Molecular and Biochemical Parasitology, 51 (1992) 165-168
165
© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01709
Short Communication
Characterization of a repetitive D N A element from Entamoeba histolytica Bertha Michel, A l e j a n d r o A l a g 6 n , Paul M. Lizardi a n d M a r i o Z u r i t a Departamento de Bioquimiea, Instituto de Biotecnologia, Universidad Nacional Aut6noma de MOxico, Cuernavaca Morelos, Mexico (Received 7 November 1991; accepted 9 November 1991)
Key words: Repetitive DNA; Entamoeba histolytica; DNA sequence
Repetitive DNA families have been described in all the organisms and are useful in the study of genome organization and evolution [1-3]. The identification of repetitive sequences located in the Entamoeba histolytiea chromosomes will be useful in the molecular analysis between different pathogenic and non-pathogenic strains. Although repetitive DNA sequences have been reported in E. histolytica, all such elements so far identified are localized in the rRNA episome [4,5]. In this work we report the physical characterization of a repetitive DNA element located elsewhere in the genome. A repetitive DNA clone named B/C was isolated from a 2gtl0 library from E. histolytica HK9, a strain which displays a 'pathogenic' phenotype [6]. This DNA clone contains an insert of around 1 kb whose sequence is present in 3% of the clones in the kgtl0 library. Digestion of DNA from E. histolytica HK9 DNA with either HindIII or BglII followed by Southern blot analysis with labeled B/C probe showed only one band of Correspondence address." Mario Zurita, Dpto. de Bioquimica, Instituto de Biotecnologia, Universidad Nacional Autdnoma de M+xico, Apartado Postal 510-3, 62271, Cuernavaca Morelos, Mexico. Tel.: 52-73-172799, 172999; Fax: 52-73172388.
Note. Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M77091.
high molecular weight. By contrast, in a digestion with the enzyme EcoRI, 2 bands were identified (Fig. 1). This signal was detected after a very short exposure and the hybridizing bands correspond to discrete restriction fragments that could be observed in an ethidium bromide staining. These bands do not co-migrate with the bands corresponding to fragments of the ribosomal DNA episome [4,5]. These results indicate that this DNA has a high copy number in the E. histolytica genome. The fact that some enzymes generate very large fragments also suggests that the B/C DNA sequences are tandemly linked, however, since ladder patterns have not yet been observed with frequent cutters, we can not discard the possibility of a circular DNA containing the B/C DNA. Interestingly, this DNA is present in all the E. histolytica strains tested, as well as in other species of Entamoeba. Fig. 1B shows a dot blot hybridization performed under high stringency conditions; the labeled B/C probe hybridizes with E. histolytica DNA from pathogenic or non-pathogenic strains and with Entamoeba moshkovski and Entamoeba invadens DNA. This suggests that B/C is a repetitive DNA family that is conserved among Entamoeba genera. No signal was detected in Northern blot experiments using the clone B/C as probe (data not shown), suggesting that B/C DNA is not transcribed in the trophozoite stage.
