Gene. 96 (1990) 197-203 Elsevier
197
GENE 03844
C h a r a c t e r i s a t i o n of a repetitive D N A family from E n t a m o e b a histolytica containing Saccharomyces cerevisiae A R S consensus sequences (Protozoan parasite; recombinant plasmid; tandem repeats; sequence analysis; replication origin; bent DNA)
Anuradha Lohia, N. Haider and B.B. Biswas Department o f Biochemistry, Bose Institute, Cakutta 700054 (India)
Received by G.N. Godson: 23 March 1990 Revised: 2 May 1990 Accepted: 27 September 1990
SUMMARY
Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously repficating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIPS,. to replicate autonomously in S. eerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.
INTRODUCTION
The protozoan parasite E. histolytica is a human pathogen which causes extra-intestinal abcesses and dysentery in its invasive form, affecting almost 10~o of the world population (Walsh, 1986). Little has been reported on DNA replication or on the chromosomal organisation of this organism. However, like the genomes of most eukaryotic org,'misms ~he Entamoeba genome has also been reported to possess large amounts of repetitive DNA (Bhattacharya et al., 1988; Huber et al., 1989). One of the repeated DNA classes has been identified as ribosomal DNA (Bhattacharya et al., 1988; Huber et al, 1989). We are describing here another class of repetitive DNA
Correspondence to: Dr. B.B. Biswas, Department of Biochemistry, Bose Institute, P 1/12 C.I.T. Scheme Vll M, Calcutta 700054 (India). Tel. (91-33)379219: Fax (91-33)343886.
Abbreviations: ARS, autonomously replicating sequence(s); bp, base pair(s); C. , Caenorabditis; CEN, centromere; E. , Entamoeba ; ESP, 0.5 M EDTA/I% Sarkosyl/2mg per ml proteinase K; EtdBr, ethidium
from E. histolytica. Sequence analysis of members of this repetitive DNA class showed the presence of multiple copies of sequences homologous to the ll-bp ARS core consensus sequence 5' - AT A A A cT A -t" A A A TA of S. cerevisiae (Broach et al., 1983; Campbell, 1988; Palzkill and Newlon, 1988). These DNA fragments were also shown to be capable of transforming S. cerevisiae at a high frequency, when linked to a selectable marker. Fortuitously, one of the DNA fragments analysed showed a 'bent DNA'-like sequence. This 'bent DNA' fragment may serve as an origin of replication in E. histolytica, since the presence of such sequences has been shown at origins of replication in other organisms (Kidane et al., 1984; Ryder et al., 1986; Snyder et al., 1986; Zahn and Blattner, 1985).
bromide; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; RF, replicative form; S., Saccharomyces; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCi/0.15 M Na3"citrate pH 7.6; SV40, simian virus 40; TYIS-33, 2% trypticase peptone/1% yeast extract/2.28 mg per 100 ml ferric ammonium citrate/ 10% bovine serum; UWGCG, University of Wisconsin Genetics Computer Gro'ap; YEPD, 0.5 % yeast extract/1% peptone/2% dextrose.
198 RESULTS AND DISCUSSION
B
A
(a) Restriction enzyme digestion patterns of Entamoeba
histolytica Total DNA from E. histolytica 200: NIH nuclei was isolated (Edman et al., 1987), digested with several restriction endonucleases and fractionated on a 1% agarose gel (Fig. 1A). Frominent discrete bands ranging from 6.5-0.4 kb were observed in the Sau3AI-digested DNA,
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0.87 0.60
0.31
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1.35
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0.75
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.
0.60
1
2
3
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Fig. 1. Restriction patterns of E. histolytica 200: NIH. (Panel A) Total DNA from E. histolytica nuclei was isolated by the method ofEdman et al. (1987) except that after isolating nuclei through sucrose cushions they were directly incubated in ESP for 2 h at 50°C, followed by treatment with phenol, chloroform and ethanol precipitate. The DNA was digested and separated by electrophoresis on a 1% agarose gel (lanes 2-4). The gel was stained with EtdBr (0.5 #g/ml) and the DNA was visualised under UV light at 300 nm. Numbers on the left margin give sizes in kb. Lanes: I, M, size markers HindIIl digest ofA DNA and HaeIII digest of OX174 RF DNA; 2, Sau3AI; 3, EcoRI; 4, HindIIl. Organisms: Trophozoites of E. histoh'tica 200 : NIH, were maintained axenically in TYIS-33 medium (Diamond et al., 1978) using 10';0 bovine serum. Usually 72-96 h cultures were used for DNA isolation. (Panel B) Autoradiographic detection of E. histolytica genomic DNA bands that hybridise to the recombinant clone pEH212. E. histolytica DNA from lane 2 (panel A) gel was tr~msferred to Nytran paper and hybridised to radioactively labelled pEH212 DNA (see Figs. 2 and 3). Radioactive DNA probes were prepared by using the technique of oligo labelling using random primers (Feinberg and Vogelstein, 1984). Hybridisation with radioactive probes was carried out in 0.5 M phosphate buffer pH 7.4/7% SDS at 65°C for 16-18 h. Washing was done at high stringency with 0.5 x SSC/0.1% SDS at 68°C. Numbers on the left margin are deduced sizes in kb of the hybridised bands.
0.31
1
2 3 4 5
6
7
Fig. 2. Restriction pattern ofpEH212 showing fragments 1-4. A genomic library of E. h~olytica 200: NIH trophozoite DNA was prepared in the yeast shuttle vector YIP5 by ligating Sau3Al.digested Entamoeba DNA at the BamHI site of YIP5 and transforming Escherichia coif HBI01. Plasmid pEH212 was a recombinant clone isolated from the E. histolygca genomic library, which hybridised to several DNA bands in Sau3AIdigested Entamoeba DNA. Plasmid pEH212 was digested with several restriction enzymes and separated by electrophoresis on a 1% agarose gel. The bands were stained with 0.5 #g EtdBr/ml, and visualised under UV light at 300 nm. Lanes: 1, HindIII digest of ADNA (see Fig. 1); 2, HaellI digest of ~X174 RF DNA; 3-7, digests of pEH212; 3, HindlIl + BamHI; 4, HindIII + Sinai; 5, BamHI + Smal; 6, BamHI + PvuI; 7, PvuI +SalI. (The top band in lane 7 at -8.5 kb is due to incomplete digestion and represents uncut plasmid.) Arrowhead shows the position of fragment 2 in lane 5. Numbers on the lef~ margin are sizes in kb.
