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Cloning and Structural Analysis of the Gene Encoding the Ribosomal Protein S6 from the ParasiteLeishmania infantum
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
248, 464–468 (1998)
RC988927
Cloning and Structural Analysis of the Gene Encoding the Ribosomal Protein S6 from the Parasite Leishmania infantum Gloria Gonzalez-Aseguinolaza, Soraya Taladriz, Alberto Marquet, and Vicente Larraga1 Centro de Investigaciones Biolo´gicas, CSIC, Velazquez 144, Madrid 2006, Spain
Received May 15, 1998
We have cloned the S6 ribosomal protein encoding gene from a Leishmania infantum cDNA library. This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changes in vitro and results a good model to study cell differentiation processes. The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes. Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites. However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues. The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite. q 1998 Academic Press
Leishmania infantum is a parasite protozoon responsible for the visceral form of leishmaniasis in the Mediterranean basin (1). Leishmania has a digenetic lifecycle, proliferating in the insect vector, the sandfly, as extracelular flagellated promastigote and in the mammalian host as intracellular non flagellated amastigote (2). Along this life-cycle the parasite is affected by environmental changes, mainly in pH and temperature, that represent signals for cell differentiation (3,4).The differentiation changes can be mimicked by in vitro culture from avirulent promastigotes in the early logarithmic phase to virulent metacyclic promastigotes ready to enter in the host macrophage, in the late loga1 Corresponding author: Fax: 341 5627518. E-mail: vlarraga@ fresno.csic.es. GenBank Accession Number for the sequence reported here: AFO45457.
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rithmic and the stationary phase (5). There have been detected changes in protein phosphorylation during this type of transformation (6-8). As protein phosphorylation is involved in many essential eukaryotic processes such as cell division, proliferation and differentiation, transport, cell activation, oncogenesis, etc. . . . Leishmania results an interesting model to study several of these processes. The S6 protein is the major substrate for protein phosphorylation in the 40 S ribosomal subunit of eukaryotic cells (9,10). Several studies have shown that S6 is multiple phosphorylated on serine residues as the result of stimulation of a variety of cells by different agents such mitogens, viruses, oncogene products, phorbol esters, progesterone or insulin (11-14). Furthermore, stimulation of protein synthesis concomitant with a stimulatory effect on elongation and with phosphorylation of the ribosomal protein S6 has also been described (15). Then, the study of this ribosomal protein in a protozoon results of interest in order to compare their structural and functional similarities with the other eukaryotes studied so far. We report here the cloning and structural analysis of the gene encoding for the S6 ribosomal protein from Leishmania infantum. The information provided by the data on structure show differences in the protein regions and phosphorylation sites with respect to the corresponding proteins from mammalians, Drosophila, Xenopus or yeasts. The possible functional relevance of these data with respect to that found in other eukaryotic ribosomal protein genes are discussed. MATERIALS AND METHODS Cloning of the LiS6 ribosomal protein encoding gene. Leishmania infantum PB75 promastigotes (kindly provided by Dr. J. Alvar. Reference Parasitology Service. Instituto de Salud Carlos III. Majadahonda. Spain) were cultured in vitro at 287C in RPMI 1640 (GIBCO, Middlesex, UK) supplemented with 10% of heat inactivated fetal calf serum (Flow Laboratories, UK). Cultures were initiated at 11106 parasites/ml and harvested from late logarithmic/early stationary
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FIG. 1. Nucleotide and deduced amino acid sequences of the L. infantum S6 ribosomal protein gene. The nucleotide sequence is displayed in the 5* to 3* direction with the first base of the ATG initiation codon at position 1 and the third base of the TAG stop codon at position 750. The derived aminoacid sequence is shown in the single letter amino acid code. The S6 signature sequence is marked by a shadowed box. Open boxes at positions 170 and 191 indicate the nucleus entrance signals. The beta casein kinase phosphorylation sites at positions 151 and 205 are marked by open squares. The open circle represents the phoshorylation residue for cGMP protein kinase. Protein kinase C residues are marked by black circles.
