Restriction site map of African swine fever virus DNA

VIROLOGY 133,2!58-270 (1984)

Restriction Site Map of African Swine Fever Virus DNA

J. M. ALMENDRAL, R. BLASCO, V. LEY,’ A. BELOSO, A. TALAVERA, AND E. VINUELA2

Cmtro de Biologic Molecular (CSIC-VAM), Universidod Autbmma, Fawltud de Ciencias, Canto B&co, Madrid-.%, Spain

Received July 7, 1983; accepted November 29, 1989

Treatment of African swine fever virus DNA (about 170 kbp) with the restriction endonucleases SolI, EcoRI, Kpn1, PvuI, and SmaI yielded 14,31,17,13, and 11 fragments, respectively. The order of the restriction fragments produced by each nuclease was established by identifying the crosslinked EcoRI and Sal1 terminal fragments and then finding overlapping fragments. The five restriction fragment maps were integrated into a single map by locating SalI, KpnI, PvuI, and SvzaI sites in cloned EcoRI fragments, and orienting each fragment in the overall map.

INTRODUCTION

African swine fever (ASF) virus is an icosahedral cytoplasmic deoxyvirus, which infects only domestic pigs and related an- imals (Hess, 1971,198l). The viral genome is a double-stranded DNA molecule with a molar mass of about 100 X 10” g mol-’ (Enjuanes et al, 197613) and terminal co- valent crosslinks (Ortin et al, 1979).

The availability of cloned fragments spanning the viral genome and the knowledge of the order of restriction fragments in the DNA molecule are prerequisites for studies of regions coding for viral poly- peptides and for analysis of genetic het- erogeneity of the virus. For these aims Ley et al (1984) have prepared a collection of cloned ASF virus DNA restriction fragments, which account for about 98% of viral DNA.

In this paper we show a map of ASF virus DNA with about 80 sites recognized by the restriction endonucleases S&I, EcoRI, KpnI, PvuI, and SmuI, with an av- erage distance of about 2 kilobase pair (kbp) between contiguous sites.

’ Present address: Abello, S.A., Juliin Camarillo, 8, Madrid-17, Spain.

* Author to whom requests for reprints should be addressed.

MATERIALS AND METHODS

Viruses and cells. ASF virus, adapted to grow in VERO cells (CCLSl, American Type Culture Collection), was cloned by plaque purification (Enjuanes et al, 1976a). Virus stocks were obtained from VERO cells infected at a multiplicity of infection (m.0.i.) of 0.001-0.01 plaque-forming units (PFU) per cell, in Dulbecco modified Ea- gle’s medium (DME) with 2% newborn calf serum. When the cytopathic effect was complete, cell debris was removed by low- speed centrifugation and the supernatant centrifuged for 6 hr at 8000 rpm at 4’ in a Sorvall GS3 rotor. The pellet was resuspended in phosphate-buffered saline (PBS) and the virus suspension was stored in portions at -70”.

Un$brm ltxbeli~ of ASF wires DNA with %P. VERO cells (about 4 X lo4 cells/cm2) were added to a 75-cm2 plastic flask in 10 ml of DME with nonessential amino acids and 10% calf serum. After cell attachment, 50 &i/ml of carrier-free [32P]phosphate was added to the medium. When the culture reached a density of about lo5 cells/ cm’, the medium was removed and the cells were infected with plaque-purified ASF virus at a m.o.i. = 10 PFU/cell, in 2 ml of medium with 2% calf serum and radioactive phosphate as above. After 2 hr ad- sorption, 6 ml of the last medium with

0042-6822/84 $3.00 258 Copyright 0 1984 by Academic Press. Inc.

All rights of reproduction in any form reserved.

RESTRICTION MAP OF ASF VIRUS DNA 259

[32P4phosphate were added and the incu- bation was continued for about 40 hr, when the cytopathic effect was almost complete.

ASF virus DNA. ASF virus DNA was isolated from partially purified virus and prepared by a modification of the method described by Black and Brown (1976). The extracellular virus from l-2 X 10’ VERO cells, infected at a m.o.i. of about 1 PFU/ cell, was concentrated by centrifugation of the culture medium as indicated before. The pellet was resuspended overnight in 5 ml of 50 miki Tris-HCl (pH 7.5), 1 mM EDTA, and 1 M NaCl (HS buffer) at 4”.

The virus suspension was treated with 0.25% Tween 80 and, immediately, centrifuged through a discontinuous gradient of 7 ml of 20% sucrose and 2 ml of 50% sucrose in HS buffer, in a Sorvall AH627 rotor at 25,000 rpm for 30 min at 4”. The virus was collected from the interphase and, after dialysis against HS buffer, centrifuged through 27 ml of a 10-50s sucrose gradient in HS buffer for 60 min, as before. In these conditions the virus banded at a density of 1.20 g/ml.

