.) 1992 Oxford University Press
2376 Nucleic Acids Research, Vol. 20, No. 9
Identification of Li Qe/Li 2e ribosomal protein genes in Babesia bovis Brian P.Dalrymple and Jennifer M.Peters CSIRO Division of Tropical Animal Production, PO Box No. 3, Indooroopilly, OLD, Australia GenBank accession no. M81359
Submitted March 24, 1992 Babesia bovis is an intraerythrocytic protozoan parasite of cattle transmitted by ticks (1). A Xgtl 1 cDNA clone encoding an 112 amino acid B. bovis ribosomal protein, L12eIA, has been isolated and sequenced (Figure 1). The LlOe/Ll2e family of ribosomal proteins have highly conserved carboxy-terminal amino acid sequences (2). Unlike the protozoan Tetrahymena thermophila, which expresses an Ll2eII protein with an archaebacterial type of carboxy-terminal sequence (3), the B. bovis L12eIA protein has a typical eukaryote final seven amino acid sequence (Figure 1). The carboxy-terminal fifteen amino acids of L12eIA were identical to the equivalent region of the partial sequence of a B. bovis protein of previously unknown function encoded by the gene KABbl (4). The published predicted amino acid sequence preceding the carboxy-terminal end did not exhibit homology with any known Ll2e/LlOe proteins. However, the DNA sequence of the first 39 nucleotides of the sequence had 70% identity with the equivalent region of the Saccharomyces cerevisiae LIOe gene (data not shown). When the sequence of KABb 1 was translated in the same frame as the S. cerevisiae gene ten of fourteen amino acids were identical (Figure 1). It is probable that KABbl encodes the B. bovis LlOe protein and that the additional nucleotide was a cloning artefact. Higher eukaryotes appear to have single genes encoding each of L12eI, L12eII and LlOe (5,6). In contrast, the yeasts S. cerevisiae and Schizosaccharomyces pombe have two genes encoding variants of L12eI and L12eII and one gene encoding LlOe (7,8). In hybridizations of the cloned L12eIA gene to B. bovis genomic DNA digested with EcoRI two major and two minor bands were observed in all isolates of B. bovis examined (Figure 2). A similar pattern was also observed for B. bovis genomic DNA digested with AccI, BamHI, BglII, ClaI and HindIII (data not shown). By analysis of genomic DNA clones the L12eIA gene was assigned to the 13.5kb EcoRl fragment (data not shown). The - 7.5kb EcoRI band contained a second closely related gene, L12eIB. The hybridization signal from the weaker bands was due to the highly conserved DNA sequence encoding the carboxy-terminal region of the family of proteins. The 4.6kb EcoRI fragment was the same size as that detected with the KABbl probe (4) and thus contained the putative LlOe gene. By analogy with other eukaryotes the L12eII gene was assigned to the fourth EcoRI band. Unless extra genes were not separated by any of the enzymes used B. bovis has two genes encoding variants of Ll2eI (like the yeasts) and one gene encoding L12eII (like other eukaryotes).
REFERENCES 1. McCosker.P.J. (1981) In Ristic,M. and Kreier,J.P. (eds.), Babesiosis. Academic Press, New York, pp 1-24. 2. Shimmin,L.C., Ramirez,C., Matheson,A.T. and Dennis,P.P. (1989) J. Mol. Evol. 29, 448-462. 3. Hansen,T.S., Andreasen,P.H., Dreisig,H., Hojrup.P., Nielsen,H., Engberg,J. and Kristiansen,K (1991) Gene 105, 143-150. 4. Gill,A., Timms,P. and Kemp,D.J. (1987) Mol. Biochem. Parasitol. 22,
5. 6. 7. 8.
195 -202. Rich,B.E. and Steitz,J.A. (1987) Mol. Cell. Biol. 7, 4065-4074. Prieto,J., Candel, E. and Coloma,A. (1991) Nucleic Acids Res. 19, 1342. Beltrame,M. and Bianchi,M.E. (1990) Mol. Cell. Biol. 10, 2341-2348. Newton,C.H., Shimmin.L.C., Yee,J. and Dennis,P.P. (1990) J. Bacteriol. 172, 579-588.