phase cultures as defined by morphology and cell concentration (16). Total RNA from promastigotes was prepared using Trizol (Gibco BRL). A cDNA library was constructed in a l-ZAP Express vector using Poly(A)/RNA isolated using Biotinylated Oligo (dT) probe, the hybrids were captured with streptavidin coupled to magnetic particles and magnetic separation stand, washed and eluted according to manufacturer’s instructions (PolyATtact mRNA Isolation systems, Promega). On the basis of an S6 conserved sequence ISGGNDKQGFPM a degenerate oligonucleotide was designed according to the codon preference showed in Leishmania (17). The oligonucleotide probe was 5* end labelled with T4 polynucleotide kinase (New England Biolabs) and [a-32P] ATP and used to screen the cDNA library. This strategy yielded seven independent clones and for each of them a pBluescript plasmid derivate was excised from the ZAP-EXPRESS cloning vector. The cDNAs were sized on agarose gels and the plasmid with the largest insert, LiRS6-2, was selected. The nucleotide sequence was determined using the dideoxy chain method on an automated DNA sequencer (ABI PRISM DyeTerminator Cycle sequencing with ampliTaq DNA Polymerase, Perkin Elmer). Sequence comparison analysis of the proteins were carried out using the Altschul et al. algorithm (18). The sequences of the different species considered have been aligned using the CLUSTALW multiple sequence alignment method using default values (19). Isolation and structural analysis of the LiS6 genomic clone. Total genomic DNA from Leishmania infantum was prepared as described (20) digested with the indicated restriction enzymes, resolved on 0.7% agarose gels and transferred to nylon membranes (Hybond-N, Amersham). Total RNA was isolated from the promastigotes culture at daily intervals as described above. Southern and Northern blot hybridizations were done following standard procedures (21). In short, the blots were hybridized at 657C for 16 h in 6 1 Ssc, 5 1 Denhart’s mix, 0.5 % SDS, 100ml/ml salmon sperm DNA with a radiolabelled cDNA probe prepared by randomly primed synthesis with Klenow DNA polymerase and [a-32P]dCTP. Filters were washed twice at room temperature for 10 min in 2 1 SSC, 0.1% SDS, followed by two washes of 15 min at 657C in 0.1 1 SSC, 0.1% SDS.
RESULTS AND DISCUSSION Cloning and sequencing of the LiS6 ribosomal protein encoding gene. The cDNA insert in the clone LiRS62 contains 852 nucleotides and includes a single open reading frame and the 3* flanking region. The length of the Poly(A) tail is of 42 nucleotides. The open reading frame of 624 nucleotides codifies for a 249 aminoacids protein, with a calculated molecular weight of 28,290 Da. The nucleotide sequence (GenBank accession number AFO45457) and the deduced amino acid sequence are shown in Fig 1. As can be seen, the ATG initiation codon is located at position one and the stop codon TAG is located at position 750. The LiS6 protein has an excess of basic residues over the acidic ones with a theoretical pI of 11.45. The protein displays regions highly conserved with respect to other S6 proteins from different origins as well as remarkable differences. The S6 signature sequence is located between the fifty-five and sixty-four amino acid positions with two non conserved changes at position 57 (SrN) and at position 60 (DrQ), (see the shadowed box). There are two sequences corresponding to nucleus entrance signals at positions 170KKDR and 191-RAKK, (open boxes in the figure). Two Casein kinase II phosphorylation sites are present in positions 151 and 205, a Tyrosine kinase phosphorylation site sequence between the 152 and the 159 positions and a cGMP kinase phosphorylation site at position 139. The ribosomal S6 protein from L.infantum presents remarkable differences with those described so far specially in higher eukaryotes (22-26). In the
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FIG. 2. L. infantum S6 ribosomal amino acid sequence and comparison to other eukaryotic species. (1) Schizosaccharomyces pombe (GenBank Z54308); (2) Saccharomyces cerevisiae (GenBank P02365); (3) Kluyveromyces marxianus (GenBank S53430); (4) Leishmania infantum (bold letters); (5) Human (GenBank Z59209); (6) Chicken (GenBank X81968); (7) Xenopus laevis (GenBank P39017); (8) Drosophila melanogaster (GenBank P29327). Missing amino acids are shown by dashes. Asterisks indicate the residues corresponding to Protein kinase C phosphorylation sites absent in L.infantum S6 and present in higher eukaryotes species. Open box at position 12 indicate a common phosphorylation residue of LiS6 with yeasts. Open box at position 139 indicates a shared cAGMP phosphorylation site with the higher eukaryotes. Shadowed box indicate the common beta tyrosine kinase phosphorylation site.