The virus solution was diluted to about 40 ml with PBS and the virus was pelleted by high-speed centrifugation as described above. The pellet was incubated overnight with 0.5 ml of a mixture containing 10 mM Tris-HCl (pH 8), 10 mM EDTA, 10 mM NaCl, 0.5% Sarkosyl NL-97, and 250 pg of autodigested proteinase K at 4”. After phenol treatment, the aqueous solution was layered onto 13 ml of a 5-20s sucrose gradient in 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, and 50 mM NaCl, and centrifuged in a Beckman SW40 rotor at 35,000 rpm for 3.5 hr at 20”. After gradient fraction- ation, DNA was assayed in each fraction by fluorometry (Kapuscinsky and Skoczy- las, 1977). The fractions containing ASF virus DNA were pooled and the DNA was precipitated with ethanol and dissolved in 10 mMTris-HCl (pH 8.0) and 1 mMEDTA (TE buffer).

Recowzbinant DNAs All recombinant DNAs, except plasmid pllSE/KC36, were described by Ley et aZ. (1984). Digestion of ASF virus DNA fragment S&-E with KpnI nuclease produced fragment SE/KC (see Fig. ll), which was ligated with the

5.0-kbp-long fragment resulting from the digestion of plasmid pKBll1 (Beckingham, 1980) with Sal1 and KpI. The reaction products were used to transform Esche- richia coli HBlOl and the recombinants, labeled with 32P by nick translation, were characterized by hybridization to immobilized Sal1 and KpnI fragments of ASF virus DNA, respectively. Recombinant plasmids and phages were named as described by Ley et al. (1984).

Restriction enzyme digests, agarose electrophoresis, blotting, and hybridization were carried out by following standard techniques (Maniatis et cd, 1982).

Materials. Restriction enzymes were purchased from New England Biolabs and Bethesda Research Laboratories, Inc. E. coli DNA polymerase and T4 DNA ligase from New England Biolabs. DNase I (type III), nuclease Sl, and agarose (type II) were from Sigma Chemical Company. Nitrocel- lulose membranes (HAWP, 304FO) were from Millipore Corporation. [a-32P]dTTP

kb

Pvu I Sal1 KpnI EcoRI

‘a; K-

I -j&f L- ./ J -i 0” M-

g=

K- R-y : J-

l- P, P’- 3=

K- u+

L- Vt- x,x-

FIG. 1. Restriction fragments of ASF virus DNA. Autoradiographs of dried agarose gels showing the hands obtained after digestion of uniformly labeled ASF virus [“LpjDNA with the restriction nucleases indicated in the figure. Approximate size values are given on the left side of the figure.

260 ALMENDRAL ET AL,

and [32P]phosphate (carrier free) were purchased from the Radiochemical Centre, Amersham. Dulbecco’s modified Eagle medium and newborn calf serum were from Gibco Europe, Ltd.

RESULTS

Selection of Restriction Endonucleases for Mapping

From more than twenty restriction nucleases tested, SmaI, Pvd, SalI, and KpnI were the enzymes which produced the smallest number of fragments in ASF virus DNA. Those enzymes were chosen for mapping purposes together with endonuclease EcoRI, because most of the fragments produced by the last enzyme had been previously cloned in plasmid or phage

vectors (Ley et al, 1984). Figure 1 shows the fragment patterns obtained with endonucleases PvuI, SalI, KpnI, and EcoRI. DNA bands were designated by capital letters in order of decreasing size. Densito- metric scanning of autoradiographs indicated that bands PvuI-A and C, SalI-F and I, and EcoRI-C, D, E, K, N, and Q contained two fragments each (data not shown). In these cases, the two fragments were designated by the same letter with or without a prime superscript. Mapping experiments described later showed that also bands KpnI-P and EcoRI-U and X contained two fragments each and that all fragments pairs with similar electrophoretic mobility were different.

Table 1 shows the size of the ASF virus DNA restriction fragments selected for

TABLE 1

SIZES OF ASF VIRUS DNA RESTRICTION FRAGMENTS, kbp

Band S?Td PVUI Sldl KpnI EcoRI

A B C D E F G H I J K L M N 0 P

Q R S T U V

>50.0 32.d” 28.0 25.0 16.0 14.02 15.3 12.5 10.2 7.9 9.1 6.1 3.7 4.5 2.9 4.0 2.5 2.8 2.1 2.5 1.9 1.8

32.3 25.0 21.0 18.7 16.0 11.7* 10.3

8.9 5.02 1.4 0.9 0.6

39 24.6 17.2 14.3 13.6 12.8 8.9 8.4 6.2 5.7 3.6 2.7 2.4 2.2 2.1 1.3e

21.2 14.5 11.5* 1o.72 8.82 8.4 7.5 6.6 5.6 5.3 4.g2 3.3 3.0 2.92 2.7 2.2 1.92 1.6 1.5 1.3 0.8’ 0.5

X o.32

Fragments 11 13 14 17 31

Total size >141.7 159.1 168.5 166.3 168.6

Note. Fragment size was determined by agarose electrophoresis of the digestion products produced by each nuclease, in parallel with fragments of known size.

a The number 2 as a superscript indicates the existence of two fragments in the corresponding band.


mapping as determined from their electrophoretic mobilities.