-
-
L12eI con
50 60 40 30 20 10 E + N DE E V L R L M--KYVAAYLLA. LsGN. . PSA. DIKKI L. SVGIE .D.. RL. . hhSEL . GK. hEDLIAEG
L12eIA Bb MALKYVSSYLLAVAAGNENPSVDDLKKILDAVGSDVDEECLQGLVDSMSGKTVHETIAAG 70 TM A
80
90
100
110
Ll2eI con
..KLASVPTG-GAsss ssssssasssssnnKnnnKKnnnnnnnnnnnMGFGLFD
L12eIA Bb
MTKLQSMPAG-GAAMPAAAAGGAPAAAEDKAEAKKPEAEPEEi.EDDMGFSLFD A A** *****
k* * *** * * ** * * * ** *** * * ** * * *****
* AA* ** ** * **
** *
* '* **'AS *~~~~~~~1 *
LlOe? Bb
RLDN-PELFAAAAP---PGAVEAVQEAAP---EAAEEPEEEEDDMGFSLFD
LlOe Sc
RIEN-PEKYAAAAP--AATSAASGD-AAPAEEAAAEEEEESDDDMGFGLFD
Figure 1. Alignment of the amino acid sequences of the B. bovis(Bb) L12eIA and putative LlOe proteins with a eukaryote L12eI consensus sequence (con) and a S. cerevisiae (Sc) L lOe sequence (8). The Ll2eI consensus sequence was derived from the protein sequences encoded by the entries in GenBank release 69. Amino acids in the consensus sequence are highly conserved, common alternatives are shown above the residues. 'h' indicates L, I or V, 's' indicates A, G or P, 'n' indicates E or D. '*' indicates identities between the B. bovis sequence and the other sequences shown. The amino acid sequence shown in italics is translated from a different frame from that previously published (4).
-
W..
4,
_0
ACKNOWLEDGEMENTS The authors wish to thank Dr K.R.Gale for the gift of the B. bovis cDNA and genomic libraries. This work was supported in part by the Commonwealth Serum Laboratories, Pitman Moore Australia and the Meat Research Corporation.
Figure 2. EcoRI digested B. bovis genomic DNA separated by gel electrophoresis, transferred to a nylon membrane and probed with the B. bovis L12eIA cDNA insert. Different isolates of B. bovis are indicated by different abbreviations.
2376 Nucleic Acids Research, Vol. 20, No. 9
Identification of Li Qe/Li 2e ribosomal protein genes in Babesia bovis Brian P.Dalrymple and Jennifer M.Peters CSIRO Division of Tropical Animal Production, PO Box No. 3, Indooroopilly, OLD, Australia GenBank accession no. M81359
Submitted March 24, 1992 Babesia bovis is an intraerythrocytic protozoan parasite of cattle transmitted by ticks (1). A Xgtl 1 cDNA clone encoding an 112 amino acid B. bovis ribosomal protein, L12eIA, has been isolated and sequenced (Figure 1). The LlOe/Ll2e family of ribosomal proteins have highly conserved carboxy-terminal amino acid sequences (2). Unlike the protozoan Tetrahymena thermophila, which expresses an Ll2eII protein with an archaebacterial type of carboxy-terminal sequence (3), the B. bovis L12eIA protein has a typical eukaryote final seven amino acid sequence (Figure 1). The carboxy-terminal fifteen amino acids of L12eIA were identical to the equivalent region of the partial sequence of a B. bovis protein of previously unknown function encoded by the gene KABbl (4). The published predicted amino acid sequence preceding the carboxy-terminal end did not exhibit homology with any known Ll2e/LlOe proteins. However, the DNA sequence of the first 39 nucleotides of the sequence had 70% identity with the equivalent region of the Saccharomyces cerevisiae LIOe gene (data not shown). When the sequence of KABb 1 was translated in the same frame as the S. cerevisiae gene ten of fourteen amino acids were identical (Figure 1). It is probable that KABbl encodes the B. bovis LlOe protein and that the additional nucleotide was a cloning artefact. Higher eukaryotes appear to have single genes encoding each of L12eI, L12eII and LlOe (5,6). In contrast, the yeasts S. cerevisiae and Schizosaccharomyces pombe have two genes encoding variants of L12eI and L12eII and one gene encoding LlOe (7,8). In hybridizations of the cloned L12eIA gene to B. bovis genomic DNA digested with EcoRI two major and two minor bands were observed in all isolates of B. bovis examined (Figure 2). A similar pattern was also observed for B. bovis genomic DNA digested with AccI, BamHI, BglII, ClaI and HindIII (data not shown). By analysis of genomic DNA clones the L12eIA gene was assigned to the 13.5kb EcoRl fragment (data not shown). The - 7.5kb EcoRI band contained a second closely related gene, L12eIB. The hybridization signal from the weaker bands was due to the highly conserved DNA sequence encoding the carboxy-terminal region of the family of proteins. The 4.6kb EcoRI fragment was the same size as that detected with the KABbl probe (4) and thus contained the putative LlOe gene. By analogy with other eukaryotes the L12eII gene was assigned to the fourth EcoRI band. Unless extra genes were not separated by any of the enzymes used B. bovis has two genes encoding variants of Ll2eI (like the yeasts) and one gene encoding L12eII (like other eukaryotes).