199
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Fig. 3. Res~r/~.'ou map :~i the E. hbtolytica repetitive DNA insert ofpEH212 and the sequencing strategy. Blackened boxes, vector DNA. Junction of blackened boxes and solid fines, BamHI cloning site of the vector. Restriction sites are designated as follows: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, Pvul; Ps, Pstl; R, Rsal; S, Sau3AI; Sa, SaiI; Sin, Sinai. The junctions of the four different fragments of the insert are shown by open arrows. The numbers above the solid line indicate the four different fragments. Numbering is in ascending order in the 5'-3' direction. The M ! 3 subclones are shown by blackened arrows. Each subclone was read in both directions up to the points marked by the arrows.
suggesting the presence of highly abundant repetitive DNA classes. No single multimeric ladder could be detected in partial digests with Sau3Al (data not shown) which would suggest the presence of a single class of tandemly repeated DNA. It is possible that different Sau3Al families of repetitive DNA exist which comprise the multiple bands in the Sau3AI digested E. histolytica DNA. It may be noted that we were unable to detect prominent bands in the EcoRI and HindIIl-digested Entamoeba DNA as reported earlier (Bhattacharya et al., 1988; Huber et al., 1989). In an attempt to characterise and study the Sau3Al repetitive DNA classes, we constructed a genomic library ofEntamoeba DNA as described in the legend to Fig. 2. We chose the yeast integrating plasmid YIP5 as our cloning vector since we were also interested in isolating putative origins of replication from E. histolytica in yeast for another study. In this study the genomic library was screened by colony hybridisation with total E. histolytica 200:NIH DNA, radioactively labelled with [0(-32p]dATP as described in the legend to Fig. lB. The colonies which showed very intense signals were picked up on the basis that clones carrying repetitive DNA fragments would light up more strongly due to the abundance of similar DNA sequences in the radioactive probe. Five such clones were picked for further analysis. They were individually labelled with [0c-3Zp]dATP and Southern blots of genomic digests of E. histolytica DNA were hybridised separately with each of the radioactively labelled clones. One ofthe clones pEH212
6.40
2.25 1.80 1.70
1.10 0.95 0.87 0.75
2
3
4
• Fig. 4. Autoradiographic detection of Sau3Al-digested E. histolytica genomic DNA bands by hybridisation with fragments 1-4 of pEH212. Sau3AI-digested E. histolytica DNA was separated by electrophoresis on a 1% agarose gel (lanes 1-4) and transferred to Nytran paper. The Southern blot was cut along the lanes and the individual lanes were hybridised with radioactively labelled fragments !-4, respectively. Lanes: 1, probe-fragment 1; 2, probe-fragment 2; 3, probe-fragment 3; 4, probe-fragment 4. Numbers on the leR margin as in Fig. lB.
200 hybridised to several prominent bands in the Sau3Aldigested DNA (Fig. 1B). This clone was selected for further analysis. Restriction analysis of pEH212 showed the presence of an approx. 3.0-kb band, which was made of four Sau3AI fragments (0.5, 0.5, 1.25 and 0.92 kb; Figs. 2 and 3). As may be seen from a partial physical map of pEH212 (Fig. 3), the size of fragment 2 is less than 0.5 kb, the latter due to its anomalous mobility as discussed in section b. It is possible that four random Sau3Al fragments were ligated together, or the multiple inserts were a result of partially digested DNA. To determine whether the four fragments belonged to the same repetitive D N A classes or different ones, we isolated the fragments numbered 1, 2, 3, 4 (numbering in ascending order from 5 ' - 3 ' , Fig. 3) from a low-melting-point agarose gel and labelled them separately by o!igo labelling as described in the legend of Fig. lB. Southern blots ofSau3Al-digested E. histolytica DNA were hybridised with the four [~t-32P]dATP-labelled fragments separately (Fig. 4). At the relatively high stringency of washing used (0.5 x SSC/0.1~o SDS/68°C) some common bands in the two sets could be identified (data not shown). All four fragments showed smears when hybridised to other restriction enzymes digested E. histolytica DNA.
This suggested that these repetitive D N A classes were dispersed in the genome. We have also seen the presence of similar bands in Sau 3AI-digested D N A of several clinical strains orE. histolytica and similar hybridisation patterns with the two repetitive D N A classes (data not shown) with slight v',u'iations in the sizes of the hybridising bands when compared to E, histolytica 200: NIH. E. histolytica strains AXI79, AX71 and E32 were obtained as a gift from Kothari Centre of Gastroenterology, Calcutta Hospital, Calcutta, where they were isolated as virulent strains with different clinical manifestations from patients.
(b) Sequence analysis of the insert in pEH212 The sequencing strategy is shown in Fig. 3. Fragments of pEH212 were subcloned in M l 3 m p l 8 and M l 3 m p l 9 and sequenced (Fig. 5). (1) It can be seen from Fig. 5a that fragment 1 is made up of several groups of direct repeats. A 12-bp sequence AATAATAAAGAT is repeated eleven times in perfect tandem except for the absence of the first AAT triplet in the second and third repeat. After a 66-bp stretch, a 9-bp sequence TATAATAAG is repeated eleven times in perfect tandem. Computer analysis of the sequence predicted an
I C;A'ICA( ; r r l T I A T A L ' I ' A A ' I ' A ( . ; A T A ( ;( ;1'
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ATACATATAA AATATATACA TrATA'rAAAG 130 140 150 ATAAGGrAI'A GAAATAACAG GC;(.;AGAGA'I'A A'I'AAGAAC.I'A TATAAAGAAT 160 170 180 19(1 2{~) AGG~rA'I'AAA(; t,~A'I'AGAAAC~A TATA'I'AAGCA CATATAGAAA I"ATAAAAGGIt
A T A ( ; ( ; A A A A A C ; A ( ; I ATAAA'I'AA(.;I'A(.;AAAIAA(.;
AAATATATAC
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AATA I A(.; I CAAG'I'ACATAAGCATAAA'I'AATAAAC;N[' AA'['AAA( ;AT AArAAAt ;AI' AA'I'AATAAA{;AT ~A'IA A r A A A L ; A T ,~ATAA'rAAA(,A'I A A T A A T A A A ( ',AT A A IAA'rAAAt;AT AATAA'rAAA(;AT AATAA'rAAA(;AT AA'rAA'rAAA[ ;ATAA'['AATAA ]~ATAAA(;(,;A ATA( ;AAACA'I'A'rA'IIAA ATATACA'rAAGTCC;TGC,TAAAAGA(.;AA( ;I' AAAGTATAA['AA(', "['ATAATAAG T A T A A ! AA(; TATAATAAG TATAATAA(; TATAATAA( ; T A T A A IAA(; TA'I'AA'['/~~ rC, TATAA'rAAr~; TATAATAAC; T A T A A T A A ( ;(;AAI ATA(.;ATA(;'I(.'IAC;(;(;(;L'A'I'A TAGTCA(.;t;I A A A T C.C A Tr(.;rL;~.riGT A GC;rr (;t ;T A A C A T A C A A GC( -1,rC A C GTF C;TC;CCC(.;C;GICG ATC
120
230
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210
220
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Fig. 5. Nucleotide sequence of fragments 1 and 2, and the position and orientation of 10/11 and 9/11 matches to the ll-bp ,4RS core sequence. (a) Sequence of fragment 1 of pEH212. (b) Sequence of fragment 2 of pEH212. In a, the 12-bp and 9-bp tandemly repeated sequences are arranged as blocks. The common24-bpoligooffragment I and 2 is underlined.The bold letteringin b denotes the sequence representingbent DNA. (c and d) Positions and orientation of the 10/1!-bp matches and 9/11-bp matches for fragments 1 and 2, with respect to the yeast 1l-bp ARS core consensus sequence. Solid symbols (boxes and arrowheads): 10/11bp match; thin arrows: 9/11 bp match; arrows indicate 5'-3' direction of ghe 10/11 and 9/11 matches; double-headed arrows: bent DNA.