166
A
1
2
3
4
10 20 3O 40 SO eO GAATTCTAT'rCCTACATCTACCTAGGTCCTCCCGATCTCTACCAACTGAGCTATQGTCQG
B
70 80 gO 100 110 120 TATCGATATCACC] I I I IATAI~rCTFITGCTTATrCTITrGATATCCTTI-rCTrAI-rCTr
Kb
130
Q
8.4 7.2 6.3 5.6 4.8 4.3 3.6
2jO
140
150
160
170
laO
TTCTrATrC i ! I I IATATCCTTTTATATTrCTATATGTGCI"rATATATG1TTCTATFCTr 190
200
210
220
230
240
YATACCTAI-r CTTTATATAC l"rC TFATrA TCTCTTCCCTGTTA TTTCTCTATACCTrA TC 250 260 270 280 2gO 300 TTTATA'rrATGTA TATATTTATATTTATATFTATAI-FATGTATCCCTTATCTI-rA TATTA
O 7
2.3
0
1.9
m
310
32O
33O
340
3~0
3¢=O
TGTATATATTI-rATATTATGTATATATI"rTATATTATGTGTAGTATA TATTTATITI'ATG 370 380 3QO 400 410 420 TATATGTGTAGTGTACTATATGTGTAGTGTACTTATGTGTAC TATATATAYATATTCTrG 430 440 450 CTAAAAAATrCCATCT~TCGAACCTAG 49O
S00
510
460 52O
470 53O
4410 TGAAAGGCT 54O
TGTATGI"rACCAAC TACACCACAATGGA1TrACCATGAC TATATrAGCCCCCTrAGACTrr
1.3 1.2
550 560 570 580 590 600 A TTC TTTA TA TTCC TTA TTA TAC TTA TTA TAC TTA T[A TAC TTATTA TAC TrA TTA TACT 610 620 630 640 650 660 TATTATACTTATTATACTTATrATACTTATTATACTrA TTATACTTA'rrAT,&.C TTTACTTC
m
0.7
670 680 6gO 700 710 720 TCTITrACGACCATTATGTATATTI'ATATAGTrTCTATTGCTTATATTATTATTATC'ITI'/~ 730 740 750 760 770 780 TTATTATCTTTATTATTATCTrTATTATTATCTTrATTA'IrATCITrATTA'IrATCTrTA 79O 8OO 810 82O 83O 84O TTA"rrATC T r r A TTATTATC TTTATTATTATCTTTATTATCTTTATTATC~TrA TrA TTT
Fig. 1. Southern blot ana]vsis of total DNA from E. histolytica (strain HK9) hybridized with the B/C clone and dot blot hybridization with different Entamoeba species. (A) Southern blot hybridization of total DNA from E. histolytica with the B/C clone, digested with BgIII (lane 2); EcoRI (lane 3) and HmdIll (lane 4); lane 1 are molecular weight markers. (B) Dot blot hybridization of the different E. histolytica strains D N A (pathogenic and non-pathogenic), E. invadens and E. moshkovski with the clone B/C. Dots 1 5, E. histolytica pathogenic strains DNA (HK9, HM38, HM1, clones 4 and 45, respectively); dot 6, E. invadens DNA; dot 7, E. moshkovski DNA; dot 8 Escherichia coli DNA; dots 9, 10 and 11 non-pathogenic strains DNA (44, 42 and 43, respectively).
The B/C clone is 976 bp in length and contains 2 types of related direct repeats (Fig. 2). The sequence CTTATTATA is tandemly repeated 12 times and the sequence CTTTATTATTAT is present 10 times. A computer search in the EMBL and GenBank data bases showed a high degree of homology with mitochondrial D N A from Paramecium and yeast (Fig. 3). This identity (80%), is present in the 2 regions that contain the tandemly repeated oligos in B/ C and the (TTA)n sequence present in the replication origin of some mitochondrial D N A [7]. In order to find out the statistical significance of this similarity, an analysis using the R D F 2 program [8], with 50 random sequences generated from the same base
86O 870 88O 8gO g~o ATGCTrATGTACI-rGACTATATACI"rAITrCTACTTAI"rTATACTCi I ! ! ICCTATACCT 910 920 gOO 940 950 960 ATACCTATCCCTATACCTATCTA1-rAGTATAAAAACTGATCCGACCAACCGGATrCGAAC 97O
CAGTGACCTAAGGAATrC
Fig. 2. Nucleotide sequence of the B/C clone. The underlined bases represent the regions containing the tandemly repeated oligonucleotides.
