn
Keywords:
Leishmania;
Pulsed-field
gel electrophoresis;
Ploidy; Homologous
1. Introduction Since genetic material of trypanosomes never condensesduring their life cycle, karyotype analysis through conventional cytogenetics is not possible. However, the advent of pulse-field gradient gel electrophoresis (PFGE) presentsan opportunity to characterize the number and organization of the chromosomes of parasitic protozoa. These studies have demonstratedthe existence of variations in the chromosome size and chromosomal distribution of selected genesamong several speciesof 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 397 4799
author.
Tel.: + 34 (1) 397 84.54; Fax: + 34 (1)
037%1097/95/$09.50 % 19YS Federation SSDI 0378.1097(YS)OO129-8
chromosomes; Gene location
It seems that the housekeeping genes usually are located in diploid chromosomes[4], whereaspart 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 provides strong evidence for the diploid condition of chromosomeXIX and showsthat chromosomesXVII and XVIII may also represent a pair of homologs.
2. Materials and methods 2.1. Strains Leishmania
and media donor>ani
(MHOM/FR/78/LEM75) of European
Microbiological
Societies.
All rights reserved
infantum LEM75 was used as reference
organism, grown at 2h” C in RPM1 medium containing 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 electrophoresis (OFAGE) [7] and contour-clamped homogeneouselectric field electrophoresis(CHEF) [8] were performed at 15” C in a Pharmacia-LKB apparatus. Different chromosomesizes were resolved by several pulse times between 60 and 500 s. Gels were
P
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 cerer.i.siaeYN295 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.
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
infanturn PSA clone was derived from the nucleotide sequence of the L. major promastigote surface antigen (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. infanturn as it results from the compositional analysis obtained using different pulse times and electrophoretic conditions (see Materials and methods
2
Letten
129
(1995)
29
27-32
for more details). In agreement with previously reported 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 separate 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 window 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 between the two estimates is due to the high degree of linear resolution which arises from the PFGE condition 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
-XIX
-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
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 chromosomal bands and based in the rationale that if a group of genes have alleles in chromosomes of similar molecular size they could be considered homologous we have determined the chromosomal location of 12 different genetic markers. Fig. 2 (lanes l-6) and Table 1 show examples of the chromosoma1 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
Table I Karyotypc Chromosomal
XXXIV XXIII XXII XXI xx XIXb XIXa XVIII XVII XVI xv XIV XIII XII XI X IX VIII VII VI V IV III 11
analysis band
of Leishmania
Letters
129 (1995)
27-32
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, histone 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 reported 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 thesetwo bandscan be considered as a pair of homologs.
infanturn Band size (kb) 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
Gene locations
hsp70, P-tubulin, ubiquitin. histone HZA. 18s rRNA, LIP, LiPO. hsp83, histone H3 PSA. LIP’
Histone
H2A,
hsp70, cr.tubulin P-Tubulin
P-tubulin,
histone H3
M. Soto el al. / FEMS
Microbiology
4. Discussion
A combination of two methodsof 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 resolved 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 Pageset al. [16] with two variations: (i) the chromosomal band XIX has been resolved in two; and (ii) the estimatedsize of the larger chromosomal band is higher than that previously reported. We have found that 75% of the genetics markers used map in chromosomeXIX. 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 consideredto be homologs. The fact that the chromosomal bands XIXa and XIXb hold homologous chromosomesis 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 homologouschromosomalloci. Since the genescoding 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 abundant. Either the parasitecan be consideredas haploid being disomic or polysomic for one or more chromosomesor as diploid having two setsof almost identical chromosomes.Although the data presented cannot 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 chromosomes.
Acknowledgements We thank Drs. E. Rondinelli and M. Izquierdo for the supply of some of the DNA probes. Grant sup-
Letters
129 (19951 27-32
31
port: Plan Regional de la Comunidad de Madrid (160/9), CICYT SAF93-0146, LET1 S.A. and Institutional grant of the Fundacion Ramon Areces.