201 ORF on the opposite strand. It was also seen h,y homology search that the ta.ademly repeated 12-bp sequence had approx. 90~ homology to the S. cerevisiae A R S 11-hp 'core' consensus sequence (5'-TAAATcATAAAT-3'). The entire fragment consisting of 486 bp was found to contain multiple copies of 10/11 and 9/11 matches to the A R S core consensus sequence. The second set oftandem 9-bp repeats was also seen to have a 9/11 match to the A R S core consensus sequence. A single 9/11 bp match was found on the opposite strand. The positioning of the different 10/11-bp and 9/11-bp matches is shown schematically in Fig. 5c. (2) The complete sequence of fragment 2 is shown in Fig. 5b. Tandem repeats as seen in fragment 1 were not seen in this fragment. However homology searches showed the presence of several dispersed and overlapping copies of 10/ll-bp and 9/11-bp matches to the yeast A R S core consensus sequence. A computer search to identify homology between fragments 1 and 2 was carried out. The dot plot of the COMPARE analysis (UWGCG program) (data not shown) indicates that the points of homology are located in those parts ofthe two sequences which carry 9/11 or 10/11 matches to the A R S consensus sequence. This suggested that fragments 1 and 2 comprised a repetitive DNA family of E. histolytica which had characteristic sequences homologous to yeast A R S core consensus sequences. A similar repetitixe DNA family has been reported to exist in the nematode C. elegans (Felsenstein and Emmons, 1988). However, unlike the C. elegans repetitive DNA family which also contained sequences homologous to CEN sequences from $. cerevisiae, we could not detect any sequences homologous to S. cerevisiae CEN sequences in this E. histolytica repetitive DNA family. It has been reported that E. histolytica DNA is 76.5% A + T rich (Golderman et al., 1971). Fragment 1 was seen to be 78% A + T rich and fragment 2 was seen to be 72% A + T rich. Even with this A + T content the probability of getting multiple copies of 9/ll-hp and 10/ll-bp matches to the core consensus sequence due to random occurrence is extremely low. Another common feature between the sequences of fragments 1 and 2 was a 24-bp oligo (Fig. 5a,b). As mentioned earlier, there was ambiguity in the molecular size of fragment 2 as determined by gel electrophoresis and nt sequence analysis. To rule out deletions, several subclones in both directions were read but in every case the data showed the presence of 365 bp in fragment 2, as opposed to approx. 500 bp by gel electrophoresis (Fig. 2, lane 5). We next took the Ml3mpl8 subclone which contained 331 bp of fragment 2 along with 486 bp of fragment 1. The size of fragment 1 as deduced from the sequence of this subclone (486 bp) matched the size of fragment 1 as deduced from gel electrophoresis (data not shown). We then digested the RF form of this Ml3mpl8 subclone with EcoRI + Smal and separated the digested
1.35 1.07 0.~7
0.60 0.31
1
2
Fig. 6. Anomalous mobility of fragment 2. The Ml3mp|8 subclone carrying 331 bp offragment 2 was digested and analysed on a 1% agarose gel, as described in section b. Lanes: 1, HaeIll digest ofcpX 174 RF DNA; 2, Sma I + EcoRl digest of the M 13mp 18 RF subclone carrying fragments 1 and 2. Arrowhead shows the position of the EcoRI.Smal 331 bp of fragment 2. Numbers on the left margin give sizes in kb.
DNA on a 1% agarose gel. It may be seen from Fig. 6 that this fragment moves at approx. 500 bp, whereas its sequence reads 331 bp. This led us to examine the sequence of fragment 2 more closely. It can be seen from Fig. 5b that there is a 75-bp sequence from nt 194-269 (bold-face letters; Fig. 5b) in which (A)4_~ runs are phased about every 10.5 bp. Wu and Crothers (1984) showed that DNA fragments containing (A)5_6 runs spaced periodically per helical repeat have an altered conformation. It was suggested that such DNA fragments have a bent helix and therefore migrate more slowly than expected for their size during electrophoresis. This behaviour is attributed to the resistance encountered by a 'bent' molecule in snaking through the gel pores (Wu and Crothers, 1984). From the evidence presented it is possible that fragment 2 has an altereu conformation. Nucleotide sequences having similar characteristics have been reported in Leishmania, phage ~., SV40, and $. cerevisiae (Kidane et al., 1984; Zahn and BlaVuer, 1985; Ryder et al., 1986; Snyder et al., 1986). 'Bent DHA' fragments have been found to serve as origins of replication and as sites where DNA-protein interactions occur (Kur et al., 1989; Zahn and Blattner, 1985; Ryder et al., 1986). No DNA transformation system exists in E. histolytica yet, by which we can test whether this DNA fragment could serve a similar role in this organism. However, in vitro studies are in progress to study the putative role of this DNA sequence in E. histolytica. Fig. 5d shows a schematic representation of the prominent features of the nt sequence of fragment 2 along with the 'bent DNA' and copies of 10/ll-bp and 9/ll-bp matches to the yeast l l-bp core consensus sequence. It can be seen that it is strikingly similar to the structure of domain B of S. cerevisiae ARS1 (Campbell, 1988). (3) Fragment 3 ofpEH212 was sequenced partially (data
202 not shown). On the basis of hybridisation studies, we have earlier suggested that this fragment comprises a different repetitive D N A family. However, upon comparing the partial sequence of fragment 3 to that of fragment 1, we could detect a small stretch of nt in fragment 3 which had homology to the 12-bp and 9-bp tandem repeats offragment 1. It is possible that this homology may be responsible for the hybridisation observed under low stringency (see section a). No 10/11 or 9/11 matches could be detected to the 11-bp A R S core consensus sequence, however. This suggests that the observed homology of fragment 3 to the tandem repeats of fragment 1 represents the degeneracy in those sequences which does not match the A R S core consensus sequence. Further characterisation of this fragment and fragment 4 is in progress to define the salient features of this family.