composition of the B/C clone was done. It showed that the similarity between the B/C clone and mitochondrial D N A was 13.4 standard deviations away from the mean. Although the similarity to mitochondrial D N A is very high, it could be due to chance, since there is a mismatch pattern which is 710 720 730 740 750 760 TAI-rAI~AI-rATC 1TrATTATTATCT T T A T T A l l A T C T l l ' A T T A T r ATC 1TrATTATTATC I IIIEIIIIIII IIIIIIIIIII IIIIIIIIIII IIIIIIIIIII IIIIIIIIlll GAI-rATTA'n'ATTATTATTATTATTATTATTATTATTAllATTA'FrATTATTATrATTATT 280 290 300 310 320 330 770 780 790 800 810 TTrATTATTATCT~ATI'ATTATCTTTATrATrATCTTrATTATTATC]3"TATTATCTTT lllllllllll IIIllllllll IIIIIIIIIII IIIIIIIIIII IIIIIIII II ATTATTAI-rATTATrATrATTATrAI"rATTA]-rATTATTATTATrATTA'I-rAT'/'ATTAI~ 34O 350 360 370 380 82O ATTATCTTTA]TAI-rTATG III II IIIIIIIIIIIII ATTAI I I I I ATrATTrATA 390
clone BiC milochondrial DNA
Fig. 3. Homology between the Paramecium mitochondrial origin of replication [7] and the B/C clone. The numbers of the bases are from the GenBank files.
167
highly conserved, suggesting that this similarity is not generated by an evolutive process. It is puzzling, however that the best similarity was with mitochondrial DNA, since E. histolytica does not contain mitochondria. Further analysis will be required to explore the possibility of a mitochondrial origin of the B/C clone. On the other hand the general features and sequence of the B/C clone suggest that it could be a so-called satellite DNA, composed of tandemly repeated oligonucleotides, as in many other organisms [9]. The analysis of the B/C sequence domain and related DNA sequences, for example, the determination of chromosomal localization, may be useful for understanding of evolutionary relationships among Entamoeba spp.
Acknowledgements We thank Alejandro Olvera for his expert assistance in the culture of E. histolytica trophozoites and other Entamoeba. We also thank to Dr. Isaura Meza for her generous gift of non-pathogenic strain DNA, Dr. Patricia Leon for helpful suggestions and Dr. Enrique Merino for his help in the computer analysis. This work was supported by a grant from DGAPA/UNAM.
References 1 Britten, R.J. and Kohue, D.E. (1968) Repeated sequences in DNA. Science 161,529-540. 2 Doolittle, W.F. and Sapienza, C. (1980) Selfish genes, the phenotype paradigm and genome evolution. Nature 284, 601-603. 3 Chen, Z.Q., Ritzel, R.G., Lin, C.C. and Hoggetts R.B. (1991) Sequence conservation in avian CRI: an interspersed repetitive DNA family evolving undel functional constrains. Proc. Natl. Acad. Sci. USA 88, 5814-5818. 4 Garfinkel, L.I. Giladi, M., Huber, M., Gitler, C., Mirelman, D., Revel, M. and Rozenblath, S. (1989) DNA Probes Specific for Entamoeba histolytica possessing pathogenic and non-pathogenic zymodemes. Infect. Immun. 57, 926-931. 5 Samuelson, J., Acufia-Soto R., Reed, S., Biagi, F. and Wirth, D. (1989) DNA hybridization probe for clinical diagnosis of Entamoeba histolytica J. Clin. Microbiol. 27, 671-678. 6 Sargeaunt, P.G., Williams, J.E. and Neal, R.E. (1980) A comparative study of Entamoeba histolytica (NIH:200, HK9, etc.) Trans. R. Soc. Trop. Med. Hyg. 74, 489-474. 7 Pritchard, A.E., Laping, J.L., Seilhamer, J.J. and Cummings, A.J. (1983) Inter-species sequence diversity in the replication initiation region of Paramecium mitochondrial DNA. J. Mol. Biol. 164, 1-15. 8 Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. 9 Singer, M.F. (1982) SINES and LINES: highly repeat short and long interspersed sequences in mammalian genomes. Cell 40, 433-434.