Identijcation of Terminal Fragments

The ends of ASF virus DNA are cova- lently closed by single-stranded DNA (Or- tin et al, 1979) as those of vaccinia virus DNA (Geshlein and Berns, 1974; Baroudy et aL, 1982). When the DNA was cleaved by a restriction nuclease and the fragments were denatured and quickly renatured, only the terminal fragments became Sl nuclease-resistant duplex molecules, which could be identified by gel electrophoresis (Ley et al., 1934). Figure 2 shows the identification of restriction fragments SaZI-F and G and EcoRI-D’ and K’ as the terminal fragments produced by those enzymes.

Restriction Maps

The order of most restriction fragments produced by each nuclease was deduced from (1) the identification of the crosslinked terminal fragments (Jaureguiberry, 1977; Wittek et aL, 1978); (2) the order of overlapping fragments, obtained by hybridization of fragments produced by another enzyme (McDonell et aL, 1977); and (3) the analysis of the digestion products of the restriction fragments produced by one enzyme, after partial digestion with another enzyme (Danna et aZ., 1973; Smith and Birnstiel, 1976).

(a) SaK fragment map. Figure 3 shows the result of the hybridizations of 32P-labeled EcoRI fragments to Sal1 fragments immobilized on nitrocellulose. The hybridization of each terminal EcoRI fragment (D’ and K’) to both terminal Sal1 fragments (F and G) indicated the existence of re- peated sequences in ASF virus DNA (Sogo et aZ., 1984).

The order of the Sal1 fragments could not be obtained from the data shown on Fig. 3, until the fragments with the same electrophoretic mobility (SaZI-F and F’ and I-I’) were distinguished from each other. Fragments SaZI-F and I?’ were differen- tiated because the first one had been characterized as a terminal fragment and the second one hybridized with fragments

FIG. 2. Sol1 and EcoRI terminal fragments of ASF virus DNA. ASF virus DNA was digested with either Sol1 (2) or EcoRI (3) endonuclease, and the fragments subjected to agarose electrophoresis. A portion of either Sal1 (1) or EcoRI (4) fragment mixture was heat denatured and cooled in ice, and digested with nuclease Sl before electrophoresis. The fragments were stained with ethidium bromide.

EcoRI-E’, M, and C (Fig. 4), which over- lapped the internal fragment KpnI-B (Fig. 6). Fragments SaZI-1 and I’ were distinguished by cloning in pBR322 (Ley et al, 1983). SaZI-1 hybridized with EcoRI-D, S, and P and SalI-I’ hybridized only with EcoRI-A (Fig. 4). From the data shown in Fig. 3 one could read from right (SaZI-F) to left, the order of the Sal1 fragments till fragment SaZI-B inclusive. This fragment should be contiguous to SaZI-I’ (5.1 kbp), because this was contained within EcoRI- A (21.2 kbp), which hybridized also with fragments SaZI-B (24.1 kbp) and C (19.5 kbp). No EcoRI fragment connecting the terminal fragment SaZI-G to other Sal1 fragment has been found. Figure lla shows the order of the Sal1 fragments of ASF virus DNA.

(b) EcoRI fragment map. Figure 4 sum- marizes the result of the hybridizations carried out between Sal1 and EcoRI fragments. These data and the lengths of the terminal fragments (Fig. 2 and Table 2)

ASFV K’ A E’ C C’ D P H E I D’

kb

32

21

12

5

1.5 J-

K-

L-

FIG. 3. Hybridization of q-labeled EcoRI fragments to Sal1 fragments of ASF virus DNA. ASF virus DNA was digested with endonuclease Sal1 and the digestion products were subjected to agarose electrophoresis. The fragments were transferred from the agarose gel to a nitrocellulose sheet and hybridized with the q-labeled EcoRI fragments indicated in the figure. The DNA in the lane labeled ASFV was hybridized with ASF virus [=P]DNA. Approximate size values are given on the left side of the figure.

