J. Mol. Biol.

(1977)

117, 447-471

Fine Structure of Polyoma Virus DNA BEVERLY E. GRIFFIN Imperial Cancer Research Fund P.O. Box 123, Lincoln’s Inn Fields London WC2A 3PX, England (Received 27 January

1977, and in revised form

2.9 J&y

1977)

A fine structure map of polyoma DNA has been made based on cleavage wit,h a number of restriction endonucleases (including Hue11 and III, Baml, Hind11 and

III,

BumI, H;oaII,

eight HpalI

and in part, H@I)

restriction

fragments

and depurinat,ion

of wild-type

made possible some correlation

with simian virus 40 DNA,

cl&ailed examination of various t,lle rc$on of t,he origin of DNA

polyoma replication

DNA, the

This analysis has

and some Hue111 fragments.

and has facilitated

strains and variants. have been examined.

Sequences

from

1. Introduction ‘I’hn two DKA tumour viruses, polyoma virus and simian virus 40 (SV40) appear to he remarkably similar in their biochemical and genetic organization (Fried & Griffin, 1977). In spite of this, they show very different responses towards their host cells. Polyoma. virus grows lytically in mouse cells and transforms a number of other ~11 types. including rat and hamster cells. SV40 grows lytically in monkey cells and tmrlsforms mouse cells, as well as rat and hamster cells. In order to try to understand the similarities and differences between the polyoma virus and SV40 genomes and their host cell interactions, and to define the variant and defective species that appear to arise with remarkable ease following polyoma virus infection (Fried, 1974; Griffin & Fried, 1975; Lund et al., 1977a,b), a fine structure analysis of the polyoma virus genome has been carried out. A large number of x&i&ion endonuclease cleavage sites have been mapped in relation to the HpaIl physical map of polyoma virus DNA (Griffin et al., 1974). Depurination fingerprints have been made of polyoma virus and XV40 DNAs, and of a number of restriction candonuclease fragments of polyoma virus DNA. Analysis in detail of the sequences itround the origin of DNA replication of polyoma virus shows certain similarities to ~quences reported for the origin of replication of SV40 (Dhar et al., 1977).

2. Materials and Methods (a) 3ZP-labelled polyoma

DNA

3TG cells were grown on 90-mm dishes at 37°C in the presence of 5q/, CO, in Eagle’s ( 1959) medium supplemented with 5% foetal calf serum. At counts of 6 x lo6 to 8 x 10” cells/dish (subconfluent) the medium was removed and the cells were infected with lon Irrultiplicities of virus (1 to 5 plaque-forming units/cell). After adsorption for 1 II at 37°C’. Inetlium (9.0 ml of Eagle’s medium supplemented with 3 ‘$6 horse serum (GIBCO-Biocult. Glasgow) was added back to tile cells and they were left 8 to 12 h at 37°C. At) tile eud of t Iris pcsriod, the medium was removed and 9.0 ml of Eagle’s medium rnin7k.s phosphate 447