protozoon protein there are nine PKC phosphorylation sites, being four of them threonine instead of serine sites, the usual amino acid residue in the other eukaryotes and are located in different positions of the protein, (black circles in the figure). Fig 2 shows the comparison between the L.infantum S6 and ribosomal S6 proteins from different sources both from higher and lower eukaryotes. The protozoon LiS6 presents the same size of the higher eukaryotes and shares the S6 signature sequence. In addition it presents one cGMP dependent kinase phosphorylation site at position 139 (open box) similar to that of the high eukaryotes, as well as the beta Tyrosine kinase phosphorylation site with human, chicken, Xenopus and Kluyveromyces but not with other yeast species and Drosophila, (shadowed box). The key functional PKC phosphorylation sites present clear differences. LiS6 shares a phosphorylation site with all the three yeast species considered at position 12 and one to three additional sites depending upon the species of yeast considered. In addition, it presents a single phosphorylation site at position 233-235 coinci-
dent with one of the displayed by the mammalian, Drosophila and Xenopus at position 235 in the carboxy terminal region of the protein where all the high eukaryotic species present serine and arginine residues related to the activity of the p70, typeI and typeII S6 kinases (9,12). However, even in this case, LiS6 displays a threonine instead of the usual serine residue at this site. The rest of the PKC phosphorylation sites are distributed along the amino terminal and the central regions of the protein. The carboxy terminal residues present in the high eukaryotic species studied so far, (marked by asterisk in the fig) seem to play an important functional role and appear phosphorylated concomitantly with processes such cell differentiation, proliferation and division as well as with the action of mitogens, phorbol esters, oncogenic agents or insulin (see above) (11-14). It has been also suggested that S6 phosphorylation may play a role in the selective binding of mRNA species to ribosomes (27,28). The amino acid homology between the protozoon LiS6 and the proteins from the other sources considered ranges from 60
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to 65 per cent (data not shown) which indicate a certain phylogenic distance from L.infantum to the other species. This fact is mainly due to differences in the caboxy terminal fragment. Restriction enzyme analysis of the LiS6 encoding gene and determination of transcripts. Southern blot of the L.infantum genomic DNA digested with different restriction enzymes and detected with the labelled S6 probe (see above) is shown in Fig. 3. In all the cases three bands of different size and similar intensity are obtained according to the densitometric analysis showing that three copies of the gene were present in the genome. Most of the Leishmania genes do not contain introns and often repeat in tandem. The presence of a common 3kb band obtained by the DNA digestion with Bc1I and PstI which present restriction sites within the Li S6 encoding region, would agree with this possibility. However, the existence of introns can not be ruled out. The determination of the transcripts by Northern blot at different days of culture from the avirulent early logarithmic to the highly infectious stationary phase (5) it is shown in Fig. 4. L.infantum showed under high stringency conditions ( see Materials and Methods), three transcripts of 1.0, 1.2 and 1.3 kbp respectively along the different phases of the culture. The transcripts differ in their relative amounts and present a sligth increase at the stationary phase according to the densitometric analysis along the different growth phases. Altogether, the data suggest the existence of three different genes encoding for the LiS6 protein in L.infantum, although the existence of pseudogenes, not described so far in Leishmania but frequent in other species (29) can not be discarded.