\,cmK’(L U V X’)(J U X) A (F K)E M C (N B GIG (0 1 N’) 0 S I= H (R 0 Ql E I D’ 311 hbp4.6 3.306050.3 530.60,32126.4 4.6 6650 II5 29146 751,527 IS 2910, 15226616 19 19 665.6lOi

; 97JL-f J 3 31 J

: 19.5 (J J J)J

I’ 51 s

B 241 J(JJ)J L 06 J

F’ 116 JJJ

A 554 J(J J J)J

D 164 J(3 f -t)J

K 09 a

I 52 J3J

J 16 JJ

J(J f J)J JJ

JJ

FIG. 4, EcoRI fragment order obtained by hybridization of immobilized EcoRI or Sal1 fragments with ‘?I’-labeled Sal1 or EcoRI fragments of ASF virus DNA. Starting with the overlapping terminal fragments EcoRI-K’ and MI-G, shown on the upper left side of the figure, the hybridization results allowed to locate most of the EcoRI fragments, as shown in the upper row. The groups of EcoRI fragments that could not be ordered from these hybridization data are shown within a parenthesis. The arrows indicate a positive hybridization and point from the radioactive to the unlabeled fragment.


la2 b

123

8

(N’) 2.9 CO)27

(T)l3

-73

-4.2 -3.8

-0.6

18.4-

ll.l- 10.2-

8.2-

6.9-

42-

2.7-

-142

,44 (PEu7 -4.2 - 3.8

-27

-06

322)

FIG. 5. Order of the fragments EcoRI-N’, 0, and T deduced from a restriction mapping of pZSD12 recombinant plasmid. (a) p2SD12 plasmid, containing fragment S&I-D of ASF virus DNA inserted in the Sal1 site of pBR322, was digested with either EcoRI (lane 1) or EcoRI and Sal1 (lane 2) and the resulting fragments were subjected to 0.7% agarose electrophoresis and stained with ethidium bromide. The double band with a size of about 8 kbp was split into four bands of sizes 7.3, 4.2, 3.8 (large fragment of pBR322), and 0.6 (small fragment of pBR322) kbp, respectively. These results indicated that the long pBR322 fragment (3.8 kbp) was linked to a 4.2-kbp-long ASF virus DNA fragment, but whether the last one was Cg or D, could not be established from these data. (b) A partial EcoRI digest of p2SD12 recombinant plasmid was digested to completion with Sal1 and, after electrophoresis, stained with ethidium bromide (lane 1). The fragments were transferred to nitrocellulose sheets and hybridized with “P-labeled DNA from either LRC351 recombinant-containing fragment EcoRI-C cloned in XWESXB, (lane 2) or p5R015 recombinant-containing fragment EcoRI-0 cloned in pBR325, (lane 3). The result of the hybridizations indicated that the 4.2-kbp- long fragment was Cp and that the EcoRI fragment order was Cb-0-T-N’D, as shown in (c), where the dashed and the solid lines indicate the vector and the ASF virus DNA SuEI-D fragment, respectively. R, EcoRI; S, S&I. A p as a subscript of the letter designating a fragment indicates the portion of the corresponding fragment present in the insert.

implied that the Sal1 and EcoRI maps were oriented in such a way that fragment EcoRI-K’ (4.8 kbp) was contained within fragment SczZI-G (9.7 kbp); otherwise, this fragment would hybridize only with fragments EcoRI-D’ and K’ (Figs. 4 and 11).

The overlapping Sal1 and EcoRI fragments allowed to establish the order of about half the EcoRI fragments but five groups of EcoRI fragments could not be ordered from the hybridization data alone (Fig. 4).

The order of fragments EcoRI-N’, 0, and T was established from an analysis of the recombinant plasmid p2SD12, which contained the fragment SalI-D inserted in the

Sal1 site of pBR322 (Ley et al, 1984). The insert contains the EcoRI fragments CP-(N’,O,T)-D,, where cl, and D, indicate the portion of the EcoRI fragments present in fragment S&I-D (Fig. 11). Figure 5 shows an analysis of the EcoRI partial digestion products of p2SD12, indicating that the order of the EcoRI fragments in the insert was C’,-0-T-N’-D,.

The order of the EcoRI fragments N-B-G and F-K was deduced from the hybridization results of KpnI and EcoRI fragments shown in Fig. 6 (see also Fig. 11).

Fragments EcoRI-Q, &‘, and R were eon- tained in plasmid pllSE/KC36, which had

264 ALMENDRAL ET AL.

RK’ RL RA RF RK RE’ RC RN PF RB SA RG RC’ SD RD SI RH RE RI RD’

I(P~I hbp 4.6 3.3 21.2 6.4 4.6 6.6 11.5 2.9 6.0 14.5 354 7.5 11.5 16.4 10.7 5.2 6.6 66 5.6 10.7