I .Ix
tuj Conclusions
(1) In this study we report the identification of different repetitive D N A families isolated by digestion ofE. histolytica D N A with the restriction enzyme Sau3AI. (2) Sequence analysis of one of these families reveals the presence of multiple copies of sequences homologous to S. cerevisiae A R S core consensus sequence, contributing to the homology between the different members of this repetitive D N A family. (3) The recombinant plasmid pEH212 isolated in this study was found to function as an A R S in S. cerevisiae. (4) One of the D N A fragments analysed in this study was found to contain sequences similar to 'bent DNA' identified in other organisms.
ACKNOWLEDGEMENTS (c) Transformation
of
Saccharomyces
cerevisiae with
pEH212
Fragments 1 and 2 of pEH212 had structural features very similar to the yeast A R S I , which prompted us to test if pEH212 would function as an A R S in yeast. We transformed S. cerevisiae 8534-10A [a, leu2, ura3, his4] with pEH212 by the lithium acetate method (Ito et al., 1983), using YIP5 as the negative co'~trol. The plasmid pCM3 which contains ARS1 was used as a positive control, pCM3 was obtained after deleting CEN5 from YCp 1 (Maine et al., i984). Plasmids pEH212 and pCM3 both showed high-frequency transformation (around 200 colonies/#g of plasmid DNA). However, it was observed that the size of the transformed colonies was five- to sevenfold smaller in case of pEH212 compared to pCM3. Therefore, pEH212 is not able to replicate as efficiently as pCM3 and carries a 'weaker' A R S than ARS1. Single colonies were isolated from the transformant~ and pla,smid stability assays were done by streaking for single colonies on YEPD plates and then replica plating on selective medium. The stability of pEH212 was two- to threefold less than pCM3. The efficiency of different A R S sequences in yeast have been observed to depend on the presence of a critical number of 11-bp core consensus sequences of perfect and near homology and also on the orientation of these sequences with respect to each other (Palzkill and Newlon, 1988). It is possible that the inefficiency of pEH212 compared to pCM3 is due to the fact that the tandem multimers of 10/I I and 9/11 matches in fragments 1 and 2 lack proper orientation with respect to each other. We next transformed S. cerevisiae with the plasmid pEH202. This was derived from pEH212 after deleting fragments 1 and 2. No transforma-_ts appeared with this plasmid. Thus, fragments 1 and 2 of pEH212 are capable of functioning as A R S elements in yeast, albeit less efficiently than the yeast ARS1.
The authors would like to thank Dr. P. Sinha and Dr. S. Roy for many helpful discussions and for critically reading the manuscript. S. cerevisiae 8534-10A and plasmids pCM3 and YIP5 were a gift from Dr. P. Sinha. This work was supporte d by the Department of Biochemistry, Bose Institute, Calcutta, India.
REFit~RENCES Bha(~harya, S., Bhattacharya, A. and Diamond, L.S.: Comparison of r,p.eatedr~ DNA from strains of Entamoeba histolytica and other
Emamoebas. Mol. Biochem. ParasitoL 27 (1988) 257-262. Broach, LR., Li, Y.Y., Feldman, J., Jayaram, M., Abraham, J., Nasmyth, K.A. and Hicks, I.B.: Localisation and sequence analysis of yeast origins of replication. Cold Spring Harbor Symp. Quant. Biol. 47
(1983) 1165-1173. Campbell, J.: Eukaryotic DNA replication: yeast bares its AR$. Trends
Biochem. Sci. 13 (1988) 212-217. Diamond, L.S., Harlow, D.R. and Cunnick, C.C.: A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoebas. Trans. Roy. Soc. Trop. Med. Hyg. 72 (1978) 431-432. Edman, U., Meza, I. and Agabian, N.: Genomic and eDNA actin sequences from a virulent strain of Entamoeba histolytica. Proc. Natl.
Acad. Sci. USA 84 (1987) 3024-3028. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeiling DNA restriction endonuclease fragments to high specific activity (Addendum). Anal. Biochem. 137 (1984) 266-267. Felsenstein, K.M. and Emmons, S.W.: Nematode rept;titive DNA with
ARS and segregationfunction in Saccharomycescerevisiae. Mol. Cell. Biol. 8 (1988) 875-883. Golderman, A.H., Bartgis, I.L., Keister, D.B. and Diamond, L.S.: A comparison ofgenome si~es and thermal denaturation derived base compositionof DNAs from several membersofEntamoeba (histolytica group). J. Parasitoi. 57 (1971) 912-916. Huber, M., Koller, B., Gitler, C,, Mirelman, D., Revel, M., Rozenblatt, S. and Garfinkel,L.: Entamoeba histolyticaribosomal RNA genes are carried on palindromic circular DNA molecules. Mol. Biochem. Parasitol. 32 (1989) 285-296. Ito, H., Fukuda, Y,, Murata, K. and Kimura,A.: Transformationofintact yeast cells treated with alkali cations. J. Bacteriol. 153 (1983) 163-168.
203 Kidane, G.Z., Hughes, D. and Simpson, L.: Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tare~ltolae. Gene 27 (1984) 265-277. Kur, .I., Hasan, N. and Szybalski, W.: Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p~ promoter and its mutants. Gone 81 (1989) 1-15. Maine, G.T., Sinha, P. and T.ve, B.K.: Mutants orS. cerevisiae defective in the maintenance of minichromosomes. Genetics 106 (1984) 365-385. Palzkill, T.G. and Newlon, C.S.: A yeast replication origin consists of multiple copies of a small conserved sequence. Cell 53 (1988) 441-450.