FIG. 4. Northern blot analysis of S6 gene expression. Total RNA samples corresponding to the different days of culture from logarithmic (day 1) to stationary (day 7) phases. For further experimental details see Materials and Methods.
In conclusion, we have cloned the gene that encodes for the S6 ribosomal protein from the parasite protozoon L.infantum. This protein is conserved from mammals to primitive unicellular eukaryotes such Trypanosomatidae. It is reasonable to think that if the structure and function of ribosomes is highly conserved through the eukaryotes, the proteins involved in the different functions are conserved too. However, the LiS6 ribosomal protein presents interesting differences with the S6 proteins described so far. The number of aminoacids 249 is identical to that of mammalian and high eukaryotes. There are also similarities in the nucleus entrance sequences and the beta tyrosine kinase phosphorylation site. However, the distribution of the key regulatory PKC phosphorylation sites is different to that of mammalian, chicken, Drosophila and Xenopus. This difference is very clear in the C terminal region of the protein whereas in the amino terminal and central regions there are some similarities with the S6 proteins from yeast. In addition, the presence of threonine residues instead of the usual serine marks another difference. The Southern and Northern blot analysis suggest the existence of three genes that do not seem to be affected in their expression by the different environmental conditions that suffers the protozoon along the differentiation stages. The data suggest that the LiS6 protein from the parasite protozoon L.infantum seems to be in an intermediate position in the S6 protein family between the higher and the lower eukaryotes. The additional fact that Leishmania is an organism that can be induced to undergo developmental changes in vitro provides additional advantages for the study of the role of this protein family in the cell development and deserves further studies for the L.infantum S6 protein. ACKNOWLEDGMENTS This research has been supported by the grant # 97/0492 from the Fondo de Investigacio´n Sanitaria (FIS) of the Ministry of Health. G.G.A. thanks the Comunidad de Madrid for a fellowship. S.T. was a fellow from the FIS.
FIG. 3. Southern blot analysis of genomic DNA from L. infantum. L.infantum genomic DNA was subjected to total digestion with the following restriction enzymes: Sca I, Sph I, Hind III, Eco RI, Bc1 I, Pst I and Bam HI. For further experimental details see Materials and Methods.
REFERENCES 1. Neva, F., and Sacks, S. (1995) Leishmaniasis in Tropical and Geographical Medicine (Warren, K., and Mahmoud, A. A. S., Eds.), pp. 296–308. McGaw–Hill, New York.
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2. Sacks, D. L., and Perkins, P. V. (1984) Science 223, 1417–1419. 3. Doyle, P. S., Engel, J. C., Pimenta, P. F., Silva, P. P., and Dwyer, D. M. (1991) Exp. Parasitol. 73, 326–334. 4. Bates, P. A., Robertson, C. D., Tetley, L., and Coombs, G. H. (1992) Parasitol. 105, 193–202. 5. da Silva, R., and Sacks, D. L. (1987) Infect. Immun. 55, 2802– 2806. 6. Dell, K. R., and Engel, J. N. (1994) Mol. Biochem. Parasitol. 64, 283–292. 7. Hermoso, T., and Jaffe, C. L. (1993) Biol. Res. 26, 267–271. 8. Mukhopdhay, N. K., Saha, A. K., Lovelace, J. K., da Silva, R., Sacks, D. L., and Glew, R. H. (1988) J. Protozool. 35, 601–607. 9. Erickson, R. L. (1991) J. Biol. Chem. 266, 6007–6010. 10. Sturgill, W., and Wu, J. (1991) Biochem. Biophys. Acta 1092, 350–357. 11. Alessi, D. R., Kozlowski, M. T., Weng, Q. P., Morrice, N., and Avruch, J. (1998) Curr. Biol. 8, 69–81. 12. Frost, V., Morley, S. J., Mercep, L., Meyer, T., Fabbro, D., and Ferrari, S. (1995) J. Biol. Chem. 270, 26698–26706. 13. Lott, J. B., and Mackie, G. A. (1988) Gene 65, 31–39. 14. Morley, S. J. and Traugh, J. A. (1993) Biochimie 75, 985–989. 15. Chang, Y. W. E., and Traugh, J. A.(1997) J. Biol. Chem. 272, 28252–28257. 16. Zarley, J. H., Britigan, B. E., and Wilson, M. E. (1991) J. Clin. Invest. 88, 1511–1522.