REFERENCES 1. McCosker.P.J. (1981) In Ristic,M. and Kreier,J.P. (eds.), Babesiosis. Academic Press, New York, pp 1-24. 2. Shimmin,L.C., Ramirez,C., Matheson,A.T. and Dennis,P.P. (1989) J. Mol. Evol. 29, 448-462. 3. Hansen,T.S., Andreasen,P.H., Dreisig,H., Hojrup.P., Nielsen,H., Engberg,J. and Kristiansen,K (1991) Gene 105, 143-150. 4. Gill,A., Timms,P. and Kemp,D.J. (1987) Mol. Biochem. Parasitol. 22,
5. 6. 7. 8.
195 -202. Rich,B.E. and Steitz,J.A. (1987) Mol. Cell. Biol. 7, 4065-4074. Prieto,J., Candel, E. and Coloma,A. (1991) Nucleic Acids Res. 19, 1342. Beltrame,M. and Bianchi,M.E. (1990) Mol. Cell. Biol. 10, 2341-2348. Newton,C.H., Shimmin.L.C., Yee,J. and Dennis,P.P. (1990) J. Bacteriol. 172, 579-588.
-
-
L12eI con
50 60 40 30 20 10 E + N DE E V L R L M--KYVAAYLLA. LsGN. . PSA. DIKKI L. SVGIE .D.. RL. . hhSEL . GK. hEDLIAEG
L12eIA Bb MALKYVSSYLLAVAAGNENPSVDDLKKILDAVGSDVDEECLQGLVDSMSGKTVHETIAAG 70 TM A
80
90
100
110
Ll2eI con
..KLASVPTG-GAsss ssssssasssssnnKnnnKKnnnnnnnnnnnMGFGLFD
L12eIA Bb
MTKLQSMPAG-GAAMPAAAAGGAPAAAEDKAEAKKPEAEPEEi.EDDMGFSLFD A A** *****
k* * *** * * ** * * * ** *** * * ** * * *****
* AA* ** ** * **
** *
* '* **'AS *~~~~~~~1 *
LlOe? Bb
RLDN-PELFAAAAP---PGAVEAVQEAAP---EAAEEPEEEEDDMGFSLFD
LlOe Sc
RIEN-PEKYAAAAP--AATSAASGD-AAPAEEAAAEEEEESDDDMGFGLFD
Figure 1. Alignment of the amino acid sequences of the B. bovis(Bb) L12eIA and putative LlOe proteins with a eukaryote L12eI consensus sequence (con) and a S. cerevisiae (Sc) L lOe sequence (8). The Ll2eI consensus sequence was derived from the protein sequences encoded by the entries in GenBank release 69. Amino acids in the consensus sequence are highly conserved, common alternatives are shown above the residues. 'h' indicates L, I or V, 's' indicates A, G or P, 'n' indicates E or D. '*' indicates identities between the B. bovis sequence and the other sequences shown. The amino acid sequence shown in italics is translated from a different frame from that previously published (4).
-
W..
4,
_0
ACKNOWLEDGEMENTS The authors wish to thank Dr K.R.Gale for the gift of the B. bovis cDNA and genomic libraries. This work was supported in part by the Commonwealth Serum Laboratories, Pitman Moore Australia and the Meat Research Corporation.
Figure 2. EcoRI digested B. bovis genomic DNA separated by gel electrophoresis, transferred to a nylon membrane and probed with the B. bovis L12eIA cDNA insert. Different isolates of B. bovis are indicated by different abbreviations.