165
© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01709
Short Communication
Characterization of a repetitive D N A element from Entamoeba histolytica Bertha Michel, A l e j a n d r o A l a g 6 n , Paul M. Lizardi a n d M a r i o Z u r i t a Departamento de Bioquimiea, Instituto de Biotecnologia, Universidad Nacional Aut6noma de MOxico, Cuernavaca Morelos, Mexico (Received 7 November 1991; accepted 9 November 1991)
Key words: Repetitive DNA; Entamoeba histolytica; DNA sequence
Repetitive DNA families have been described in all the organisms and are useful in the study of genome organization and evolution [1-3]. The identification of repetitive sequences located in the Entamoeba histolytiea chromosomes will be useful in the molecular analysis between different pathogenic and non-pathogenic strains. Although repetitive DNA sequences have been reported in E. histolytica, all such elements so far identified are localized in the rRNA episome [4,5]. In this work we report the physical characterization of a repetitive DNA element located elsewhere in the genome. A repetitive DNA clone named B/C was isolated from a 2gtl0 library from E. histolytica HK9, a strain which displays a 'pathogenic' phenotype [6]. This DNA clone contains an insert of around 1 kb whose sequence is present in 3% of the clones in the kgtl0 library. Digestion of DNA from E. histolytica HK9 DNA with either HindIII or BglII followed by Southern blot analysis with labeled B/C probe showed only one band of Correspondence address." Mario Zurita, Dpto. de Bioquimica, Instituto de Biotecnologia, Universidad Nacional Autdnoma de M+xico, Apartado Postal 510-3, 62271, Cuernavaca Morelos, Mexico. Tel.: 52-73-172799, 172999; Fax: 52-73172388.
Note. Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M77091.
high molecular weight. By contrast, in a digestion with the enzyme EcoRI, 2 bands were identified (Fig. 1). This signal was detected after a very short exposure and the hybridizing bands correspond to discrete restriction fragments that could be observed in an ethidium bromide staining. These bands do not co-migrate with the bands corresponding to fragments of the ribosomal DNA episome [4,5]. These results indicate that this DNA has a high copy number in the E. histolytica genome. The fact that some enzymes generate very large fragments also suggests that the B/C DNA sequences are tandemly linked, however, since ladder patterns have not yet been observed with frequent cutters, we can not discard the possibility of a circular DNA containing the B/C DNA. Interestingly, this DNA is present in all the E. histolytica strains tested, as well as in other species of Entamoeba. Fig. 1B shows a dot blot hybridization performed under high stringency conditions; the labeled B/C probe hybridizes with E. histolytica DNA from pathogenic or non-pathogenic strains and with Entamoeba moshkovski and Entamoeba invadens DNA. This suggests that B/C is a repetitive DNA family that is conserved among Entamoeba genera. No signal was detected in Northern blot experiments using the clone B/C as probe (data not shown), suggesting that B/C DNA is not transcribed in the trophozoite stage.