References [1] Van der Ploeg. L.H.T., Schwartz, D.C.. Cantor. CR. and Borst. P. (19841 Antigenic variation in Trypanosom~a hrucei analyzed by clectrophoretic separation of chromosome-sized DNA molecules. Cell 37, 77-84. [2] Gibson, W.C., Osinga, K., Michels, P.A.M. and Borst, P. (1985) Trypanosomcs of the subgenus trypanozoon arc diploid for housekeeping genes. Mol. Biochem. Parasitol. 16, 23 l-242. [3] Gibson. W.C. and Miles, M.A. (19861 The karyotype and ploidy of Trypanotama cruzi. EMBO J. 5, 1299-1305. [4] Lighthall, G.K. and Gianini. S.H. (19921 The chromosomes of L&hmania. Parasitol. Today 8, lY2-199. [5] Bastien, P.. Blaineau. C. and Pages. M. (19921 Lrishmania: sex. lies and karyotypes. Parasitol. Today 8, 174-177. [h] Galindo, I. and Ramirez-Ochoa, J.L. (lYS91 Study of Lrishmaniu mexicana electrokaryotypc by clamped homogeneous electric field elcctrophoresis. Mol. Biochcm. Parasitol. 34, 245-251. [7] Carlc, G.F. and Olson. M.V. (lY841 Separation of chromosoma1 DNA molecules from yeast by orthogonal-ficld-alternation gel clcctrophorcsis. Nucleic Acids Res. 12. 5647-5664. [Xl Chu. G.. Vollrath, D. and Davies, R.W. (19861 Separation of large DNA molecules by contour-clamped homogcnous electric fields. Science 234, 15X2-1585. [Y] Soarcs, C.M.A., De Carvalho, E.F., Urmcnti, T.P., Carvalho, J.F.O., DC Castro. F.T. and Rondinelli, E. (19891 Alpha- and beta-tubulin mRNAs of Trypanmoma cruzi originated from a single multicistronic transcript. FEBS Lett. 250, 497-502. [IO] Izquicrdo, M., Arribas, C., Galceran, J., Burke, .J. and Cabrera. V.M. (19841 Characterization of a Drosophila repeat mapping at the early-ecdysonc puff 63F and present in many eucaryotic gcnomes. Biochim. Biophys. Acta 783, 114-121. [ll] Soto, M.. Requcna, J.M.. G6me.z. L.C., Navarretc, I. and Alonxo, C. (19921 Molecular characterization of a Leishmania donmani irlfantum antigen identified as histone H2A. Eur. J. Biochem. 205, 211-216. [12] Soto, M., Requena, J.M. and Alonso. C. (1993) Isolation, characterization and analysis of the expression of the I.&hmrrnicr rihosomal PO protein genes. Mol. Biol. Parasitol. 6 1, 265-274. [13] Soto. M.. Requena, J.M., Garcia. M.. Gomcz, L.C., Navarrcte. 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. Chcm. 268, 21835-21843. [14] Soto, M., Requena, J.M.. Morales, G. and Alonso, C. (1994) The Lrishmania infanturn histone H3 possesses an extremely divergent N-terminal domain. Biochim. Biophys. Acta 1219, 533-535.
32
M. Soto et al. / FEMS
Microbiology
[15] Murray, P.J. and Spithill, T.W. (1991) Variants of a L&tmania surface antigen derived from a multigenic family. J. Biol. Chem. 266, 24477-24484. [16] Pages, M., Bastien, P., Veas, F., Rossi, V., Bellis, M., Wincker, P., Rioux, J.A. and Roizes, G. (1989) Chromosome size and number polymorphisms in Leishmania infantum
Letters
129 (19951 27-32
suggest amplification/deletion and possible genetic exchange. Mol. Biochem. Parasitol. 36, 161-168. [17] Spithill, T.W. and Samaras, N. (1985) The molecular karyotype of Leishmania major and mapping of (Y and p tubulin gene families to multiple unlinked chromosomal loci. Nucleic Acids Res. 13, 4155-4169.
FEMS
Microbiology
Letters
129 (1995)
27-32
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’
Received
‘, Unimmrdad
20 March
Authoma
de Madrid,
1995; accepted 23 March
Cantoblanco,
28049 Madrid,
Spain
1995
Abstract The use of various
pulsed-field electrophoresis methodologies under different conditions allowed us to determine the 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.
