Gene, 88 (1990) 141-147 Elsevier
141
GENE 03439
Hypersensitive m u n g bean nuclease cleavage sites in Plasmodium knowlesi D N A (Recombinant DNA; homopurine/homopyrimidine stretches)
Przemyslaw Szafrat~ski" and G. Nigel Godson u ° Polish Academy of Sciences, Institute of Biochemiswy and Biophysics. ul. Rakowiecka 36, 02-532 Warsaw (Poland) Tel. 49-04-03, and b Department of Biochemistry, New York University Medical Center, New York, N Y 10016 (U.S.A.) Received by D.M. Skinner: June 1, 1989 Revised: 23 August 1989 Accepted: 25 August 1989
SUMMARY
Nucleotide sequences of Plasmodium knowlesi DNA that are cleaved by mung bean nuclease (Mbn) at low enzyme concentration (0.2 units enzyme per/~g DNA) are listed. They are tandemly repeated purine/pyrimidine (RpY) stretches of DNA with (APT) dimers predominating. Most cut sites are within almost 100% RpY tracts. The enzyme cleaves at many points within the RpY stretch and usually hydrolyzes the 5'-ApT-Y linkage. These alternating RpY target sites are flanked by homopurine and homopyrimidine stretches. At least one Mbn target site lies next to an in vivo transcribed region.
INTRODUCTION
Five years ago McCutchan et al. (1984) reported that under denaturing conditions employing 45% formamide and 50°C, Mbn cleaved Plasmodiumfalciparum genomic DNA into 'gene size' pieces. These cleavages appeared to be at AT-rich regions. Since then several studies employing these digestion conditions have been published, attempting to identify the nt specificity of this phenomenon. Most of these reports, however, give different and in many ways incompatible results. Muhich and Simpson (1986) found that Leishrnania mrentolae kinetoplast minicircle DNA was cleaved by Mbn in 40~o formamide, in many places. Twenty-seven cleavage Correspondenceto: Dr. G.N. Godson, Department of Biochemistry, New York University Medical Center, New York, NY 10016 (U.S.A.) Tel. (212)340-5622; Fax (212)340-8166. Abbreviations: bp, base pair(s); BS, blood stage; cpm, counts per minute; cs, gene encoding the circumsporozoite protein; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani medium; Mbn, mung bean nuclease; nt, nuclcotide(s); ORF, open reading frame; P., Plasmodiwn; Poll, Escherichiacoli DNA polymerase I; R, purine; SP, sporozcite (stage); u, unit(s); Y, pyrimidine. 0378-I1191901503.50© 1990Elsevier SciencePublishers BN. (BiomedicalDivision)
sites were sequenced; few were AT-rich and many were GC-rich for 10 bp on either side of the actual cleavage site. Brown et al. (1986)examined Mbn cleavage sites flanking the Trypanosoma brucei VSG gene at various formamide concentrations and found that they were within an AT-rich sequence, consisting of runs of A and runs of T residues. In plasmid systems, Sheflin and Kowalski (1984) showed that in the absence of formamide Mbn cuts PM2 DNA within the 135-bp random AT-rich region (74~o AT), but 32 different cleavages revealed no specific sequence requirements. Recently, Vernick et al. (1988) have reported new digestion conditions that result in cleavage ofP. falcipamm DNA precisely at sites outside coding regions. These sites are all AT-rich, some consisting of (APT) dimers. The experiments described in these papers were all carried out at high enzyme concentrations (5-50u/~g DNA). It is apparent, however, from the published data that Mbn cleaves DNA efficiently at low enzyme concentrations (Brown et al., 1986, Muhich and Simpson, 1986). It may be possible, therefore, that the enzyme cleaves at different sites under different digestion conditions and enzyme concentrations. In this paper, we have examined the cutting of Mbn at
142 low enzyme concentration (0.2u/pg
DNA), using cloned into ~EMBIA. These 'hypersensitive' sites appear to be fairly specific, consisting of alternating RpY dimers, with (APT) predominating, and flanked by oligo(R) and oligo(Y) stretches.
P. Imowlesi DNA fragments (15-20kb)
MATERIALS AND METHODS
(a) Bacterial strains and phages Escherichia coli JMI01 was used for M13 transfection and Q358 was used for propagation of recombinant A phages. LB broth (Miller, 1974) was used as growth medium.
(b) ,tEMBIA genomic DNA library P. knowlesi genomic DNA was obtained from blood
stage parasites as previously described (Ozaki et al., 1983). The DNA was partially digested with Sau3A and introduced into AEMBI.A (Frischauf et al., 1983) as described by Ozaki and Cseko (1984). The blood stage and sporozoite mRNA used to screen the library was isolated and labelled with 32p as described by Rulz i Altaba et al. (1987) and Ozaki et al. (1983).
(c) Subeloning and sequencing DNA fragments, cut with either Mbn or restriction enzymes, were separated on agarose gels and cloned into Ml3mp8, mp9 and mpl9 (Messing, 1983). Recombinant DNA procedures were as described by Maniatis et ai. (1982). The ntsequences were determined using the dideoxy chain termination method (Sanger et al., 1977) and, where necessary, the DNA "1"4polymerase treatment was used to produce overlapping DNA fragments (Dale et al., 1985).
(d) Nucleic acid hybridization DNA digests were fractionated on agarose gels, transferred to nitrocellulose (Mania~is et al., 1982) and probed with either parasite mRNA fraginents end-labelled with [7-32P]ATP (Ruiz i Altaba et al., 1987) or with DNA fragments nick translated with [0c-32p]dATP using E. col[ Poll.