166
A
1
2
3
4
10 20 3O 40 SO eO GAATTCTAT'rCCTACATCTACCTAGGTCCTCCCGATCTCTACCAACTGAGCTATQGTCQG
B
70 80 gO 100 110 120 TATCGATATCACC] I I I IATAI~rCTFITGCTTATrCTITrGATATCCTTI-rCTrAI-rCTr
Kb
130
Q
8.4 7.2 6.3 5.6 4.8 4.3 3.6
2jO
140
150
160
170
laO
TTCTrATrC i ! I I IATATCCTTTTATATTrCTATATGTGCI"rATATATG1TTCTATFCTr 190
200
210
220
230
240
YATACCTAI-r CTTTATATAC l"rC TFATrA TCTCTTCCCTGTTA TTTCTCTATACCTrA TC 250 260 270 280 2gO 300 TTTATA'rrATGTA TATATTTATATTTATATFTATAI-FATGTATCCCTTATCTI-rA TATTA
O 7
2.3
0
1.9
m
310
32O
33O
340
3~0
3¢=O
TGTATATATTI-rATATTATGTATATATI"rTATATTATGTGTAGTATA TATTTATITI'ATG 370 380 3QO 400 410 420 TATATGTGTAGTGTACTATATGTGTAGTGTACTTATGTGTAC TATATATAYATATTCTrG 430 440 450 CTAAAAAATrCCATCT~TCGAACCTAG 49O
S00
510
460 52O
470 53O
4410 TGAAAGGCT 54O
TGTATGI"rACCAAC TACACCACAATGGA1TrACCATGAC TATATrAGCCCCCTrAGACTrr
1.3 1.2
550 560 570 580 590 600 A TTC TTTA TA TTCC TTA TTA TAC TTA TTA TAC TTA T[A TAC TTATTA TAC TrA TTA TACT 610 620 630 640 650 660 TATTATACTTATTATACTTATrATACTTATTATACTrA TTATACTTA'rrAT,&.C TTTACTTC
m
0.7
670 680 6gO 700 710 720 TCTITrACGACCATTATGTATATTI'ATATAGTrTCTATTGCTTATATTATTATTATC'ITI'/~ 730 740 750 760 770 780 TTATTATCTTTATTATTATCTrTATTATTATCTTrATTA'IrATCITrATTA'IrATCTrTA 79O 8OO 810 82O 83O 84O TTA"rrATC T r r A TTATTATC TTTATTATTATCTTTATTATCTTTATTATC~TrA TrA TTT
Fig. 1. Southern blot ana]vsis of total DNA from E. histolytica (strain HK9) hybridized with the B/C clone and dot blot hybridization with different Entamoeba species. (A) Southern blot hybridization of total DNA from E. histolytica with the B/C clone, digested with BgIII (lane 2); EcoRI (lane 3) and HmdIll (lane 4); lane 1 are molecular weight markers. (B) Dot blot hybridization of the different E. histolytica strains D N A (pathogenic and non-pathogenic), E. invadens and E. moshkovski with the clone B/C. Dots 1 5, E. histolytica pathogenic strains DNA (HK9, HM38, HM1, clones 4 and 45, respectively); dot 6, E. invadens DNA; dot 7, E. moshkovski DNA; dot 8 Escherichia coli DNA; dots 9, 10 and 11 non-pathogenic strains DNA (44, 42 and 43, respectively).
The B/C clone is 976 bp in length and contains 2 types of related direct repeats (Fig. 2). The sequence CTTATTATA is tandemly repeated 12 times and the sequence CTTTATTATTAT is present 10 times. A computer search in the EMBL and GenBank data bases showed a high degree of homology with mitochondrial D N A from Paramecium and yeast (Fig. 3). This identity (80%), is present in the 2 regions that contain the tandemly repeated oligos in B/ C and the (TTA)n sequence present in the replication origin of some mitochondrial D N A [7]. In order to find out the statistical significance of this similarity, an analysis using the R D F 2 program [8], with 50 random sequences generated from the same base
86O 870 88O 8gO g~o ATGCTrATGTACI-rGACTATATACI"rAITrCTACTTAI"rTATACTCi I ! ! ICCTATACCT 910 920 gOO 940 950 960 ATACCTATCCCTATACCTATCTA1-rAGTATAAAAACTGATCCGACCAACCGGATrCGAAC 97O
CAGTGACCTAAGGAATrC
Fig. 2. Nucleotide sequence of the B/C clone. The underlined bases represent the regions containing the tandemly repeated oligonucleotides.