A 40.3 J J J J J

p 1.3 J

B 235 cl J J J

D 13.7 JJJJJ

E 6.3 J J J

p’ 1.3 22

N 2.2 ss_

y 2.0 J+-JJ

M 2.4 J J

G 9.9 J J J

F 12.1 J J J

L 2.7 J J

J 6.6 J J J

c 19.2 JJJJ

J J E 13.3

J K 4.6

1 6.2 A J

FIG. 6. KpnI fragment order obtained by hybridization of immobilized KpnI fragments with ‘zp- labeled EcoRI, PvuI, and Sal1 fragments of ASF virus DNA. Starting with the overlapping terminal fragments KpnI-A and KooRI-K’, shown on the upper left side of the figure, the hybridization results allowed to order most of the KpnI fragments as shown in the left column. Fragments KpnI- N and P’, which could not be ordered from the hybridization data alone, are shown within a parenthesis. The arrows indicate a positive hybridization and point from the radioactive to the unlabeled fragment. The fragments in the upper row are named with two letters; the first one indicates the enzyme (R, EcoRI; P, PvuI; S, SalI) and the second one the fragment.

the DNA sequence common to fragments S&I-E and KpnI-C inserted in the corresponding sites of plasmid pKBll1 (Beck- ingham, 1980). The insert spanned fragments EcoRI-H through E (Fig. llb). An analysis of the partial digestion products of pllSE/KC36 with EcoRI, allowed to establish the order R-Q-Q (data not shown).

The order of fragments EcoRI (L, U, V, and X’), on one hand, and EcoRI-(J, U’, and X) on the other, both groups near the left end of ASF virus DNA (Figs. 4 and llb), has not been solved yet.

Figure llb shows the EcoRI fragments which have been ordered along ASF virus DNA.

(c) KpnIfrwgment map. Figure 6 shows the result of the hybridizations carried out between 32P-labeled EcoRI, SaZI, and PvuI fragments and unlabeled KpnI fragments. These data provided enough information

to order all KpnI fragments, except fragments N and P’.

The order of fragments KpnI-N and P’ was established from an analysis of the plasmid p5RB5, which contains the fragment EcoRI-B inserted in the EcoRI site of pBR325 (Ley et aL, 1983). The insert contains the KpnI fragments D,-H-(N,P’)-0,, where D, and 0, indicate the portion of those KpI fragments present in fragment EcoRI-B (Fig. 11). Figure ‘7 shows that an analysis of partial digestion products of the recombinant with KpnI indicated that the order of the fragments in the insert was D,-H-P’-N-O, (Fig. 11~).

(d) PvuIfragment map. Figure 8 shows the result of the hybridizations of selected 32P-labeled probes with PvuI fragments immobilized on nitrocellulose. These data, together with the previously established


TABLE 2

COMPARISON OF FRAGMENT SIZES OBTAINED BY AGAROSE ELECTROPHORESIS (I) AND DEDUCED FROM MAPPING DATA (II)

Fragment

A A B C C D E F F G H I I’ J K L M N 0 P P

SWULI PVUI Sal1 KpnI

(1) (11) (1) (11) (1) (11) (1) (11)

>50 76.6 32.0 38.0” 32.3 35.4 39.0 40.3 32.0 35.8

28.0 29.6 25.0 24.8 25.0 24.1 24.6 23.5 16.0 16.2 14.0 13.4 21.0 19.5 17.2 19.2”

14.0 14.6 15.3 14.5 12.5 12.3 18.7 18.4 14.3 13.7 10.2 9.9 7.9 8.0 16.0 16.5 13.6 13.3 9.1 9.2 6.1 6.0 11.7 11.4 12.8 12.1

11.7 11.6 3.7 3.7 4.5 4.5 10.3 9.7 8.9 9.9” 2.9 2.9 4.0 4.2 8.9 8.6 8.4 8.3 2.5 2.6 2.8 2.8 5.0 5.2 6.2 6.2

5.0 5.1 2.1 2.2 2.5 2.5 1.4 1.6 5.7 5.6 1.9 2.0 1.8 1.7 0.9 0.9 3.6 4.5”

0.6 0.6 2.7 2.7 2.4 2.4 2.2 2.2 2.1 2.0 1.3 1.3 1.3 1.3

a Values with more than 10% deviation.

maps, led to the complete map of PvuI fragments (Fig. lld).

(e) Smalfragment map. Figure 9 shows the result of the hybridizations of selected 32P-labeled probes with SmaI fragments. These data and the previously established maps allowed to establish the order of all SmaI fragments (Fig. lle).

Fragment SmaI-A, with a length larger than 50 kbp (Table l), hybridized with both terminal SaZI-F and G fragments, because of the terminal repetition, and also with SalI-A and B, among other Sal1 fragments. Therefore, fragment SmaI-A should be the left terminal fragment, containing fragment SaZI-G (Fig. lle). The fact that fragment SmaI-A hybridized with fragment SalI-A but not with EcoRI-B, indicated that fragment SmaI-A ended within either fragment EcoRI-C or N. The first possi- bility was discarded because EcoRI-N has

one SmaI site and EcoRI-C has none (Fig. 10). The size of fragment SmaI-A, obtained from the addition of the lengths of all the EcoRI fragment to the left of the SmuI site in fragment EcoRI-N (Fig. 11 and Table 2) plus 2.0 kbp of fragment EcoRI-N (Fig. lo), was 76.6 kbp.