RESULTS AND DISCUSSION
(a) Clones used and strategy for this study If Mbn cleaves DNA at specific nt sequences that flank gene coding sequences, it would be expected that the enzyme would cleave cloned Plasmodlum DNA in the same way as genomic Plasmodlum DNA. Using cloned genomic DNA fragments has the advantage that P. knowlesi DNA
containing transcribed sequences can be identified before Mbn cleavage and that each DNA fragment will have only a few (one or two) cleavage sites, which can be isolated and then studied in detail. A partial Sau3A library of/'. knowlesi DNA was therefore constructed in ~EMBIA. The library was screened for clones that contained expressed genomic DNA sequences by hybridizing with labelled BS poly(A) + mRNA, and SP poly(A) + mRNA. These clones were given random numbers prefixed by BS and SP respectively. AEMBL4 clones containing the already sequenced and characterized P. knowlesi cs gene were also isolated using as a probe a 0.9-kb HindIII fragment of the cloned gene which contains only the coding sequences (Ozaki et al., 1983). KBS01 obtained in this way contains a 15-kb genomic DNA insert. (b) Digestion of ~EMBIA clones with Mbn: sensitive and hypersensitive cleavage sites The ~EMBIA KBS01 clone was chosen to establish the best conditions for Mbn cleavage. This was because the
u/,u.g ONA
X0.21 2 3 I I I I I 17 kb 9,4kb 6.6kb 4.6kb 4,4kb
23kb 2.0kb
*"CS ----BS 1 "--BS2
1.Skb
Fig, 1, Effect of Mbn concentration on AKBS01 b~4A {:l~avase.2/~g of DNA was resuspet~ded in 10pl of 45% formamide containing 0.2 M NaCI/I mM ZnSO4/30 mM Na" acetate pH 4.6 and 0.05 to 3.00 u of Mbn (Sigma chemicals)was added per/48 of DNA. The mixture was the.n incubated at 50°C for 30 mitt (McCutchan et al., 1984).Lane !, ~ marker; lanes 2-5, indicated number of Mbn u//AgDNA. The products were run on a 0.7% agarose gel, stained with ethidium bromide and photographed. CS refers to a band containing the cs gene, and BS to bands that hybridised to BS parasite mRNA.
143 15-kb genomic DNA insert contains at least one transcribed gene, the cs gene. Using the original digestion conditions of McCutchan etal. (1984), 2/~g KBS01 DNA sample was digested with increasing amounts of Mbn (Fig. 1). With 0.2 u/#g DNA, no gene-sized pieces were excised from the DNA, although a series of large ( < 5 kb) DNA bands were apparent. Increasing the concentration of enzyme to I u/#g DNA released 2.8-kb and 2. l-kb bands. At higher Mbn concentrations (2 u//~g and 3 u/ttg DNA), the 2.8-kb band disappeared, the 2. l-kb band increased in intensity and lower Mr bands appeared. The high Mr bands ( > 4.4 kb) that appeared after digestion with 0.2 u//~g DNA were present at I u/#g DNA, but disappeared with 2 or more u Mbn/#g. Probing a Southern blot ofthe gel with the 0.9-kb HindlIl probe containing cs gene coding region, showed that only the 2.8-kb band contained the cs gene. Two bands of 2.1 and 1.6-kb, respectively, hybridized strongly with 32p end-labelled BS parasite mRNA. These bands contain sequences from genes transcribed in the blood stages and were therefore labelled B S I and BS2 respectively. The high Mr bands did not hybridize to either probe.
or) ( n
(n u) (n
v v v ~
I IIIII
,,
Mbn I
Thus the cs and B S I , B S 2 genes are cleaved out at different enzyme concentrations. This effect was observed with other ~ clones, where different discrete bands appear at different enzyme concentrations (data not shown). The low Mbn concentration (0.2 u/~tg DNA), however, efficiently cleaved out discrete gene-sized fragments from many of the ~. clones. Thus Plasrnodium DNA has sites that are more or less sensitive to digestion with Mbn. For convenience, those sites that cleave at low concentrations of enzyme ( < 1.0 u/#g DNA) are termed 'hypersensitive' sites and those that require > 1.0 u//~g DNA are termed "sensitive' sites.
(e) Sequence of hypersensitive Mbn cleavage sites Five random clones that hybridized to BS mRNA and that were digested efficiently with Mbn at 0.2 u/#g DNA, are shown in Fig. 2. The sizes of the partial S a u 3 A DNA insert of these five clones were 9.5 kb (KBS23), 16.4 kb (KBS25), 17.8 kb (Keg26), 13.7 kb (KBSS0)and 16.1 kb (KBS55). All clones gave different patterns of Mbn digestion products. KBS25 and KBS50 had two identical lower M r bands (Mbnl, 3.5 kb and Mbn2, 1.0 kb) but differed in sizes of the high-Mr bands. This suggests that they may be partially overlapping clones. KBS55 gave a single low-Mr band (Mbn3, 1.7 kb). Mbnl, 2, and 3 hybridized with BS mRNA indicating they were from transcribed genes, Both KBSS0 and KBS55 however do contain the sporozoite cs gene, but this is present in the undigested DNA and is only excised at higher enzyme concentrations (see section h above). Mbnl, 2 and 3 therefore represent hypersensitive cleavage sites and the undigested cs gene sensitive sites, KBS50 was chosen for further study. First a restriction enzyme cleavage map of the 13.7-kb insert of KBS50 was constructed (Fig. 3). Then the Mbn digestion products (0.2 u/ttg DNA) were cloned into M 13mp 18 and sequenced, either completely (Mbn2) or just the 5' and 3' ends (Mbnl). Mbnl and Mbn2 DNA fragments were then used as radioactive probes on Southern blots of restriction enzyme digests of the KBSS0 DNA insert and were found to lie in the approximate positions shown in Fig. 3. The insert DNA was then digested with EcoRl alone, EcoRI + HtndIll and Hindlll + PstI, and
Mbn 3 ,,, I k b
,,
,
Mbn 2
I
0 Fig. 2. Mbn digestionpattern of several~,EMBIArecombinantclones, at 0.2 u Mbn/pg DNA. Conditions are as describedin Fig. 1 legend. Bands markedMbnl, 2 and 3 wereisolatedfromthe gel and clonedfor further study.