Ryder, K., Silver, S., DeLucia, A.L., Fanning, E. and Tegtmeyer, P.: An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. Cell 44 (1986) 719-725. Snyder, M., Buchman, A.R. and Davis, R.W.: Bent DNA at a yeast replicating sequence. Nature 324 (1986) 87-89. Walsh, J.A.: Problems in recognition and diagnosis of amebiasis. Estimation of the global magnitude of morbidity and mortality. Rev. Infect. Dis. 8 (1986) 228-238. Wu, H.M. and Crothers, D.M.: The locus of sequence directed and protein induced DNA bending. Nature 308 (1984) 509-513. Zahn, K. and Blattner, F.R.: Sequence induced DNA curvature at the bacteriophage lambda origin of replication. Nature 317 (1985) 451-453.
197
GENE 03844
C h a r a c t e r i s a t i o n of a repetitive D N A family from E n t a m o e b a histolytica containing Saccharomyces cerevisiae A R S consensus sequences (Protozoan parasite; recombinant plasmid; tandem repeats; sequence analysis; replication origin; bent DNA)
Anuradha Lohia, N. Haider and B.B. Biswas Department o f Biochemistry, Bose Institute, Cakutta 700054 (India)
Received by G.N. Godson: 23 March 1990 Revised: 2 May 1990 Accepted: 27 September 1990
SUMMARY
Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously repficating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIPS,. to replicate autonomously in S. eerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.
INTRODUCTION
The protozoan parasite E. histolytica is a human pathogen which causes extra-intestinal abcesses and dysentery in its invasive form, affecting almost 10~o of the world population (Walsh, 1986). Little has been reported on DNA replication or on the chromosomal organisation of this organism. However, like the genomes of most eukaryotic org,'misms ~he Entamoeba genome has also been reported to possess large amounts of repetitive DNA (Bhattacharya et al., 1988; Huber et al., 1989). One of the repeated DNA classes has been identified as ribosomal DNA (Bhattacharya et al., 1988; Huber et al, 1989). We are describing here another class of repetitive DNA
Correspondence to: Dr. B.B. Biswas, Department of Biochemistry, Bose Institute, P 1/12 C.I.T. Scheme Vll M, Calcutta 700054 (India). Tel. (91-33)379219: Fax (91-33)343886.
Abbreviations: ARS, autonomously replicating sequence(s); bp, base pair(s); C. , Caenorabditis; CEN, centromere; E. , Entamoeba ; ESP, 0.5 M EDTA/I% Sarkosyl/2mg per ml proteinase K; EtdBr, ethidium
from E. histolytica. Sequence analysis of members of this repetitive DNA class showed the presence of multiple copies of sequences homologous to the ll-bp ARS core consensus sequence 5' - AT A A A cT A -t" A A A TA of S. cerevisiae (Broach et al., 1983; Campbell, 1988; Palzkill and Newlon, 1988). These DNA fragments were also shown to be capable of transforming S. cerevisiae at a high frequency, when linked to a selectable marker. Fortuitously, one of the DNA fragments analysed showed a 'bent DNA'-like sequence. This 'bent DNA' fragment may serve as an origin of replication in E. histolytica, since the presence of such sequences has been shown at origins of replication in other organisms (Kidane et al., 1984; Ryder et al., 1986; Snyder et al., 1986; Zahn and Blattner, 1985).
bromide; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; RF, replicative form; S., Saccharomyces; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCi/0.15 M Na3"citrate pH 7.6; SV40, simian virus 40; TYIS-33, 2% trypticase peptone/1% yeast extract/2.28 mg per 100 ml ferric ammonium citrate/ 10% bovine serum; UWGCG, University of Wisconsin Genetics Computer Gro'ap; YEPD, 0.5 % yeast extract/1% peptone/2% dextrose.
198 RESULTS AND DISCUSSION
B
A
(a) Restriction enzyme digestion patterns of Entamoeba
histolytica Total DNA from E. histolytica 200: NIH nuclei was isolated (Edman et al., 1987), digested with several restriction endonucleases and fractionated on a 1% agarose gel (Fig. 1A). Frominent discrete bands ranging from 6.5-0.4 kb were observed in the Sau3AI-digested DNA,
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2
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Fig. 1. Restriction patterns of E. histolytica 200: NIH. (Panel A) Total DNA from E. histolytica nuclei was isolated by the method ofEdman et al. (1987) except that after isolating nuclei through sucrose cushions they were directly incubated in ESP for 2 h at 50°C, followed by treatment with phenol, chloroform and ethanol precipitate. The DNA was digested and separated by electrophoresis on a 1% agarose gel (lanes 2-4). The gel was stained with EtdBr (0.5 #g/ml) and the DNA was visualised under UV light at 300 nm. Numbers on the left margin give sizes in kb. Lanes: I, M, size markers HindIIl digest ofA DNA and HaeIII digest of OX174 RF DNA; 2, Sau3AI; 3, EcoRI; 4, HindIIl. Organisms: Trophozoites of E. histoh'tica 200 : NIH, were maintained axenically in TYIS-33 medium (Diamond et al., 1978) using 10';0 bovine serum. Usually 72-96 h cultures were used for DNA isolation. (Panel B) Autoradiographic detection of E. histolytica genomic DNA bands that hybridise to the recombinant clone pEH212. E. histolytica DNA from lane 2 (panel A) gel was tr~msferred to Nytran paper and hybridised to radioactively labelled pEH212 DNA (see Figs. 2 and 3). Radioactive DNA probes were prepared by using the technique of oligo labelling using random primers (Feinberg and Vogelstein, 1984). Hybridisation with radioactive probes was carried out in 0.5 M phosphate buffer pH 7.4/7% SDS at 65°C for 16-18 h. Washing was done at high stringency with 0.5 x SSC/0.1% SDS at 68°C. Numbers on the left margin are deduced sizes in kb of the hybridised bands.
0.31
1
2 3 4 5
6
7
Fig. 2. Restriction pattern ofpEH212 showing fragments 1-4. A genomic library of E. h~olytica 200: NIH trophozoite DNA was prepared in the yeast shuttle vector YIP5 by ligating Sau3Al.digested Entamoeba DNA at the BamHI site of YIP5 and transforming Escherichia coif HBI01. Plasmid pEH212 was a recombinant clone isolated from the E. histolygca genomic library, which hybridised to several DNA bands in Sau3AIdigested Entamoeba DNA. Plasmid pEH212 was digested with several restriction enzymes and separated by electrophoresis on a 1% agarose gel. The bands were stained with 0.5 #g EtdBr/ml, and visualised under UV light at 300 nm. Lanes: 1, HindIII digest of ADNA (see Fig. 1); 2, HaellI digest of ~X174 RF DNA; 3-7, digests of pEH212; 3, HindlIl + BamHI; 4, HindIII + Sinai; 5, BamHI + Smal; 6, BamHI + PvuI; 7, PvuI +SalI. (The top band in lane 7 at -8.5 kb is due to incomplete digestion and represents uncut plasmid.) Arrowhead shows the position of fragment 2 in lane 5. Numbers on the lef~ margin are sizes in kb.