448

B. E. GRIFFIN

supplemented with 3% horse serum and containing 0.25 mCi [32P]phosphate (PBS-l, Radiochemical Centre, Amersham) was added to each dish. Cells were left at 37°C until they showed signs of a cytopathic effect (usually 48 to 72 11 after infection) at which time medium was removed, and the viral DNA extracted by the Hirt (1967) procedure. After 8 to 12 h at 4°C the Hirt supernatant was collected by centrifugation and form I (superhelical) 32P-labelled polyoma DNA isolated from it by caesium chloride equilibrium gradient centrifugation (Radloff et al., 1967). In a typical experiment, 6.7 ml Hirt supernatant containing 0.2 ml ethidium bromide solution (10 mg/ml) and 6.0 g CsCl was placed in a cellulose nitrate tube (3 in x 5/S in) and spun for 16 to 20 11 at 47,000 revs/min in a Beckman type 65 rotor. Superhelical DNA, which bands at a higher density than other DNA (form II), was visualised as a red band in U.V. light and collected from the bottom of the centrifuge tube. Ethidium bromide was removed from the DNA by extraction (3 to 4 times) with equal volumes of caesium chloride-saturated 2-propanol. The aqueous layer was diluted 3-fold and DNA precipitated by addition of 2 to 2.5 vol. cold ethanol. To complete the precipitation, the alcohol solution was left overnight at - 20°C and DNA was collected by centrifugation. In typical experiments, 5 to 6 pg of superhelical polyoma DNA were obtained from each dish (about & the yield obtained when no isotope was used and the infected cells were grown in medium with foetal calf serum) with specific activities of 5 x lo5 cts/min per pg. Higher specific activity material (up to approx. 2 x lo6 cts/min per pg) could be obtained by increasing the amount of [32P]phosphate up to 0.5 to 1.0 mCi/ dish, but this was at the expense of the yield of superhelical DNA. 32P-labelled polyoma DNA for these studies was mainly made from large-plaque (A2 strain) virus, but some studies were also made with DNA from A3, 208 and P16 strains (Fried et al., 1974). For studies with restriction endonucleases which cleave polyoma DNA at a few sites only (for example, HindIII, HhaI, etc.), DNA prepared as above (-70 to 90% pure) was used directly. The main contaminating species were found to be a mixture of DNA and RNA, approx. 5 S in size. For studies with multicleavage restriction endonuoleases, or the depurination studies (below), polyoma DNA was further purified by sucrose (5% to 20%) velocity gradient centrifugation (Fried, 1974). (b) Analysis

with restriction

endonucleases

The restriction endonucleases from Haemophilus parainJuenzae (HpaII) was prepared as described by Sharp et al. (1973) and that from Haemophilus aegyptius (HaeIII) was prepared as described by Takanami & Kojo (1973). Other endonucleases were generous gifts from Drs Rich Roberts, Bob Old, Bob Kamen, Nigel Brown and Mr Alan Robbins. Endonucleases used in this study, in addition to the above, were derived from Bacillus awbyloliquefaciens (BarnI), Brevibacterium umbra (BumI), Escherichia coli RY13 (EcoRI), HaemophiZu8 aegyptius (HaeII), Haemophilus influenaae Rd (Hind11 and HindIII), Haemophilus haemolyticus (HhaI), Haemophilus parahaemolyticus (HphI)t. For each endonuclease preparation, it was necessary to carry out small-scale assays to determine the amount of endonuclease and the period of digestion necessary for limit cleavage of polyoma virus DNA. All digestions (except with EcoRI) were carried out at 37°C in 0.01 M-Tris buffer (pH 7+5), 0.01 M magnesium ion, 0.001 M-dithiothreitol (low salt conditions) ; with EcoRI, the digestion buffer was made up to 0.1 M with sodium chloride (high salt conditions). Assays were carried out on 0.1 to 0.5 pg DNA and digestions were analysed by electrophoresis of the products on 1.4% agarose gels (containing ethidium bromide) in Tris-acetate (pH 7.5) buffer (Sharp et al., 1973). Agarose (SeaKem) was purchased from Marine Colloids Inc., Rockville, Maine. Polyoma DNA restricted fragments were separated either by electrophoresis on agarose slab or cylindrical gels or on polyacrylamide slab gels, using either Tris-acetate (pH 7.5; Hayward & Smith, 1972) or Tris-borate (pH 8.3; Dingman &Z Peacock, 1968) buffers. For t DNA name of polyoma cleavage denoted

fragments obtained by cleavage with restriction endonuclease are the endonuclease and a number which shows the size of the fragment A2 DNA. Thus HpaII-6 is the fifth largest fragment obtained from etc. Restriction with an enzyme, HpoII, from H. parainfluenzae, as suggested by Smith & Nathans (1973).