A
III
BCO
I
E
Fig. 3. Restrictionmap of the 13.7-kbinsert of ,tKBS$0 showingthe approximatelocationof bands Mbnl and Mbn2shownin Fig,2. The Mbn cleavageproducts,Mbul and Mbn2, wereplacedon the map by blotting protocols.The black areas lettered A-E refer to the Mbn cleavagesites describedin Fig.4.
144 the D N A fragments were cloned into Ml3mpl9. Using Mbn I and Mbn2 DNA fragments as probes, clones of the insert D N A were isolated that contained all and part of Mbn I and Mbn2. These clones were sequenced. Genomic sequences (2.5 kb) encompassing Mbn2 and its cleavage sites, and genomic sequences (0.5 kb) surrounding Mbnl cleavage sites were established. The 5' and 3' cleavage sites flanking Mbnl are designated A and B and those flanking Mbn2 are designated D
~0
20
10
40
50
60
70
80
~0
IOl
110
120
11o
14o
ll5o
,T O
18o
230
240
and E (Fig. 3). The Mbn cutting sites and sun'ounding sequences are shown in Fig. 4. Fifteen M13 clones containing Mbn2 and two clones containing Mbnl were sequenced in both orientations. The 5' and 3' termini of the M 13 clones are marked with an arrow in Fig. 4. Within each AT-rich region, several independent clones were found to cut at a single site. Other cuts within the AT-rich region were observed, but their exact location was ambiguous due to the repeating ApT dimers. Between Mbn 1 and Mbn2, there is believed to be an extra Mbn site (marked C in Fig. 3). This site was first identified from sequence comparison, using data from A, B, D and E cleavage sites. Its existence would explain why the 0.5-kb band that separates Mbnl and Mbn2 was never observed.
Site A
IT?
200
21o
220
irT
2.
270 V
280
290
300
GT~A~CCCT CCT~CCAGT ACAATC'~-~TT~._GTTTC.A.~CGA~O GAAGAAC~AU
310
320
330
340
350
360
GACGAT" A A A Am,
420
TACTC~-~C~A ATGTCTOT~GGGAAAGAAAA A ~ T ~ G ~ o C A G T A TTTACAACAA
TC~TTGTA~
TCT~AC~
^ C ~ A A ~ ~ ~ ~ - - - ~
370
380
390
400
430
440
4S0
460
I0
20
30
70
8o
90
13o
140
190
|00
23o
260
410
~AA~TTGG C~TAATCATGGTC^TAOCTGRTTCC'~'CTCI".CA_A_~TOTT^~-~CTCACA "~CAC^ AC^TACGAGCc ~ - - ~ ^ ~ ' ~ T ~ - ~
470
1490
1500
1510
1520
1530
1540
1550
1560
1570
1580
1590
1600
1610
1620
1630
1640
1650
1660
1670
1680
1690
1700
1710
1720
1730
1740
1750
1760
1770
1780
1790
1800
1910
1820
1830
1040
1950
1060
,s7o
isso
189o
10oo
llll
io2o
193o
1940
1950
IO60
Io70
1980
1900
,00
,OlO
IU °
,040
2050
2060
2070
2080
2000
2100
il 1°
'1'"
Ull
,15o.
,1.o
.~'*CCGC~---"~GCC"I~A~~A CGTG"I~ACd31
AGCACgA-~CAACTGCTGA T ~ C a G ~ C O ACTT~T^TTA^CAGA~'A'A~A'I'~__..~.
ioo
11o
12o
TACACCA~C~--A~AG AATAAT~A----~'~TATTGC~-~----A~CAATGC~'~ACAOT
150
160
170
180
OCTGCT^G{FCC'~'!CTTTTGA IT'~AGC~-'~CAATAAq~-----~-'~TAACIGAA---~AOCT
210
220
230
240
220
2so
290
sou
~^^C~TTT..~ ^^~A^^^O^ ^O^^^e^^~6. y?y^~OC~T]
~IQ m2o ~}o p40 ~o 50o LUU~atLL~,:A AAO4~O~kA~4~AOAAGAAAA^OAAOOTTCTTT AAA~AGOAOOAAA.AAOAA~ ~
390
400 ~
dlO
4,0
.4o
1H
lfl
,,o
490
500
510
320
$~0
560
570
~00
590
~on
610
620
6~0
640
630
660
~ ^ T O T A
1480
60
~AAO^^OAAOAAOOTT~TAA AAI~'~AAO AAA-AATAAGGTT~CCYbAG(]OA^~AAAG'
~r~A~TAOC~AAA
1470
50
40 . . . .
_~--A.AC~--AA'AK~AAOAAAOAAGGTTT¢TTAAC~AGGAAGOAAAAOAAGAAAGAA~TTTC~
~..~=.~0
1460
C~'~GAGAAAAAA~AATGAC~-~---_A..~ATATGA~TCT~TA~CGA TGATAATATC
cc~-----~oc c*^
QA^~AAAAO^AGA^AOAAO GTTTAAA~AG~aGAAAAOAAO A ~ O A A ~ T ?T~TT^AC~A
'r~3~r~^^^o
1450
Site B
,,o
~SAATqlQAAAOOA~rATAT "
AAAAAAAAAAAAAAAAAAAGJTO
l
~
'
~
2170
~
T A T O T A A T O 0 ~ : L ~
V
2190
2 | 0 0 ~ 0
::30
::40
2230
2250
2290
2300
2310
2320
^^Ao0^^^^ocmu^^^^oo^ ^ ~ H C l ~ ^ , ^ o ^ o __
o^^oo^,^~^ o^^r.,o^^^^o ^^^~Aop~'~'l~o^^~^~ __
Site E
|||0 22?0
2280
^^^G^^^o^o ^^oGo~r~ 2330
~AAGO~
2340
AC~-.a40~A~
SlteC 6NO
670
690
700
710
233o
720
730 740 750 [~O~CAC AATTATOGCGO C A T C ~ T O T T
~9o
8oo
8
3
760
slo
770
780
8m0
54o
0 930
940
930
900
1030
1040
1030
1060
1090
I00
II10
1120
1150
1160
1170
1180
1190
122o
12~o
124o
1230
126o
1300
1310
1320
1070
10RO
A~aC~JC~[TC TGUAGAAA~ATCTC~TCTGTGTACTGAC~C~CTT ~ ' ~ G ~
AATATTTI~'--~TC~TGq~--G~C~CAq~TC~r A C ~ £ C121o C..~^^ 1270
TCG~C
1200 ^OACCAACTA
A~rTACGACCT~C~ATGA T~--~L"TTAT OCACGCTGTA 1280
1290
1340
1350
1360
1370
1380
1400
,410
1420
1430
1440
~TI~TC~T~T
^CC.~^OATCA T T O C ~ C ^
^TCG'UCTAATGT~-~AATA AAT~CCACTT~AGC[~OC
~ ?