17. Langford, C. K., Ullman, B., and Landfear, S. M. (1992) Exp. Parasitol. 74, 360–361. 18. Altschul, S. F., Warren, M., Webb, W., Eugene, M., and Lipman, D. L. (1990) J. Mol. Biol. 215, 403–410. 19. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic. Acids Res. 22, 4673–4680. 20. Gonzalez-Aseguinolaza, G., Almazan, F., Rodriguez, J. F., Marquet, A., and Larraga, V. (1997) Biochem. Biophys. Acta 1361, 92–102. 21. Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 22. Antoine, M., and Fried, M. (1992) Hum. Mol. Genet. 1, 565–570. 23. Lalanne, J., Lucero, M., and le Moullec, J. (1987) Nucleic Acids Res. 15, 4990. 24. Chan, Y. and Wool, I. G. (1988) J. Biol. Chem. 263, 2891–2896. 25. Odenwald, Jr., P. W., Jones, K. L., and di Mario, P. J. (1993) Nucleic Acids Res. 21, 4981. 26. Watson, K. L., Konrad, K. D., Woods, D. F., and Bryant, P. J. (1992) Proc. Natl. Acad. Sci. USA 89, 11302–11306. 27. Jefferies, H. B. J., Thomas, G., and Thomas, G. (1994) J. Biol. Chem. 269, 4367–4372. 28. Jefferies, H. B. J., Reinhard, C., Kozma, S. C., and Thomas, G. (1994) Proc. Natl. Acad. Sci.USA 91, 4441–4445. 29. Bandi, H. R., Ferrari, S., Krieg, J., Meyer, H. E., and Tomas, G. (1993) J. Biol. Chem. 268, 4530–4533.
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248, 464–468 (1998)
RC988927
Cloning and Structural Analysis of the Gene Encoding the Ribosomal Protein S6 from the Parasite Leishmania infantum Gloria Gonzalez-Aseguinolaza, Soraya Taladriz, Alberto Marquet, and Vicente Larraga1 Centro de Investigaciones Biolo´gicas, CSIC, Velazquez 144, Madrid 2006, Spain
Received May 15, 1998
We have cloned the S6 ribosomal protein encoding gene from a Leishmania infantum cDNA library. This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changes in vitro and results a good model to study cell differentiation processes. The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes. Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites. However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues. The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite. q 1998 Academic Press
Leishmania infantum is a parasite protozoon responsible for the visceral form of leishmaniasis in the Mediterranean basin (1). Leishmania has a digenetic lifecycle, proliferating in the insect vector, the sandfly, as extracelular flagellated promastigote and in the mammalian host as intracellular non flagellated amastigote (2). Along this life-cycle the parasite is affected by environmental changes, mainly in pH and temperature, that represent signals for cell differentiation (3,4).The differentiation changes can be mimicked by in vitro culture from avirulent promastigotes in the early logarithmic phase to virulent metacyclic promastigotes ready to enter in the host macrophage, in the late loga1 Corresponding author: Fax: 341 5627518. E-mail: vlarraga@ fresno.csic.es. GenBank Accession Number for the sequence reported here: AFO45457.