199
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Fig. 3. Res~r/~.'ou map :~i the E. hbtolytica repetitive DNA insert ofpEH212 and the sequencing strategy. Blackened boxes, vector DNA. Junction of blackened boxes and solid fines, BamHI cloning site of the vector. Restriction sites are designated as follows: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; P, Pvul; Ps, Pstl; R, Rsal; S, Sau3AI; Sa, SaiI; Sin, Sinai. The junctions of the four different fragments of the insert are shown by open arrows. The numbers above the solid line indicate the four different fragments. Numbering is in ascending order in the 5'-3' direction. The M ! 3 subclones are shown by blackened arrows. Each subclone was read in both directions up to the points marked by the arrows.
suggesting the presence of highly abundant repetitive DNA classes. No single multimeric ladder could be detected in partial digests with Sau3Al (data not shown) which would suggest the presence of a single class of tandemly repeated DNA. It is possible that different Sau3Al families of repetitive DNA exist which comprise the multiple bands in the Sau3AI digested E. histolytica DNA. It may be noted that we were unable to detect prominent bands in the EcoRI and HindIIl-digested Entamoeba DNA as reported earlier (Bhattacharya et al., 1988; Huber et al., 1989). In an attempt to characterise and study the Sau3Al repetitive DNA classes, we constructed a genomic library ofEntamoeba DNA as described in the legend to Fig. 2. We chose the yeast integrating plasmid YIP5 as our cloning vector since we were also interested in isolating putative origins of replication from E. histolytica in yeast for another study. In this study the genomic library was screened by colony hybridisation with total E. histolytica 200:NIH DNA, radioactively labelled with [0(-32p]dATP as described in the legend to Fig. lB. The colonies which showed very intense signals were picked up on the basis that clones carrying repetitive DNA fragments would light up more strongly due to the abundance of similar DNA sequences in the radioactive probe. Five such clones were picked for further analysis. They were individually labelled with [0c-3Zp]dATP and Southern blots of genomic digests of E. histolytica DNA were hybridised separately with each of the radioactively labelled clones. One ofthe clones pEH212
6.40
2.25 1.80 1.70
1.10 0.95 0.87 0.75
2
3
4
• Fig. 4. Autoradiographic detection of Sau3Al-digested E. histolytica genomic DNA bands by hybridisation with fragments 1-4 of pEH212. Sau3AI-digested E. histolytica DNA was separated by electrophoresis on a 1% agarose gel (lanes 1-4) and transferred to Nytran paper. The Southern blot was cut along the lanes and the individual lanes were hybridised with radioactively labelled fragments !-4, respectively. Lanes: 1, probe-fragment 1; 2, probe-fragment 2; 3, probe-fragment 3; 4, probe-fragment 4. Numbers on the leR margin as in Fig. lB.
200 hybridised to several prominent bands in the Sau3Aldigested DNA (Fig. 1B). This clone was selected for further analysis. Restriction analysis of pEH212 showed the presence of an approx. 3.0-kb band, which was made of four Sau3AI fragments (0.5, 0.5, 1.25 and 0.92 kb; Figs. 2 and 3). As may be seen from a partial physical map of pEH212 (Fig. 3), the size of fragment 2 is less than 0.5 kb, the latter due to its anomalous mobility as discussed in section b. It is possible that four random Sau3Al fragments were ligated together, or the multiple inserts were a result of partially digested DNA. To determine whether the four fragments belonged to the same repetitive D N A classes or different ones, we isolated the fragments numbered 1, 2, 3, 4 (numbering in ascending order from 5 ' - 3 ' , Fig. 3) from a low-melting-point agarose gel and labelled them separately by o!igo labelling as described in the legend of Fig. lB. Southern blots ofSau3Al-digested E. histolytica DNA were hybridised with the four [~t-32P]dATP-labelled fragments separately (Fig. 4). At the relatively high stringency of washing used (0.5 x SSC/0.1~o SDS/68°C) some common bands in the two sets could be identified (data not shown). All four fragments showed smears when hybridised to other restriction enzymes digested E. histolytica DNA.
This suggested that these repetitive D N A classes were dispersed in the genome. We have also seen the presence of similar bands in Sau 3AI-digested D N A of several clinical strains orE. histolytica and similar hybridisation patterns with the two repetitive D N A classes (data not shown) with slight v',u'iations in the sizes of the hybridising bands when compared to E, histolytica 200: NIH. E. histolytica strains AXI79, AX71 and E32 were obtained as a gift from Kothari Centre of Gastroenterology, Calcutta Hospital, Calcutta, where they were isolated as virulent strains with different clinical manifestations from patients.