2430
2410
2480
2490
2530
2540
2550
2380
2400
OTO~TTTAG
2440
2430
2460
2500
2510
2520
2360
2570
2380
AAAAV.IGAAAAGGAAAAVaGAAAA~'OAAAAr~
2.0
2600
2010
TAGTAACC~GTAI"~CCGGATGGTA
2620
TGAACCC~AT
CTTTCCTTCC TTTCTTTTC~21^TC~=TC'~_C
970 980 990 IO00 I010 1020 [A[A[A[A[A[AJ~'~T~3,AAC~AAOAT.TyAOAOAAAqC ^ ~ ~ A A O A T T _ C C C C . ~ T C T G .,
2420
235o
AIrJ~AOAAOAO G A ~ O O ~
~AAGOA^^A ~AAAGOA^~ O ~ A ~ A O A A ~ ^ ~ ~ C : C A O A T O ^
~
Sited V920
otQ
2410
l~AR~AAAA GOQAAA~A^ A A ~ A A ~
^TAC~AATOTL=~-'-A~TACCOTTAC~-G-~GCTGC OAmmTGC"GA O C ~ ' ~ CC~TAAOTGA
2370
~=~]~..~sGGAA GGA~'[DAAGG^AGGOAAAGG OAAA(~0OAAO GA^AOGAA^O ~^^AOGOAG[
GC^OCCTTC~ TT~Cp~'A'A"~
520
2300
AAACmC836AAG ACS(3AAA~OA A A ~ A ^ A ~
i1 U 1U.~ GGTTOAC'GAC
TACC
Fig. 4. Nt sequence of Mbn.cutting sites present in ~KBSS0. Sites A - E refer to the IVlbn-cutting sites shown in Fig. 3. The alternating RpY regions containing the cut sites are shaded and the actual cleavage sites are marked with filled arrowheads. One independent Mbn-cutting point ending at the arrow was located in site A, two at site B, five at site D and tour at site E. Other cleavage sites in these regions were observed, but their location was ambiguous due to the repeating ApT dinners. The oligu(R) stretches flanking the alternating RpY region are boxed with solid lines and the oligo(Y) stretches are boxed with a solid line above and dotted line below. (a) sequence ofsite A; (b) sequence of sites B, C, D andE.
145 TABLE I
•
,o,
,
70
Site b
Dimers
Total nt
//
I l l
RpY dimers present in the Mbn cleavage sites = nt not in RpY
80
,o
,o
,o
100
I I0
•~ ~[ t *.G~¢ L.,C~CC.AC~_.AX'~ACACACSite G
90
120
130 140 150 160 170 IIIO ATCGAGTI~G" _~GAA.~T~o~ ~ X ~ A T G C A~.~.~.~.~.~.~.~.~AC~ATTA C A G T ~ G AATA'Ir~-~rGGT
dime, s
A B C D E i/2F l/2G
ApT
GpT
ApC
GpC
73 27 45 23 80 6 13
21 4 4 8 22 0 2
$ 0 0 5 6 ! 0
3 0 ! 0 0 1 0
279 62 114 80 295 22 32
77 0 12 7 76 5 !
No specific points of enzyme cleavage for this hypothetical site are available. It is evident from Fig. 4, that Mbn cuts in regions of DNA containing alternating RpY bases. These dimers are predominantly (APT) pairs, but a significant number (25~) of (GpT) pairs, plus a few (ApC) and (GpC) pairs make these regions approximately 100~o in alternating RpY nt (see Table I). This can also be seen in Fig. 5, where the number of(ApT) dimers and number of total RpY dimers is plotted for the 2-kb sequence shown in Fig. 4. To further characterize the Mbn hypersensitive cutting sites, Mbn3 was isolated from KBS55 and the ends sequenced. The genomic DNA fragments containing Mbn3 were not however sequenced and the 5' and 3' Mbn3 termini therefore represent half-sites. The exact cutting site of all clones (seven for G site and five for F si~e) plus flanking Mbn3 sequence is given in Fig. 6.
C
~
o
80
90
I00
110
120
140
150
160
170
II10
19@
200
210
220
23(1
:)40
zso z6o 2~0 GLG..~_T..T]I,'~TCCc ^ ~ , u u t , , ~ c ~ tj.~~. •2,0
a The (APT), (GpT), (ApC) and (GpC) dimers were counted throughout the Mbn cleavage sites A, B, C, D, E and F that are boxed and shaded in Figs. 4 and 6. The total nt and nt not present in dimers are counted within the same boxes, respectively. b Sites A - E and I/2F and l/2G refer to the Mbn cleavage sites described in Figs. 4 and6.
B
70 130
® 15
l
D
E
I---'4
I
SileF
? 6
. . . . . . . . .