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rithmic and the stationary phase (5). There have been detected changes in protein phosphorylation during this type of transformation (6-8). As protein phosphorylation is involved in many essential eukaryotic processes such as cell division, proliferation and differentiation, transport, cell activation, oncogenesis, etc. . . . Leishmania results an interesting model to study several of these processes. The S6 protein is the major substrate for protein phosphorylation in the 40 S ribosomal subunit of eukaryotic cells (9,10). Several studies have shown that S6 is multiple phosphorylated on serine residues as the result of stimulation of a variety of cells by different agents such mitogens, viruses, oncogene products, phorbol esters, progesterone or insulin (11-14). Furthermore, stimulation of protein synthesis concomitant with a stimulatory effect on elongation and with phosphorylation of the ribosomal protein S6 has also been described (15). Then, the study of this ribosomal protein in a protozoon results of interest in order to compare their structural and functional similarities with the other eukaryotes studied so far. We report here the cloning and structural analysis of the gene encoding for the S6 ribosomal protein from Leishmania infantum. The information provided by the data on structure show differences in the protein regions and phosphorylation sites with respect to the corresponding proteins from mammalians, Drosophila, Xenopus or yeasts. The possible functional relevance of these data with respect to that found in other eukaryotic ribosomal protein genes are discussed. MATERIALS AND METHODS Cloning of the LiS6 ribosomal protein encoding gene. Leishmania infantum PB75 promastigotes (kindly provided by Dr. J. Alvar. Reference Parasitology Service. Instituto de Salud Carlos III. Majadahonda. Spain) were cultured in vitro at 287C in RPMI 1640 (GIBCO, Middlesex, UK) supplemented with 10% of heat inactivated fetal calf serum (Flow Laboratories, UK). Cultures were initiated at 11106 parasites/ml and harvested from late logarithmic/early stationary
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FIG. 1. Nucleotide and deduced amino acid sequences of the L. infantum S6 ribosomal protein gene. The nucleotide sequence is displayed in the 5* to 3* direction with the first base of the ATG initiation codon at position 1 and the third base of the TAG stop codon at position 750. The derived aminoacid sequence is shown in the single letter amino acid code. The S6 signature sequence is marked by a shadowed box. Open boxes at positions 170 and 191 indicate the nucleus entrance signals. The beta casein kinase phosphorylation sites at positions 151 and 205 are marked by open squares. The open circle represents the phoshorylation residue for cGMP protein kinase. Protein kinase C residues are marked by black circles.
phase cultures as defined by morphology and cell concentration (16). Total RNA from promastigotes was prepared using Trizol (Gibco BRL). A cDNA library was constructed in a l-ZAP Express vector using Poly(A)/RNA isolated using Biotinylated Oligo (dT) probe, the hybrids were captured with streptavidin coupled to magnetic particles and magnetic separation stand, washed and eluted according to manufacturer’s instructions (PolyATtact mRNA Isolation systems, Promega). On the basis of an S6 conserved sequence ISGGNDKQGFPM a degenerate oligonucleotide was designed according to the codon preference showed in Leishmania (17). The oligonucleotide probe was 5* end labelled with T4 polynucleotide kinase (New England Biolabs) and [a-32P] ATP and used to screen the cDNA library. This strategy yielded seven independent clones and for each of them a pBluescript plasmid derivate was excised from the ZAP-EXPRESS cloning vector. The cDNAs were sized on agarose gels and the plasmid with the largest insert, LiRS6-2, was selected. The nucleotide sequence was determined using the dideoxy chain method on an automated DNA sequencer (ABI PRISM DyeTerminator Cycle sequencing with ampliTaq DNA Polymerase, Perkin Elmer). Sequence comparison analysis of the proteins were carried out using the Altschul et al. algorithm (18). The sequences of the different species considered have been aligned using the CLUSTALW multiple sequence alignment method using default values (19). Isolation and structural analysis of the LiS6 genomic clone. Total genomic DNA from Leishmania infantum was prepared as described (20) digested with the indicated restriction enzymes, resolved on 0.7% agarose gels and transferred to nylon membranes (Hybond-N, Amersham). Total RNA was isolated from the promastigotes culture at daily intervals as described above. Southern and Northern blot hybridizations were done following standard procedures (21). In short, the blots were hybridized at 657C for 16 h in 6 1 Ssc, 5 1 Denhart’s mix, 0.5 % SDS, 100ml/ml salmon sperm DNA with a radiolabelled cDNA probe prepared by randomly primed synthesis with Klenow DNA polymerase and [a-32P]dCTP. Filters were washed twice at room temperature for 10 min in 2 1 SSC, 0.1% SDS, followed by two washes of 15 min at 657C in 0.1 1 SSC, 0.1% SDS.