(b) Sequence analysis of the insert in pEH212 The sequencing strategy is shown in Fig. 3. Fragments of pEH212 were subcloned in M l 3 m p l 8 and M l 3 m p l 9 and sequenced (Fig. 5). (1) It can be seen from Fig. 5a that fragment 1 is made up of several groups of direct repeats. A 12-bp sequence AATAATAAAGAT is repeated eleven times in perfect tandem except for the absence of the first AAT triplet in the second and third repeat. After a 66-bp stretch, a 9-bp sequence TATAATAAG is repeated eleven times in perfect tandem. Computer analysis of the sequence predicted an
I C;A'ICA( ; r r l T I A T A L ' I ' A A ' I ' A ( . ; A T A ( ;( ;1'
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ATACATATAA AATATATACA TrATA'rAAAG 130 140 150 ATAAGGrAI'A GAAATAACAG GC;(.;AGAGA'I'A A'I'AAGAAC.I'A TATAAAGAAT 160 170 180 19(1 2{~) AGG~rA'I'AAA(; t,~A'I'AGAAAC~A TATA'I'AAGCA CATATAGAAA I"ATAAAAGGIt
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AATA I A(.; I CAAG'I'ACATAAGCATAAA'I'AATAAAC;N[' AA'['AAA( ;AT AArAAAt ;AI' AA'I'AATAAA{;AT ~A'IA A r A A A L ; A T ,~ATAA'rAAA(,A'I A A T A A T A A A ( ',AT A A IAA'rAAAt;AT AATAA'rAAA(;AT AATAA'rAAA(;AT AA'rAA'rAAA[ ;ATAA'['AATAA ]~ATAAA(;(,;A ATA( ;AAACA'I'A'rA'IIAA ATATACA'rAAGTCC;TGC,TAAAAGA(.;AA( ;I' AAAGTATAA['AA(', "['ATAATAAG T A T A A ! AA(; TATAATAAG TATAATAA(; TATAATAA( ; T A T A A IAA(; TA'I'AA'['/~~ rC, TATAA'rAAr~; TATAATAAC; T A T A A T A A ( ;(;AAI ATA(.;ATA(;'I(.'IAC;(;(;(;L'A'I'A TAGTCA(.;t;I A A A T C.C A Tr(.;rL;~.riGT A GC;rr (;t ;T A A C A T A C A A GC( -1,rC A C GTF C;TC;CCC(.;C;GICG ATC
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Fig. 5. Nucleotide sequence of fragments 1 and 2, and the position and orientation of 10/11 and 9/11 matches to the ll-bp ,4RS core sequence. (a) Sequence of fragment 1 of pEH212. (b) Sequence of fragment 2 of pEH212. In a, the 12-bp and 9-bp tandemly repeated sequences are arranged as blocks. The common24-bpoligooffragment I and 2 is underlined.The bold letteringin b denotes the sequence representingbent DNA. (c and d) Positions and orientation of the 10/1!-bp matches and 9/11-bp matches for fragments 1 and 2, with respect to the yeast 1l-bp ARS core consensus sequence. Solid symbols (boxes and arrowheads): 10/11bp match; thin arrows: 9/11 bp match; arrows indicate 5'-3' direction of ghe 10/11 and 9/11 matches; double-headed arrows: bent DNA.
201 ORF on the opposite strand. It was also seen h,y homology search that the ta.ademly repeated 12-bp sequence had approx. 90~ homology to the S. cerevisiae A R S 11-hp 'core' consensus sequence (5'-TAAATcATAAAT-3'). The entire fragment consisting of 486 bp was found to contain multiple copies of 10/11 and 9/11 matches to the A R S core consensus sequence. The second set oftandem 9-bp repeats was also seen to have a 9/11 match to the A R S core consensus sequence. A single 9/11 bp match was found on the opposite strand. The positioning of the different 10/11-bp and 9/11-bp matches is shown schematically in Fig. 5c. (2) The complete sequence of fragment 2 is shown in Fig. 5b. Tandem repeats as seen in fragment 1 were not seen in this fragment. However homology searches showed the presence of several dispersed and overlapping copies of 10/ll-bp and 9/11-bp matches to the yeast A R S core consensus sequence. A computer search to identify homology between fragments 1 and 2 was carried out. The dot plot of the COMPARE analysis (UWGCG program) (data not shown) indicates that the points of homology are located in those parts ofthe two sequences which carry 9/11 or 10/11 matches to the A R S consensus sequence. This suggested that fragments 1 and 2 comprised a repetitive DNA family of E. histolytica which had characteristic sequences homologous to yeast A R S core consensus sequences. A similar repetitixe DNA family has been reported to exist in the nematode C. elegans (Felsenstein and Emmons, 1988). However, unlike the C. elegans repetitive DNA family which also contained sequences homologous to CEN sequences from $. cerevisiae, we could not detect any sequences homologous to S. cerevisiae CEN sequences in this E. histolytica repetitive DNA family. It has been reported that E. histolytica DNA is 76.5% A + T rich (Golderman et al., 1971). Fragment 1 was seen to be 78% A + T rich and fragment 2 was seen to be 72% A + T rich. Even with this A + T content the probability of getting multiple copies of 9/ll-hp and 10/ll-bp matches to the core consensus sequence due to random occurrence is extremely low. Another common feature between the sequences of fragments 1 and 2 was a 24-bp oligo (Fig. 5a,b). As mentioned earlier, there was ambiguity in the molecular size of fragment 2 as determined by gel electrophoresis and nt sequence analysis. To rule out deletions, several subclones in both directions were read but in every case the data showed the presence of 365 bp in fragment 2, as opposed to approx. 500 bp by gel electrophoresis (Fig. 2, lane 5). We next took the Ml3mpl8 subclone which contained 331 bp of fragment 2 along with 486 bp of fragment 1. The size of fragment 1 as deduced from the sequence of this subclone (486 bp) matched the size of fragment 1 as deduced from gel electrophoresis (data not shown). We then digested the RF form of this Ml3mpl8 subclone with EcoRI + Smal and separated the digested
1.35 1.07 0.~7
0.60 0.31
1
2
Fig. 6. Anomalous mobility of fragment 2. The Ml3mp|8 subclone carrying 331 bp offragment 2 was digested and analysed on a 1% agarose gel, as described in section b. Lanes: 1, HaeIll digest ofcpX 174 RF DNA; 2, Sma I + EcoRl digest of the M 13mp 18 RF subclone carrying fragments 1 and 2. Arrowhead shows the position of the EcoRI.Smal 331 bp of fragment 2. Numbers on the left margin give sizes in kb.
DNA on a 1% agarose gel. It may be seen from Fig. 6 that this fragment moves at approx. 500 bp, whereas its sequence reads 331 bp. This led us to examine the sequence of fragment 2 more closely. It can be seen from Fig. 5b that there is a 75-bp sequence from nt 194-269 (bold-face letters; Fig. 5b) in which (A)4_~ runs are phased about every 10.5 bp. Wu and Crothers (1984) showed that DNA fragments containing (A)5_6 runs spaced periodically per helical repeat have an altered conformation. It was suggested that such DNA fragments have a bent helix and therefore migrate more slowly than expected for their size during electrophoresis. This behaviour is attributed to the resistance encountered by a 'bent' molecule in snaking through the gel pores (Wu and Crothers, 1984). From the evidence presented it is possible that fragment 2 has an altereu conformation. Nucleotide sequences having similar characteristics have been reported in Leishmania, phage ~., SV40, and $. cerevisiae (Kidane et al., 1984; Zahn and BlaVuer, 1985; Ryder et al., 1986; Snyder et al., 1986). 'Bent DHA' fragments have been found to serve as origins of replication and as sites where DNA-protein interactions occur (Kur et al., 1989; Zahn and Blattner, 1985; Ryder et al., 1986). No DNA transformation system exists in E. histolytica yet, by which we can test whether this DNA fragment could serve a similar role in this organism. However, in vitro studies are in progress to study the putative role of this DNA sequence in E. histolytica. Fig. 5d shows a schematic representation of the prominent features of the nt sequence of fragment 2 along with the 'bent DNA' and copies of 10/ll-bp and 9/ll-bp matches to the yeast l l-bp core consensus sequence. It can be seen that it is strikingly similar to the structure of domain B of S. cerevisiae ARS1 (Campbell, 1988). (3) Fragment 3 ofpEH212 was sequenced partially (data
202 not shown). On the basis of hybridisation studies, we have earlier suggested that this fragment comprises a different repetitive D N A family. However, upon comparing the partial sequence of fragment 3 to that of fragment 1, we could detect a small stretch of nt in fragment 3 which had homology to the 12-bp and 9-bp tandem repeats offragment 1. It is possible that this homology may be responsible for the hybridisation observed under low stringency (see section a). No 10/11 or 9/11 matches could be detected to the 11-bp A R S core consensus sequence, however. This suggests that the observed homology of fragment 3 to the tandem repeats of fragment 1 represents the degeneracy in those sequences which does not match the A R S core consensus sequence. Further characterisation of this fragment and fragment 4 is in progress to define the salient features of this family.