Composite Map of SalI, EcoRI, KpnI, Paul, and Smd Sites

Once the restriction site map for each nuclease had been obtained (Figs. lla-e), a composite map showing the relative order of the fragments produced by the five nucleases and the distances between most of the restriction sites was established. For this purpose SalI, KmI, SmaI, and PvuI sites were mapped within the EcoRI fragments cloned in XWES.XB or pBR325 (Fig. 10). A similar mapping was done on some

266

a 1

Dp-pBR-Dp\ I H/

N-

P’-

I

ALMENDRAL ET AL.

b 2 3

H-

N-P-

N-

P’ -

K

FIG. 7. Order of the fragments KpnI-N and P’ deduced from a restriction mapping of p5RB5 recombinant plasmid. (a) p5RB5 plasmid-containing fragment KcoRI-B of ASF virus DNA inserted in the EcoRI site of pBR325, was digested with KpnI to completion (lane 1) or partially digested with KpnI and then fully digested with Sal1 (lane 2) and the fragments were subjected to 0.7% agarose electrophoresis and stained with ethidium bromide. Since fragment EcoRI-B lacked WI sites (Fig. 10) the only site in the recombinant plasmid sensitive to WI is that present in the vector. The fragments in lane 2 were transferred to nitrocellulose and hybridized to the O.&kbp- long fragment SalI-Hind111 from pBR325, which lacks the inverted repetition present in the plasmid (lane 3). The result of the hybridization indicated that the orientation of the insert in the vector is that shown in (b) and that the order of the KpnI fragments was D,-H-P’-N-O,. (b) Diagram of p5RB5 recombinant plasmid, where the continuous and the discontinuous lines indicate the insert and the vector, respectively, and the thick line indicates the pBR325 sequence used as radioactive probe. K, Kpd; S, SalI; R, EcoRI; H, Hind111 sites. A letter with p as a subscript indicates the portion of the fragment present in the insert.

Sal1 fragments cloned in pBR322, after digestion with SmaI or PvuI (data not shown). Often, it was possible to map the restriction sites from the restriction patterns and the knowledge of the location of restriction sites within the vectors XWESXB (Daniels and Blattner, 1982), pBR325 (Prentki et aL, 1981), and pBR322 (Sutcliffe, 1979). When the insert contained one or two sites recognized by the endonuclease, their locations were deduced from the electrophoretic mobilities of the fragments containing vector sequences linked to insert sequences. When the number of sites in the insert was larger, the order of internal fragments was deduced from the mapping data shown before. As plasmids pBR322 and pBR325 contain no SmaI site, the mapping of these sites in the inserts

was done by double digestion of the recombinants with either Sal1 or EcoRI and SmaI (data not shown). Figure 10 shows the restriction site map of the EcoRI fragments for each enzyme.

The orientation of each individual map shown in Fig. 10 in the overall map, was done in either of two ways.

(a) Some times, the orientation was obtained from hybridization data. For instance, fragment EcoRI-C was divided by a Sal1 site in two segments of different size, the left corresponding to fragment MI-A and the right to MI-D (see Fig. 5). Similarly, a KpI site, which cut EcoRI- C’ in two different segments, marked the boundary between fragments KmI-G on the left side and F on the right side. The segment between both sites should be


J J J J J J J J J

J ’ J J

J J

FIG. 8. PvuI fragment order obtained by hybridization of immobilized PvuI fragments with “?-labeled KpnI, SalI, and EcoRI fragments of ASF virus DNA. Starting with the overlapping terminal fragments PvuI-C and KpnI-A, shown on the upper left side of the figure, the hybridization results allowed to order all the PvuI fragments, as shown in the left column. The arrows indicate a positive hybridization and point from the radioactive to the unlabeled fragment. The fragments in the upper row are named with two letters; the first one indicates the enzyme (K, KpnI; S, S&I; R, EcoRI) and the second one the fragment.

common to SalI-A and KpnI-F in one orientation or to KpnI-G and SaZI-D in the other. Only the orientation given in Fig. 11 was consistent with the positive hybridization of fragments SalI-A and KpnI- F and the negative hybridization of fragments SalI-D and KpI-G (Fig. 6).

(b) In other cases, the orientation of a fragment was deduced from the distances between successive restriction sites in this and the next fragment. For instance, the left KpnI site in fragment EcoRI-G (previously oriented) was at a distance of 1.5 kbp from the left end of the fragment (Fig. 10). This segment was a part of fragment KmI-0, the rest of which extended into fragment EcoRI-B (Fig. 11). This fragment could be located on the left of fragment EcoRI-G in two orientations. One of them would implicate that fragment KpnI-0 had a total length of 3.7 kbp, which differed from the 2.1-kbp value calculated from the electrophoretic mobility of this fragment (Table 1). The correct orientation of EcoRI- B, shown in Fig. 10, resulted in a total

length for fragment KpnI-0 of 2.0 kbp, a value which agrees better with that of 2.1 kbp obtained from its electrophoretic mobility (Table 1).