POSSIBLE
OIMERS
:
.q
-
141
GENE 03439
Hypersensitive m u n g bean nuclease cleavage sites in Plasmodium knowlesi D N A (Recombinant DNA; homopurine/homopyrimidine stretches)
Przemyslaw Szafrat~ski" and G. Nigel Godson u ° Polish Academy of Sciences, Institute of Biochemiswy and Biophysics. ul. Rakowiecka 36, 02-532 Warsaw (Poland) Tel. 49-04-03, and b Department of Biochemistry, New York University Medical Center, New York, N Y 10016 (U.S.A.) Received by D.M. Skinner: June 1, 1989 Revised: 23 August 1989 Accepted: 25 August 1989
SUMMARY
Nucleotide sequences of Plasmodium knowlesi DNA that are cleaved by mung bean nuclease (Mbn) at low enzyme concentration (0.2 units enzyme per/~g DNA) are listed. They are tandemly repeated purine/pyrimidine (RpY) stretches of DNA with (APT) dimers predominating. Most cut sites are within almost 100% RpY tracts. The enzyme cleaves at many points within the RpY stretch and usually hydrolyzes the 5'-ApT-Y linkage. These alternating RpY target sites are flanked by homopurine and homopyrimidine stretches. At least one Mbn target site lies next to an in vivo transcribed region.
INTRODUCTION
Five years ago McCutchan et al. (1984) reported that under denaturing conditions employing 45% formamide and 50°C, Mbn cleaved Plasmodiumfalciparum genomic DNA into 'gene size' pieces. These cleavages appeared to be at AT-rich regions. Since then several studies employing these digestion conditions have been published, attempting to identify the nt specificity of this phenomenon. Most of these reports, however, give different and in many ways incompatible results. Muhich and Simpson (1986) found that Leishrnania mrentolae kinetoplast minicircle DNA was cleaved by Mbn in 40~o formamide, in many places. Twenty-seven cleavage Correspondenceto: Dr. G.N. Godson, Department of Biochemistry, New York University Medical Center, New York, NY 10016 (U.S.A.) Tel. (212)340-5622; Fax (212)340-8166. Abbreviations: bp, base pair(s); BS, blood stage; cpm, counts per minute; cs, gene encoding the circumsporozoite protein; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani medium; Mbn, mung bean nuclease; nt, nuclcotide(s); ORF, open reading frame; P., Plasmodiwn; Poll, Escherichiacoli DNA polymerase I; R, purine; SP, sporozcite (stage); u, unit(s); Y, pyrimidine. 0378-I1191901503.50© 1990Elsevier SciencePublishers BN. (BiomedicalDivision)
sites were sequenced; few were AT-rich and many were GC-rich for 10 bp on either side of the actual cleavage site. Brown et al. (1986)examined Mbn cleavage sites flanking the Trypanosoma brucei VSG gene at various formamide concentrations and found that they were within an AT-rich sequence, consisting of runs of A and runs of T residues. In plasmid systems, Sheflin and Kowalski (1984) showed that in the absence of formamide Mbn cuts PM2 DNA within the 135-bp random AT-rich region (74~o AT), but 32 different cleavages revealed no specific sequence requirements. Recently, Vernick et al. (1988) have reported new digestion conditions that result in cleavage ofP. falcipamm DNA precisely at sites outside coding regions. These sites are all AT-rich, some consisting of (APT) dimers. The experiments described in these papers were all carried out at high enzyme concentrations (5-50u/~g DNA). It is apparent, however, from the published data that Mbn cleaves DNA efficiently at low enzyme concentrations (Brown et al., 1986, Muhich and Simpson, 1986). It may be possible, therefore, that the enzyme cleaves at different sites under different digestion conditions and enzyme concentrations. In this paper, we have examined the cutting of Mbn at
142 low enzyme concentration (0.2u/pg
DNA), using cloned into ~EMBIA. These 'hypersensitive' sites appear to be fairly specific, consisting of alternating RpY dimers, with (APT) predominating, and flanked by oligo(R) and oligo(Y) stretches.
P. Imowlesi DNA fragments (15-20kb)
MATERIALS AND METHODS
(a) Bacterial strains and phages Escherichia coli JMI01 was used for M13 transfection and Q358 was used for propagation of recombinant A phages. LB broth (Miller, 1974) was used as growth medium.
(b) ,tEMBIA genomic DNA library P. knowlesi genomic DNA was obtained from blood
stage parasites as previously described (Ozaki et al., 1983). The DNA was partially digested with Sau3A and introduced into AEMBI.A (Frischauf et al., 1983) as described by Ozaki and Cseko (1984). The blood stage and sporozoite mRNA used to screen the library was isolated and labelled with 32p as described by Rulz i Altaba et al. (1987) and Ozaki et al. (1983).
(c) Subeloning and sequencing DNA fragments, cut with either Mbn or restriction enzymes, were separated on agarose gels and cloned into Ml3mp8, mp9 and mpl9 (Messing, 1983). Recombinant DNA procedures were as described by Maniatis et ai. (1982). The ntsequences were determined using the dideoxy chain termination method (Sanger et al., 1977) and, where necessary, the DNA "1"4polymerase treatment was used to produce overlapping DNA fragments (Dale et al., 1985).
(d) Nucleic acid hybridization DNA digests were fractionated on agarose gels, transferred to nitrocellulose (Mania~is et al., 1982) and probed with either parasite mRNA fraginents end-labelled with [7-32P]ATP (Ruiz i Altaba et al., 1987) or with DNA fragments nick translated with [0c-32p]dATP using E. col[ Poll.