Leishmania
infanturn
Keywords:
Leishmania;
Pulsed-field
gel electrophoresis;
Ploidy; Homologous
1. Introduction Since genetic material of trypanosomes never condensesduring their life cycle, karyotype analysis through conventional cytogenetics is not possible. However, the advent of pulse-field gradient gel electrophoresis (PFGE) presentsan opportunity to characterize the number and organization of the chromosomes of parasitic protozoa. These studies have demonstratedthe existence of variations in the chromosome size and chromosomal distribution of selected genesamong several speciesof 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 397 4799
author.
Tel.: + 34 (1) 397 84.54; Fax: + 34 (1)
037%1097/95/$09.50 % 19YS Federation SSDI 0378.1097(YS)OO129-8
chromosomes; Gene location
It seems that the housekeeping genes usually are located in diploid chromosomes[4], whereaspart 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 provides strong evidence for the diploid condition of chromosomeXIX and showsthat chromosomesXVII and XVIII may also represent a pair of homologs.
2. Materials and methods 2.1. Strains Leishmania
and media donor>ani
(MHOM/FR/78/LEM75) of European
Microbiological
Societies.
All rights reserved
infantum LEM75 was used as reference
organism, grown at 2h” C in RPM1 medium containing 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 electrophoresis (OFAGE) [7] and contour-clamped homogeneouselectric field electrophoresis(CHEF) [8] were performed at 15” C in a Pharmacia-LKB apparatus. Different chromosomesizes were resolved by several pulse times between 60 and 500 s. Gels were
P
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 cerer.i.siaeYN295 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.
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
infanturn PSA clone was derived from the nucleotide sequence of the L. major promastigote surface antigen (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. infanturn as it results from the compositional analysis obtained using different pulse times and electrophoretic conditions (see Materials and methods
2
Letten
129
(1995)
29
27-32
for more details). In agreement with previously reported 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 separate 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 window 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 between the two estimates is due to the high degree of linear resolution which arises from the PFGE condition 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
-XIX
-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
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 chromosomal bands and based in the rationale that if a group of genes have alleles in chromosomes of similar molecular size they could be considered homologous we have determined the chromosomal location of 12 different genetic markers. Fig. 2 (lanes l-6) and Table 1 show examples of the chromosoma1 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
Table I Karyotypc Chromosomal
XXXIV XXIII XXII XXI xx XIXb XIXa XVIII XVII XVI xv XIV XIII XII XI X IX VIII VII VI V IV III 11
analysis band
of Leishmania
Letters
129 (1995)
27-32
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, histone 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 reported 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 thesetwo bandscan be considered as a pair of homologs.
infanturn Band size (kb) 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
Gene locations
hsp70, P-tubulin, ubiquitin. histone HZA. 18s rRNA, LIP, LiPO. hsp83, histone H3 PSA. LIP’
Histone
H2A,
hsp70, cr.tubulin P-Tubulin
P-tubulin,
histone H3
M. Soto el al. / FEMS
Microbiology
4. Discussion
A combination of two methodsof 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 resolved 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 Pageset al. [16] with two variations: (i) the chromosomal band XIX has been resolved in two; and (ii) the estimatedsize of the larger chromosomal band is higher than that previously reported. We have found that 75% of the genetics markers used map in chromosomeXIX. 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 consideredto be homologs. The fact that the chromosomal bands XIXa and XIXb hold homologous chromosomesis 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 homologouschromosomalloci. Since the genescoding 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 abundant. Either the parasitecan be consideredas haploid being disomic or polysomic for one or more chromosomesor as diploid having two setsof almost identical chromosomes.Although the data presented cannot 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 chromosomes.
Acknowledgements We thank Drs. E. Rondinelli and M. Izquierdo for the supply of some of the DNA probes. Grant sup-
Letters
129 (19951 27-32
31
port: Plan Regional de la Comunidad de Madrid (160/9), CICYT SAF93-0146, LET1 S.A. and Institutional grant of the Fundacion Ramon Areces.
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