RESULTS AND DISCUSSION Cloning and sequencing of the LiS6 ribosomal protein encoding gene. The cDNA insert in the clone LiRS62 contains 852 nucleotides and includes a single open reading frame and the 3* flanking region. The length of the Poly(A) tail is of 42 nucleotides. The open reading frame of 624 nucleotides codifies for a 249 aminoacids protein, with a calculated molecular weight of 28,290 Da. The nucleotide sequence (GenBank accession number AFO45457) and the deduced amino acid sequence are shown in Fig 1. As can be seen, the ATG initiation codon is located at position one and the stop codon TAG is located at position 750. The LiS6 protein has an excess of basic residues over the acidic ones with a theoretical pI of 11.45. The protein displays regions highly conserved with respect to other S6 proteins from different origins as well as remarkable differences. The S6 signature sequence is located between the fifty-five and sixty-four amino acid positions with two non conserved changes at position 57 (SrN) and at position 60 (DrQ), (see the shadowed box). There are two sequences corresponding to nucleus entrance signals at positions 170KKDR and 191-RAKK, (open boxes in the figure). Two Casein kinase II phosphorylation sites are present in positions 151 and 205, a Tyrosine kinase phosphorylation site sequence between the 152 and the 159 positions and a cGMP kinase phosphorylation site at position 139. The ribosomal S6 protein from L.infantum presents remarkable differences with those described so far specially in higher eukaryotes (22-26). In the
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FIG. 2. L. infantum S6 ribosomal amino acid sequence and comparison to other eukaryotic species. (1) Schizosaccharomyces pombe (GenBank Z54308); (2) Saccharomyces cerevisiae (GenBank P02365); (3) Kluyveromyces marxianus (GenBank S53430); (4) Leishmania infantum (bold letters); (5) Human (GenBank Z59209); (6) Chicken (GenBank X81968); (7) Xenopus laevis (GenBank P39017); (8) Drosophila melanogaster (GenBank P29327). Missing amino acids are shown by dashes. Asterisks indicate the residues corresponding to Protein kinase C phosphorylation sites absent in L.infantum S6 and present in higher eukaryotes species. Open box at position 12 indicate a common phosphorylation residue of LiS6 with yeasts. Open box at position 139 indicates a shared cAGMP phosphorylation site with the higher eukaryotes. Shadowed box indicate the common beta tyrosine kinase phosphorylation site.
protozoon protein there are nine PKC phosphorylation sites, being four of them threonine instead of serine sites, the usual amino acid residue in the other eukaryotes and are located in different positions of the protein, (black circles in the figure). Fig 2 shows the comparison between the L.infantum S6 and ribosomal S6 proteins from different sources both from higher and lower eukaryotes. The protozoon LiS6 presents the same size of the higher eukaryotes and shares the S6 signature sequence. In addition it presents one cGMP dependent kinase phosphorylation site at position 139 (open box) similar to that of the high eukaryotes, as well as the beta Tyrosine kinase phosphorylation site with human, chicken, Xenopus and Kluyveromyces but not with other yeast species and Drosophila, (shadowed box). The key functional PKC phosphorylation sites present clear differences. LiS6 shares a phosphorylation site with all the three yeast species considered at position 12 and one to three additional sites depending upon the species of yeast considered. In addition, it presents a single phosphorylation site at position 233-235 coinci-
dent with one of the displayed by the mammalian, Drosophila and Xenopus at position 235 in the carboxy terminal region of the protein where all the high eukaryotic species present serine and arginine residues related to the activity of the p70, typeI and typeII S6 kinases (9,12). However, even in this case, LiS6 displays a threonine instead of the usual serine residue at this site. The rest of the PKC phosphorylation sites are distributed along the amino terminal and the central regions of the protein. The carboxy terminal residues present in the high eukaryotic species studied so far, (marked by asterisk in the fig) seem to play an important functional role and appear phosphorylated concomitantly with processes such cell differentiation, proliferation and division as well as with the action of mitogens, phorbol esters, oncogenic agents or insulin (see above) (11-14). It has been also suggested that S6 phosphorylation may play a role in the selective binding of mRNA species to ribosomes (27,28). The amino acid homology between the protozoon LiS6 and the proteins from the other sources considered ranges from 60