RESULTS AND DISCUSSION
(a) Clones used and strategy for this study If Mbn cleaves DNA at specific nt sequences that flank gene coding sequences, it would be expected that the enzyme would cleave cloned Plasmodlum DNA in the same way as genomic Plasmodlum DNA. Using cloned genomic DNA fragments has the advantage that P. knowlesi DNA
containing transcribed sequences can be identified before Mbn cleavage and that each DNA fragment will have only a few (one or two) cleavage sites, which can be isolated and then studied in detail. A partial Sau3A library of/'. knowlesi DNA was therefore constructed in ~EMBIA. The library was screened for clones that contained expressed genomic DNA sequences by hybridizing with labelled BS poly(A) + mRNA, and SP poly(A) + mRNA. These clones were given random numbers prefixed by BS and SP respectively. AEMBL4 clones containing the already sequenced and characterized P. knowlesi cs gene were also isolated using as a probe a 0.9-kb HindIII fragment of the cloned gene which contains only the coding sequences (Ozaki et al., 1983). KBS01 obtained in this way contains a 15-kb genomic DNA insert. (b) Digestion of ~EMBIA clones with Mbn: sensitive and hypersensitive cleavage sites The ~EMBIA KBS01 clone was chosen to establish the best conditions for Mbn cleavage. This was because the
u/,u.g ONA
X0.21 2 3 I I I I I 17 kb 9,4kb 6.6kb 4.6kb 4,4kb
23kb 2.0kb
*"CS ----BS 1 "--BS2
1.Skb
Fig, 1, Effect of Mbn concentration on AKBS01 b~4A {:l~avase.2/~g of DNA was resuspet~ded in 10pl of 45% formamide containing 0.2 M NaCI/I mM ZnSO4/30 mM Na" acetate pH 4.6 and 0.05 to 3.00 u of Mbn (Sigma chemicals)was added per/48 of DNA. The mixture was the.n incubated at 50°C for 30 mitt (McCutchan et al., 1984).Lane !, ~ marker; lanes 2-5, indicated number of Mbn u//AgDNA. The products were run on a 0.7% agarose gel, stained with ethidium bromide and photographed. CS refers to a band containing the cs gene, and BS to bands that hybridised to BS parasite mRNA.
143 15-kb genomic DNA insert contains at least one transcribed gene, the cs gene. Using the original digestion conditions of McCutchan etal. (1984), 2/~g KBS01 DNA sample was digested with increasing amounts of Mbn (Fig. 1). With 0.2 u/#g DNA, no gene-sized pieces were excised from the DNA, although a series of large ( < 5 kb) DNA bands were apparent. Increasing the concentration of enzyme to I u/#g DNA released 2.8-kb and 2. l-kb bands. At higher Mbn concentrations (2 u//~g and 3 u/ttg DNA), the 2.8-kb band disappeared, the 2. l-kb band increased in intensity and lower Mr bands appeared. The high Mr bands ( > 4.4 kb) that appeared after digestion with 0.2 u//~g DNA were present at I u/#g DNA, but disappeared with 2 or more u Mbn/#g. Probing a Southern blot ofthe gel with the 0.9-kb HindlIl probe containing cs gene coding region, showed that only the 2.8-kb band contained the cs gene. Two bands of 2.1 and 1.6-kb, respectively, hybridized strongly with 32p end-labelled BS parasite mRNA. These bands contain sequences from genes transcribed in the blood stages and were therefore labelled B S I and BS2 respectively. The high Mr bands did not hybridize to either probe.
or) ( n
(n u) (n
v v v ~
I IIIII
,,
Mbn I
Thus the cs and B S I , B S 2 genes are cleaved out at different enzyme concentrations. This effect was observed with other ~ clones, where different discrete bands appear at different enzyme concentrations (data not shown). The low Mbn concentration (0.2 u/~tg DNA), however, efficiently cleaved out discrete gene-sized fragments from many of the ~. clones. Thus Plasrnodium DNA has sites that are more or less sensitive to digestion with Mbn. For convenience, those sites that cleave at low concentrations of enzyme ( < 1.0 u/#g DNA) are termed 'hypersensitive' sites and those that require > 1.0 u//~g DNA are termed "sensitive' sites.
(e) Sequence of hypersensitive Mbn cleavage sites Five random clones that hybridized to BS mRNA and that were digested efficiently with Mbn at 0.2 u/#g DNA, are shown in Fig. 2. The sizes of the partial S a u 3 A DNA insert of these five clones were 9.5 kb (KBS23), 16.4 kb (KBS25), 17.8 kb (Keg26), 13.7 kb (KBSS0)and 16.1 kb (KBS55). All clones gave different patterns of Mbn digestion products. KBS25 and KBS50 had two identical lower M r bands (Mbnl, 3.5 kb and Mbn2, 1.0 kb) but differed in sizes of the high-Mr bands. This suggests that they may be partially overlapping clones. KBS55 gave a single low-Mr band (Mbn3, 1.7 kb). Mbnl, 2, and 3 hybridized with BS mRNA indicating they were from transcribed genes, Both KBSS0 and KBS55 however do contain the sporozoite cs gene, but this is present in the undigested DNA and is only excised at higher enzyme concentrations (see section h above). Mbnl, 2 and 3 therefore represent hypersensitive cleavage sites and the undigested cs gene sensitive sites, KBS50 was chosen for further study. First a restriction enzyme cleavage map of the 13.7-kb insert of KBS50 was constructed (Fig. 3). Then the Mbn digestion products (0.2 u/ttg DNA) were cloned into M 13mp 18 and sequenced, either completely (Mbn2) or just the 5' and 3' ends (Mbnl). Mbnl and Mbn2 DNA fragments were then used as radioactive probes on Southern blots of restriction enzyme digests of the KBSS0 DNA insert and were found to lie in the approximate positions shown in Fig. 3. The insert DNA was then digested with EcoRl alone, EcoRI + HtndIll and Hindlll + PstI, and
Mbn 3 ,,, I k b
,,
,
Mbn 2
I
0 Fig. 2. Mbn digestionpattern of several~,EMBIArecombinantclones, at 0.2 u Mbn/pg DNA. Conditions are as describedin Fig. 1 legend. Bands markedMbnl, 2 and 3 wereisolatedfromthe gel and clonedfor further study.