After orienting all the mapped fragments in a single map (Fig. 11), the length of the overlapping fragments was recal- culated as indicated before for fragment KpI-0. The length of the fragments, located in internal positions in the mapped clones, was obtained from their electrophoretic mobilities. Table 2 shows the sizes obtained by this method, compared with the values obtained by direct calibration of limit digests of total ASF virus DNA with the different nucleases. The size of fragment KmI-K could not be calculated by recombinant mapping because the only recombinant containing fragment EcoRI- D’, which should include fragment KpnI-, K at least in part, exhibited a 1.4 kbp dele- tion on the left side of fragment EcoRI-D’ (Ley et ah, 1983), where the left end of

-1 2 SB SA RB RG RC’ SI RH SE/KC RE KI

amI Lb 24.1 3,4 145 75 115 52 66 123 88 6.2

J A K. J J 0 H.5 E 99

;

J J J J J J

B 29.6 A J A K 20 J J I 2.6 J J F 92 J J J J 22 J J Ii 2.9 J

c 162 J J G 3, J

~. ~.- ~-- ~__--

FIG. 9. SWULI fragment order obtained by hybridization of immobilized SmaI fragments with q-labeled SolI, EcoRI, KpI fragments and the double restriction fragment SalI-E/KpnI-C of ASF virus DNA. Starting with the overlapping fragments SWULI- A and S&I-B, shown in the upper left side of the figure, the hybridization results allowed to order all the SmaI fragments, as shown in the left column. The arrows indicate a positive hybridization and point from the radioactive to the unlabeled fragment. The fragments in the upper row, except the doubly re- stricted one, are named with two letters; the first one indicates the enzyme (S, SalI; R, EcoRI; K, KpnI) and the second one the fragment. Fragment SE/KC is the common part of fragment S&-E and KpnI-C (see Fig. 11).


EcoRI,kbp

B. 14.5

c, 11.5

c’, 11.5

cl ( 10.7

D’, 10.7

E , 8.8

E’, 8.6

F, 6.4

G, 7.5

H, 6.6

I, 5.6

J , 5.3

K, 4.0

M, 3.0

N , 2.9

P, 2.2

Sal I Kpn I Pvu I Sma I Map --

N. S. Dp-H -PI-N-Op Fp-K-Ep DP-EP Z.l-B.4-13-22-05 5.4-1.7-24 13.6-0.9

Fb-Ap BP-DP 8.3 -3.2 2.0-0.7

AP-DP GP-FP 7.3-42 6.3- 5.2

Dp-K-Ip Fp-L- J-Cp 7 3-09-2 5 <O.l-2.7-5.6-2 4

N S. Ep-K-I <O.l-45-62

EP-HP CP-EP 5.1-37 1.1-77

Bp-L-Fb N S. 79-0.6-0.3

N.S. Ap-P-BP 3.0-1.3-4.1

N.S. Op-M-Gp 1.5-24-3.6

JP-EP N.S. 0.6 - 6.0

HP-FP N.S. 4 9-0.7

NS N.S

N.S. N.S

N.S. N.S.

N.S N.S.

IP-JP N.S. 1.2- 1.0

N.S. N.S

HP-BP EP- BP 1.6 -9.7 1.5-10.0

BP-AP NS. 8.2-2.5

Ap-J-I Cp- G 5.4-25.28 7 Q-3.7

N.S. Fp-J-H-Cp 0.1-2.2-2.9-36

N.S N.S.

N.S. N.S.

Ep-G - Hp NS 0.6-4.5-24

N.S. Kp-I-Fp 0.3-26-3.7

N.S. N.S.

cp-dp 2.6-2.7

dp-op 3.5-1.3

Dp-C’p 2.2- 0.0

Cb-Fp 2 3-06

N.S.

N.S.

N.S.

N.S.

AP-DP 2.0-0.9

BP-KP 05-1.7

b

FIG. 10. Restriction site maps of EcoRI fragments (kbp) of ASF virus DNA. The maps were obtained from an analysis of the digestion products of XWESXB or pBR325 recombinants with the indicated enzymes. The EcoRI fragments not shown in the figure, except fragment EcoRI-A, did not contain restriction sites for the indicated enzymes. Fragment EcoRI-A contained two Sal1 sites (Ley et aL, 1933) and no K@, PvuI, and SmaI sites. The map of the terminal EcoRI-D’was described by Ley et al (1933). S, SaZI; K, KpnI; P, Ptnd; M, SwutI. A letter with p as a subscript indicates the subfragment within the EcoRI fragment. N.S. indicates that the fragment does not contain any site sensitive to the indicated endonuciease.

fragment KpI-K should be located, ac- cording to the size of the fragment calculated by electrophoresis. The negative hybridizations found between fragments EooRI-D’ and KpnI-E or between EcoRI-I and KpnI-K (Fig. 6) and the absence of KpnI sites in fragment EcoRI-I (Fig. 10) suggested that the left end of fragment KpnI-K should be inside EcoRI-D’ and very close (~0.1 kbp) to its left end. This led to a maximal length of 4.5 kbp for fragment KpnI-K, instead of the 3.6 kbp calibrated by electrophoresis of whole KpnI digests of ASF virus DNA. The reason for this

discrepancy, as well as the one affecting the size of fragment KpnI-G (Table 2), has not been further investigated.