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to 65 per cent (data not shown) which indicate a certain phylogenic distance from L.infantum to the other species. This fact is mainly due to differences in the caboxy terminal fragment. Restriction enzyme analysis of the LiS6 encoding gene and determination of transcripts. Southern blot of the L.infantum genomic DNA digested with different restriction enzymes and detected with the labelled S6 probe (see above) is shown in Fig. 3. In all the cases three bands of different size and similar intensity are obtained according to the densitometric analysis showing that three copies of the gene were present in the genome. Most of the Leishmania genes do not contain introns and often repeat in tandem. The presence of a common 3kb band obtained by the DNA digestion with Bc1I and PstI which present restriction sites within the Li S6 encoding region, would agree with this possibility. However, the existence of introns can not be ruled out. The determination of the transcripts by Northern blot at different days of culture from the avirulent early logarithmic to the highly infectious stationary phase (5) it is shown in Fig. 4. L.infantum showed under high stringency conditions ( see Materials and Methods), three transcripts of 1.0, 1.2 and 1.3 kbp respectively along the different phases of the culture. The transcripts differ in their relative amounts and present a sligth increase at the stationary phase according to the densitometric analysis along the different growth phases. Altogether, the data suggest the existence of three different genes encoding for the LiS6 protein in L.infantum, although the existence of pseudogenes, not described so far in Leishmania but frequent in other species (29) can not be discarded.
FIG. 4. Northern blot analysis of S6 gene expression. Total RNA samples corresponding to the different days of culture from logarithmic (day 1) to stationary (day 7) phases. For further experimental details see Materials and Methods.
In conclusion, we have cloned the gene that encodes for the S6 ribosomal protein from the parasite protozoon L.infantum. This protein is conserved from mammals to primitive unicellular eukaryotes such Trypanosomatidae. It is reasonable to think that if the structure and function of ribosomes is highly conserved through the eukaryotes, the proteins involved in the different functions are conserved too. However, the LiS6 ribosomal protein presents interesting differences with the S6 proteins described so far. The number of aminoacids 249 is identical to that of mammalian and high eukaryotes. There are also similarities in the nucleus entrance sequences and the beta tyrosine kinase phosphorylation site. However, the distribution of the key regulatory PKC phosphorylation sites is different to that of mammalian, chicken, Drosophila and Xenopus. This difference is very clear in the C terminal region of the protein whereas in the amino terminal and central regions there are some similarities with the S6 proteins from yeast. In addition, the presence of threonine residues instead of the usual serine marks another difference. The Southern and Northern blot analysis suggest the existence of three genes that do not seem to be affected in their expression by the different environmental conditions that suffers the protozoon along the differentiation stages. The data suggest that the LiS6 protein from the parasite protozoon L.infantum seems to be in an intermediate position in the S6 protein family between the higher and the lower eukaryotes. The additional fact that Leishmania is an organism that can be induced to undergo developmental changes in vitro provides additional advantages for the study of the role of this protein family in the cell development and deserves further studies for the L.infantum S6 protein. ACKNOWLEDGMENTS This research has been supported by the grant # 97/0492 from the Fondo de Investigacio´n Sanitaria (FIS) of the Ministry of Health. G.G.A. thanks the Comunidad de Madrid for a fellowship. S.T. was a fellow from the FIS.
FIG. 3. Southern blot analysis of genomic DNA from L. infantum. L.infantum genomic DNA was subjected to total digestion with the following restriction enzymes: Sca I, Sph I, Hind III, Eco RI, Bc1 I, Pst I and Bam HI. For further experimental details see Materials and Methods.
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