denoted with the as a percentage of polyoma DNA by endonucleases are

FINE

STRUCTURE

OF POLYOMA

VIRI’S

I)SA

449

preparative gels the former buffer system was preferred, and for analytical gels, the la,tter. T11e procedures used for separating and quantitating polyoma DNA fragments arc described in detail by Griffin & Fried (1976). Samples were visualised either by autoradiomaterial or by fluorescence with ethidium bromide for nongraphg for 32P-labelled lahelled materiel (see Sharp et al., 1973). Fragment size was ascertained on 32P-labelletl DNA by determining the mobility of a fra,gment on the gel and the number of ctsirnitr 1971; Danna et al., 1973; ((.‘&enkov radiation) in the fragment (Danna & Nathans, Griffin et al., 1974). and cleavage aitll 2 (or more) Most, cndonucleases were found to be “compatible” ctltlonl~clc~ascs was carried out as described above. Thcro were some excrptions, lloa-c:~~~~~. (i) For double cleavage with EcoRI and Hpall, polyoma DNA was first, cleavrd witlt H~jall under low-salt conditions until digestion was complete (as assayed by clect,roplloresis on agarose gels). The solution was then made 0.1 M with respect to NaCI, EcoKl c~r~douucloase added and digestion continued. Higher salt condit.ions were necessary t,c, suppress secondary activities in the endonuclease preparat,iou (Polisky et al., 1975). HpjaI I cndonl~clmsc~ was folmd to be sensitive to high-salt concerltrations. (ii) \Vhe~r ow of the endonucleases to be used for cleavage was HaellI, the procedurewas adopted whereby the other endonuclease was first used to cleave polyoma DNA antI \vllen digestion was essentially complete, Hue111 endonuclease was added and the digestiorl continued to completion with this endonucleaso. In a typical HaellI preparation, incuhati+,n with I ~1 of HaelIl MM-ould cleave 1 pg of polyoma DNA to completion at 37°C aftc,t 1 I). HaelI seems remarkably insensitive to the prasencr of other pndonucleascs. salt conditions, or even gel contaminants (see Discussion). (iii) For cleavagr of fragments isolated from gels (see below), it was usltal to purify tilt. fra~grnetrt 1);~ sucrosr velocity gradient centrifugation (576 to 20%) (see above) brforc digestion with a second endonuclease since many cndonucleases appeared sensitive to prclducts obtained from the gels during fragment isolation. When the secorld endonucleasc: to be used was HaelTT. this was usually found to be unnecessary. (c) Isolation

of restriction

endow&ease

fragments

Products were eluted from gels by the electrophoretic method described by Galibrart et ul. (1974). and isolated by ethanol precipitation, as described above. Carrier t,ransfcLl, RNA (10 to 50 pg) was usually added to aid product precipitation. (d) Depurinution

analysis

Polyoma virus DNA (32P-labelled) was cleaved by the formic acid/diphenylamitrc: (depurination) procedure of Burton & Petersen (1960) and Burton et al. (1963) and th resulting “tracts” of pyrimidines separated by the 2-dimensional procedure developed b> Brownlee & Sanger (1969) and adapted to analysis of DNA by Ling (1972). The firstdimensional separation was carried out by electrophoresis on cellulose acetate strips (Schleicher and Schiill, Dassel, W. Germany, 33 mm x 550 mm) at pH 3.5 (pyridine/acetic acid in 7 M-urea) until the distance between the blue and yellow dye markers (see Barre]], 1971) was approx. 15 cm. The products were bransferred to DEAE-cellulose t,hin.laycr shret (Macherey-Nagel and Co., Diiren, West Germany, CEL 300 DEAE/HR-~/IS 20 cm x 40 cm, purchased from CamLab, Cambridge) and devrloped by llomochromato. graphy (3% homomix, 30 min hydrolyeed (Brownlee & Sangcr, 1969)) for npprox. 6 11 at 60°C. The fingerprints were visualised by autoradiograph),.