A
III
BCO
I
E
Fig. 3. Restrictionmap of the 13.7-kbinsert of ,tKBS$0 showingthe approximatelocationof bands Mbnl and Mbn2shownin Fig,2. The Mbn cleavageproducts,Mbul and Mbn2, wereplacedon the map by blotting protocols.The black areas lettered A-E refer to the Mbn cleavagesites describedin Fig.4.
144 the D N A fragments were cloned into Ml3mpl9. Using Mbn I and Mbn2 DNA fragments as probes, clones of the insert D N A were isolated that contained all and part of Mbn I and Mbn2. These clones were sequenced. Genomic sequences (2.5 kb) encompassing Mbn2 and its cleavage sites, and genomic sequences (0.5 kb) surrounding Mbnl cleavage sites were established. The 5' and 3' cleavage sites flanking Mbnl are designated A and B and those flanking Mbn2 are designated D
~0
20
10
40
50
60
70
80
~0
IOl
110
120
11o
14o
ll5o
,T O
18o
230
240
and E (Fig. 3). The Mbn cutting sites and sun'ounding sequences are shown in Fig. 4. Fifteen M13 clones containing Mbn2 and two clones containing Mbnl were sequenced in both orientations. The 5' and 3' termini of the M 13 clones are marked with an arrow in Fig. 4. Within each AT-rich region, several independent clones were found to cut at a single site. Other cuts within the AT-rich region were observed, but their exact location was ambiguous due to the repeating ApT dimers. Between Mbn 1 and Mbn2, there is believed to be an extra Mbn site (marked C in Fig. 3). This site was first identified from sequence comparison, using data from A, B, D and E cleavage sites. Its existence would explain why the 0.5-kb band that separates Mbnl and Mbn2 was never observed.
Site A
IT?
200
21o
220
irT
2.
270 V
280
290
300
GT~A~CCCT CCT~CCAGT ACAATC'~-~TT~._GTTTC.A.~CGA~O GAAGAAC~AU
310
320
330
340
350
360
GACGAT" A A A Am,
420
TACTC~-~C~A ATGTCTOT~GGGAAAGAAAA A ~ T ~ G ~ o C A G T A TTTACAACAA
TC~TTGTA~
TCT~AC~
^ C ~ A A ~ ~ ~ ~ - - - ~
370
380
390
400
430
440
4S0
460
I0
20
30
70
8o
90
13o
140
190
|00
23o
260
410
~AA~TTGG C~TAATCATGGTC^TAOCTGRTTCC'~'CTCI".CA_A_~TOTT^~-~CTCACA "~CAC^ AC^TACGAGCc ~ - - ~ ^ ~ ' ~ T ~ - ~
470
1490
1500
1510
1520
1530
1540
1550
1560
1570
1580
1590
1600
1610
1620
1630
1640
1650
1660
1670
1680
1690
1700
1710
1720
1730
1740
1750
1760
1770
1780
1790
1800
1910
1820
1830
1040
1950
1060
,s7o
isso
189o
10oo
llll
io2o
193o
1940
1950
IO60
Io70
1980
1900
,00
,OlO
IU °
,040
2050
2060
2070
2080
2000
2100
il 1°
'1'"
Ull
,15o.
,1.o
.~'*CCGC~---"~GCC"I~A~~A CGTG"I~ACd31
AGCACgA-~CAACTGCTGA T ~ C a G ~ C O ACTT~T^TTA^CAGA~'A'A~A'I'~__..~.
ioo
11o
12o
TACACCA~C~--A~AG AATAAT~A----~'~TATTGC~-~----A~CAATGC~'~ACAOT
150
160
170
180
OCTGCT^G{FCC'~'!CTTTTGA IT'~AGC~-'~CAATAAq~-----~-'~TAACIGAA---~AOCT
210
220
230
240
220
2so
290
sou
~^^C~TTT..~ ^^~A^^^O^ ^O^^^e^^~6. y?y^~OC~T]
~IQ m2o ~}o p40 ~o 50o LUU~atLL~,:A AAO4~O~kA~4~AOAAGAAAA^OAAOOTTCTTT AAA~AGOAOOAAA.AAOAA~ ~
390
400 ~
dlO
4,0
.4o
1H
lfl
,,o
490
500
510
320
$~0
560
570
~00
590
~on
610
620
6~0
640
630
660
~ ^ T O T A
1480
60
~AAO^^OAAOAAOOTT~TAA AAI~'~AAO AAA-AATAAGGTT~CCYbAG(]OA^~AAAG'
~r~A~TAOC~AAA
1470
50
40 . . . .
_~--A.AC~--AA'AK~AAOAAAOAAGGTTT¢TTAAC~AGGAAGOAAAAOAAGAAAGAA~TTTC~
~..~=.~0
1460
C~'~GAGAAAAAA~AATGAC~-~---_A..~ATATGA~TCT~TA~CGA TGATAATATC
cc~-----~oc c*^
QA^~AAAAO^AGA^AOAAO GTTTAAA~AG~aGAAAAOAAO A ~ O A A ~ T ?T~TT^AC~A
'r~3~r~^^^o
1450
Site B
,,o
~SAATqlQAAAOOA~rATAT "
AAAAAAAAAAAAAAAAAAAGJTO
l
~
'
~
2170
~
T A T O T A A T O 0 ~ : L ~
V
2190
2 | 0 0 ~ 0
::30
::40
2230
2250
2290
2300
2310
2320
^^Ao0^^^^ocmu^^^^oo^ ^ ~ H C l ~ ^ , ^ o ^ o __
o^^oo^,^~^ o^^r.,o^^^^o ^^^~Aop~'~'l~o^^~^~ __
Site E
|||0 22?0
2280
^^^G^^^o^o ^^oGo~r~ 2330
~AAGO~
2340
AC~-.a40~A~
SlteC 6NO
670
690
700
710
233o
720
730 740 750 [~O~CAC AATTATOGCGO C A T C ~ T O T T
~9o
8oo
8
3
760
slo
770
780
8m0
54o
0 930
940
930
900
1030
1040
1030
1060
1090
I00
II10
1120
1150
1160
1170
1180
1190
122o
12~o
124o
1230
126o
1300
1310
1320
1070
10RO
A~aC~JC~[TC TGUAGAAA~ATCTC~TCTGTGTACTGAC~C~CTT ~ ' ~ G ~
AATATTTI~'--~TC~TGq~--G~C~CAq~TC~r A C ~ £ C121o C..~^^ 1270
TCG~C
1200 ^OACCAACTA
A~rTACGACCT~C~ATGA T~--~L"TTAT OCACGCTGTA 1280
1290
1340
1350
1360
1370
1380
1400
,410
1420
1430
1440
~TI~TC~T~T
^CC.~^OATCA T T O C ~ C ^
^TCG'UCTAATGT~-~AATA AAT~CCACTT~AGC[~OC
~ ?