DISCUSSION

Most of the restriction enzymes used in this work were chosen because the recognition sequences were hexanucleotides and it was expected that these enzymes would produce less fragments than those recognizing not completely defined hexanucleotides (AccI, HgiAI, etc.) or tetra- nucleotides (&a1 or TaqI) sequences (Rob-


0 10 20 30 40 50 60 70 80 90 loo 110 120 130 140 150 160 170

kbp ‘I 1 I I I I I I I I I I I I 1

a) Sal1 ( I) , G C I’ B L F’ A D KIJ E H F

:I: I I II I I II II I I I

lL,U.V,Xl(J.U:X)

-A b) EcoRI(t1 ,K1lll IIJI

FKE’MCN ‘3 G C’ OTN’ D SPH RQO’E I D’ I II II 4

c) KpnI(P) , A P B D H P’NOM G F LJ C E K I

1

d) PvuI(l) k ’ 1 d D C’ FKE GH B A JI

I I I,

e) SmuI(ll , A D E B Kl F JH C G

I I I III III 4

FIG. 11. Restriction site map of ASF virus DNA. Lines a-e show the SalI, EcoRI, KpI, PvuI, and SmoI maps derived partially or totally from the hybridization data shown in Figs. 4-10. The upper line, below the length scale, shows the composite map for all the restriction sites indicated in the individual maps.

erts, 1982). From the 22 restriction endo- the case of Sal1 and EcoRI endonucleases. nucleases tested on ASF virus DNA, SmaI, In general, the other enzymes only pro- PvuI, SalI, and KpnI produced 11, 13, 14, duced one of the two terminal fragments and 17 fragments, respectively, and they and, in each case, the fragment identified were selected for DNA mapping (Table 1). was the smallest one. It is possible that Endonuclease EcoRI cleaved ASF virus the larger fragments contained, randomly, DNA into 31 one fragments (Table 1) and more nicks or gaps than the shorter ones, it was also used for mapping, because most giving place, after denaturation and fast of the EcoRI fragments had been cloned renaturation, to crosslinked structures in plasmids or lambda phage (Ley et al, with single-strand tails, sensitive to nu- 1984). clease Sl.

The fragment sizes ranged from 0.3 (EcoRI-X’) to 76.6 (SmaI-A) kbp. The length of the largest fragments was obtained by addition of the smallest overlapping fragments (Table 2). The addition of consec- utive restriction fragment sizes gave for the complete DNA a value of about 170 kbp, in agreement with the previously re- ported values for ASF virus DNA (En- juanes et al, 1976b).

The reciprocal hybridization of the terminal fragments indicated the existence of repetitions at the ends of the DNA molecule. As the shortest terminal fragment produced by the enzymes considered in this paper was 2.8 kbp long (PvuI-I) and the contiguous fragment PvuI-J did not hybridize with the other DNA end, the re- peated sequences should be equal or shorter than 2.8 kbp (Sogo et al., 1984).

The identification of both terminal restriction fragments was only achieved in

The order of fragments produced by the five nucleases was established by standard


techniques. However, the integration of all the restriction sites in a single map, consistent with all the hybridization data, re- quired a further mapping of individual EcoRI and Sal1 fragments, cloned in ap- propriate vectors, and the knowledge of the orientation of most of the fragments in the complete map. This was done by either hybridization of overlapping fragments produced by different nucleases or comparison of the lengths obtained by addition of the length of partially overlapping fragments and those obtained by electrophoresis. The comparison of the fragment sizes obtained by either method showed that they agreed, in general, within 10% of the value obtained by gel electrophoresis.

The longest distance between consecu- tive restriction sites in the map was 13.8 kbp, for fragment EcoRI-A/SaZI-C. Nev- ertheless, as this region has been cloned (Ley et aL, 1984), it should be easy to locate additional restriction sites for other endonucleases.

ACKNOWLEDGMENTS

We are grateful to Dr. R. Beckingham for providing pKBll1 plasmid. This investigation has been aided by grants from Comision Asesora para el Desarrollo de la Investigacibn Cientifica y TQcnica and Fondo de Investigaciones Sanitarias.

REFERENCES

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Restriction site map of African swine fever virus DNA