composition of the B/C clone was done. It showed that the similarity between the B/C clone and mitochondrial D N A was 13.4 standard deviations away from the mean. Although the similarity to mitochondrial D N A is very high, it could be due to chance, since there is a mismatch pattern which is 710 720 730 740 750 760 TAI-rAI~AI-rATC 1TrATTATTATCT T T A T T A l l A T C T l l ' A T T A T r ATC 1TrATTATTATC I IIIEIIIIIII IIIIIIIIIII IIIIIIIIIII IIIIIIIIIII IIIIIIIIlll GAI-rATTA'n'ATTATTATTATTATTATTATTATTATTAllATTA'FrATTATTATrATTATT 280 290 300 310 320 330 770 780 790 800 810 TTrATTATTATCT~ATI'ATTATCTTTATrATrATCTTrATTATTATC]3"TATTATCTTT lllllllllll IIIllllllll IIIIIIIIIII IIIIIIIIIII IIIIIIII II ATTATTAI-rATTATrATrATTATrAI"rATTA]-rATTATTATTATrATTA'I-rAT'/'ATTAI~ 34O 350 360 370 380 82O ATTATCTTTA]TAI-rTATG III II IIIIIIIIIIIII ATTAI I I I I ATrATTrATA 390
clone BiC milochondrial DNA
Fig. 3. Homology between the Paramecium mitochondrial origin of replication [7] and the B/C clone. The numbers of the bases are from the GenBank files.
167
highly conserved, suggesting that this similarity is not generated by an evolutive process. It is puzzling, however that the best similarity was with mitochondrial DNA, since E. histolytica does not contain mitochondria. Further analysis will be required to explore the possibility of a mitochondrial origin of the B/C clone. On the other hand the general features and sequence of the B/C clone suggest that it could be a so-called satellite DNA, composed of tandemly repeated oligonucleotides, as in many other organisms [9]. The analysis of the B/C sequence domain and related DNA sequences, for example, the determination of chromosomal localization, may be useful for understanding of evolutionary relationships among Entamoeba spp.
Acknowledgements We thank Alejandro Olvera for his expert assistance in the culture of E. histolytica trophozoites and other Entamoeba. We also thank to Dr. Isaura Meza for her generous gift of non-pathogenic strain DNA, Dr. Patricia Leon for helpful suggestions and Dr. Enrique Merino for his help in the computer analysis. This work was supported by a grant from DGAPA/UNAM.
References 1 Britten, R.J. and Kohue, D.E. (1968) Repeated sequences in DNA. Science 161,529-540. 2 Doolittle, W.F. and Sapienza, C. (1980) Selfish genes, the phenotype paradigm and genome evolution. Nature 284, 601-603. 3 Chen, Z.Q., Ritzel, R.G., Lin, C.C. and Hoggetts R.B. (1991) Sequence conservation in avian CRI: an interspersed repetitive DNA family evolving undel functional constrains. Proc. Natl. Acad. Sci. USA 88, 5814-5818. 4 Garfinkel, L.I. Giladi, M., Huber, M., Gitler, C., Mirelman, D., Revel, M. and Rozenblath, S. (1989) DNA Probes Specific for Entamoeba histolytica possessing pathogenic and non-pathogenic zymodemes. Infect. Immun. 57, 926-931. 5 Samuelson, J., Acufia-Soto R., Reed, S., Biagi, F. and Wirth, D. (1989) DNA hybridization probe for clinical diagnosis of Entamoeba histolytica J. Clin. Microbiol. 27, 671-678. 6 Sargeaunt, P.G., Williams, J.E. and Neal, R.E. (1980) A comparative study of Entamoeba histolytica (NIH:200, HK9, etc.) Trans. R. Soc. Trop. Med. Hyg. 74, 489-474. 7 Pritchard, A.E., Laping, J.L., Seilhamer, J.J. and Cummings, A.J. (1983) Inter-species sequence diversity in the replication initiation region of Paramecium mitochondrial DNA. J. Mol. Biol. 164, 1-15. 8 Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. 9 Singer, M.F. (1982) SINES and LINES: highly repeat short and long interspersed sequences in mammalian genomes. Cell 40, 433-434.