2430
2410
2480
2490
2530
2540
2550
2380
2400
OTO~TTTAG
2440
2430
2460
2500
2510
2520
2360
2570
2380
AAAAV.IGAAAAGGAAAAVaGAAAA~'OAAAAr~
2.0
2600
2010
TAGTAACC~GTAI"~CCGGATGGTA
2620
TGAACCC~AT
CTTTCCTTCC TTTCTTTTC~21^TC~=TC'~_C
970 980 990 IO00 I010 1020 [A[A[A[A[A[AJ~'~T~3,AAC~AAOAT.TyAOAOAAAqC ^ ~ ~ A A O A T T _ C C C C . ~ T C T G .,
2420
235o
AIrJ~AOAAOAO G A ~ O O ~
~AAGOA^^A ~AAAGOA^~ O ~ A ~ A O A A ~ ^ ~ ~ C : C A O A T O ^
~
Sited V920
otQ
2410
l~AR~AAAA GOQAAA~A^ A A ~ A A ~
^TAC~AATOTL=~-'-A~TACCOTTAC~-G-~GCTGC OAmmTGC"GA O C ~ ' ~ CC~TAAOTGA
2370
~=~]~..~sGGAA GGA~'[DAAGG^AGGOAAAGG OAAA(~0OAAO GA^AOGAA^O ~^^AOGOAG[
GC^OCCTTC~ TT~Cp~'A'A"~
520
2300
AAACmC836AAG ACS(3AAA~OA A A ~ A ^ A ~
i1 U 1U.~ GGTTOAC'GAC
TACC
Fig. 4. Nt sequence of Mbn.cutting sites present in ~KBSS0. Sites A - E refer to the IVlbn-cutting sites shown in Fig. 3. The alternating RpY regions containing the cut sites are shaded and the actual cleavage sites are marked with filled arrowheads. One independent Mbn-cutting point ending at the arrow was located in site A, two at site B, five at site D and tour at site E. Other cleavage sites in these regions were observed, but their location was ambiguous due to the repeating ApT dinners. The oligu(R) stretches flanking the alternating RpY region are boxed with solid lines and the oligo(Y) stretches are boxed with a solid line above and dotted line below. (a) sequence ofsite A; (b) sequence of sites B, C, D andE.
145 TABLE I
•
,o,
,
70
Site b
Dimers
Total nt
//
I l l
RpY dimers present in the Mbn cleavage sites = nt not in RpY
80
,o
,o
,o
100
I I0
•~ ~[ t *.G~¢ L.,C~CC.AC~_.AX'~ACACACSite G
90
120
130 140 150 160 170 IIIO ATCGAGTI~G" _~GAA.~T~o~ ~ X ~ A T G C A~.~.~.~.~.~.~.~.~AC~ATTA C A G T ~ G AATA'Ir~-~rGGT
dime, s
A B C D E i/2F l/2G
ApT
GpT
ApC
GpC
73 27 45 23 80 6 13
21 4 4 8 22 0 2
$ 0 0 5 6 ! 0
3 0 ! 0 0 1 0
279 62 114 80 295 22 32
77 0 12 7 76 5 !
No specific points of enzyme cleavage for this hypothetical site are available. It is evident from Fig. 4, that Mbn cuts in regions of DNA containing alternating RpY bases. These dimers are predominantly (APT) pairs, but a significant number (25~) of (GpT) pairs, plus a few (ApC) and (GpC) pairs make these regions approximately 100~o in alternating RpY nt (see Table I). This can also be seen in Fig. 5, where the number of(ApT) dimers and number of total RpY dimers is plotted for the 2-kb sequence shown in Fig. 4. To further characterize the Mbn hypersensitive cutting sites, Mbn3 was isolated from KBS55 and the ends sequenced. The genomic DNA fragments containing Mbn3 were not however sequenced and the 5' and 3' Mbn3 termini therefore represent half-sites. The exact cutting site of all clones (seven for G site and five for F si~e) plus flanking Mbn3 sequence is given in Fig. 6.
C
~
o
80
90
I00
110
120
140
150
160
170
II10
19@
200
210
220
23(1
:)40
zso z6o 2~0 GLG..~_T..T]I,'~TCCc ^ ~ , u u t , , ~ c ~ tj.~~. •2,0
a The (APT), (GpT), (ApC) and (GpC) dimers were counted throughout the Mbn cleavage sites A, B, C, D, E and F that are boxed and shaded in Figs. 4 and 6. The total nt and nt not present in dimers are counted within the same boxes, respectively. b Sites A - E and I/2F and l/2G refer to the Mbn cleavage sites described in Figs. 4 and6.
B
70 130
® 15
l
D
E
I---'4
I
SileF
? 6
. . . . . . . . .
POSSIBLE
OIMERS
:
.q
-