I .Ix
tuj Conclusions
(1) In this study we report the identification of different repetitive D N A families isolated by digestion ofE. histolytica D N A with the restriction enzyme Sau3AI. (2) Sequence analysis of one of these families reveals the presence of multiple copies of sequences homologous to S. cerevisiae A R S core consensus sequence, contributing to the homology between the different members of this repetitive D N A family. (3) The recombinant plasmid pEH212 isolated in this study was found to function as an A R S in S. cerevisiae. (4) One of the D N A fragments analysed in this study was found to contain sequences similar to 'bent DNA' identified in other organisms.
ACKNOWLEDGEMENTS (c) Transformation
of
Saccharomyces
cerevisiae with
pEH212
Fragments 1 and 2 of pEH212 had structural features very similar to the yeast A R S I , which prompted us to test if pEH212 would function as an A R S in yeast. We transformed S. cerevisiae 8534-10A [a, leu2, ura3, his4] with pEH212 by the lithium acetate method (Ito et al., 1983), using YIP5 as the negative co'~trol. The plasmid pCM3 which contains ARS1 was used as a positive control, pCM3 was obtained after deleting CEN5 from YCp 1 (Maine et al., i984). Plasmids pEH212 and pCM3 both showed high-frequency transformation (around 200 colonies/#g of plasmid DNA). However, it was observed that the size of the transformed colonies was five- to sevenfold smaller in case of pEH212 compared to pCM3. Therefore, pEH212 is not able to replicate as efficiently as pCM3 and carries a 'weaker' A R S than ARS1. Single colonies were isolated from the transformant~ and pla,smid stability assays were done by streaking for single colonies on YEPD plates and then replica plating on selective medium. The stability of pEH212 was two- to threefold less than pCM3. The efficiency of different A R S sequences in yeast have been observed to depend on the presence of a critical number of 11-bp core consensus sequences of perfect and near homology and also on the orientation of these sequences with respect to each other (Palzkill and Newlon, 1988). It is possible that the inefficiency of pEH212 compared to pCM3 is due to the fact that the tandem multimers of 10/I I and 9/11 matches in fragments 1 and 2 lack proper orientation with respect to each other. We next transformed S. cerevisiae with the plasmid pEH202. This was derived from pEH212 after deleting fragments 1 and 2. No transforma-_ts appeared with this plasmid. Thus, fragments 1 and 2 of pEH212 are capable of functioning as A R S elements in yeast, albeit less efficiently than the yeast ARS1.
The authors would like to thank Dr. P. Sinha and Dr. S. Roy for many helpful discussions and for critically reading the manuscript. S. cerevisiae 8534-10A and plasmids pCM3 and YIP5 were a gift from Dr. P. Sinha. This work was supporte d by the Department of Biochemistry, Bose Institute, Calcutta, India.
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(1983) 1165-1173. Campbell, J.: Eukaryotic DNA replication: yeast bares its AR$. Trends
Biochem. Sci. 13 (1988) 212-217. Diamond, L.S., Harlow, D.R. and Cunnick, C.C.: A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoebas. Trans. Roy. Soc. Trop. Med. Hyg. 72 (1978) 431-432. Edman, U., Meza, I. and Agabian, N.: Genomic and eDNA actin sequences from a virulent strain of Entamoeba histolytica. Proc. Natl.
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ARS and segregationfunction in Saccharomycescerevisiae. Mol. Cell. Biol. 8 (1988) 875-883. Golderman, A.H., Bartgis, I.L., Keister, D.B. and Diamond, L.S.: A comparison ofgenome si~es and thermal denaturation derived base compositionof DNAs from several membersofEntamoeba (histolytica group). J. Parasitoi. 57 (1971) 912-916. Huber, M., Koller, B., Gitler, C,, Mirelman, D., Revel, M., Rozenblatt, S. and Garfinkel,L.: Entamoeba histolyticaribosomal RNA genes are carried on palindromic circular DNA molecules. Mol. Biochem. Parasitol. 32 (1989) 285-296. Ito, H., Fukuda, Y,, Murata, K. and Kimura,A.: Transformationofintact yeast cells treated with alkali cations. J. Bacteriol. 153 (1983) 163-168.
203 Kidane, G.Z., Hughes, D. and Simpson, L.: Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tare~ltolae. Gene 27 (1984) 265-277. Kur, .I., Hasan, N. and Szybalski, W.: Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p~ promoter and its mutants. Gone 81 (1989) 1-15. Maine, G.T., Sinha, P. and T.ve, B.K.: Mutants orS. cerevisiae defective in the maintenance of minichromosomes. Genetics 106 (1984) 365-385. Palzkill, T.G. and Newlon, C.S.: A yeast replication origin consists of multiple copies of a small conserved sequence. Cell 53 (1988) 441-450.
Ryder, K., Silver, S., DeLucia, A.L., Fanning, E. and Tegtmeyer, P.: An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. Cell 44 (1986) 719-725. Snyder, M., Buchman, A.R. and Davis, R.W.: Bent DNA at a yeast replicating sequence. Nature 324 (1986) 87-89. Walsh, J.A.: Problems in recognition and diagnosis of amebiasis. Estimation of the global magnitude of morbidity and mortality. Rev. Infect. Dis. 8 (1986) 228-238. Wu, H.M. and Crothers, D.M.: The locus of sequence directed and protein induced DNA bending. Nature 308 (1984) 509-513. Zahn, K. and Blattner, F.R.: Sequence induced DNA curvature at the bacteriophage lambda origin of replication. Nature 317 (1985) 451-453.