(a) Cleavage of polyoma

3. Results DNA (A.2 strain) with restriction

endonucleases

A physical map of polyoma virus DNA (A2 stmin) has previously heen described (Griffin et al., 1974). It includes the cleavage with restriction endonucleases HpaIJ (eight sites), EcoRI (one site) andHilzdII1 (two sites). In subsequent communications. clenvagc with HindII (two sites) (Griffin & Fried, 1975; Folk et al., 1975), Hhal

460

B. E. GRIFFIN

(three sites) (Griffin & Fried, 1975), Hue11 (one site), Bum1 (one site) (GritEn $ Fried, 1976) and Bum1 (four sites) (Fried & Griffin, 1977) has been reported and cleavage sites placed on the HpaII physical map of polyoma A2 DNA. Hind111 (Roy & Smith, 1973) cleaves polyoma DNA to fragments with sizes equal to 56% and 44% of the full-length DNA. Hind11 (Smith & Wilcox, 1970) gives fragments with sizes 90.5% and 9.5%. HhaI (Roberts et al., 1976) gives fragments with sizes 46%, 41.5% and 12.5%. Bum1 gives fragments with sizes 585%, 22%, 16.5% and 3% (see Table 4). Hue111 (Middleton et al., 1972) cleaves polyoma DNA at 24 sites to give products which range from 135 to 0.2% the size of the full-length DNA (see Table 1 and Fig. l(a) and (b)). Twenty-two of these fragments were easily separable by gel electrophoresis, but two pairs (HueIII-14 and 14’ from A2 DNA or HueIII-15 and 15’ from A3 DNA, and HueIII-18 and 18’) were not clearly separated. (The Hue111 products have been numbered 1 to 22 in order of decreasing size, see Fig. 1.) In a plot of the logarithm 32P counts per minute versus migration (in cm) (results not shown, see Griffin & Fried, 1976) all fragments except A2 HueIII-14 (or HueIII-15 in A3 DNA) and HueIII-18 were shown to be present in one-molar yield. HueIII-14 (or HueIII-15) and HueIII-18 were present in two-molar yields. As a first approximation in locating the Hue111 fragments on the physical map of A2 polyoma DNA, the full-length linear species (produced by digestion with either EcoRI, Bum1 (Wilson & Young, 1975) or Hue11 (Roberts et al., 1975) were each cleaved with HueIII. By this procedure the EcoRI site was found in HueIII-8 and the Bum1 site in HueIII-5 (Fig. 2) ; the HueII site was found in one of the two A2 HueIII-14 fragments. By similar procedures, the HindII, HindIII, HhuI, Bum1 and HpuII sites were each located within particular Hue111 fragments. The data for these experiments are summarised in Table 1. To locate Hue111 fragments within particular segments of the DNA, the two Hind111 fragments were isolated and each cleaved with HueIII. By this procedure, Hue111 fragments 3,6,7,10,11,20,21 and 22 were located in the HindIII 44% fragment; new Hue111 fragments about 9.7% and 2.2% in size were found. Similarly, Hue111 fragments 1, 2, 5, 9, 12, 13, 14 (two-molar), 15, 16, 17, 18 (two-molar) and 19 were located in the Hind111 56% fragment; new Hue111 fragments about 1.7% and 0.3% in size were found. Hind111 sites were found in Hue111 fragments 4 and 8. The three HhaI fragments were also separated and each cleaved with HueIII. HhuI 41.5% gave fragments which were electrophoretically indistinguishable from HueIII-1, 2, 3, 8 and 18 (one-molar); two new fragments were produced which were similar in size but not identical to HaeIII-22. HhaI 46% gave HueIII-4, 5, 9 to 20 (one-molar each) and 22; new fragments were obtained with sizes 1.8% and 1.60/,. HhuI 12.5% gave only HaeIII-21 as an identifiable HaeIII fragment; two new fragments with nearly identical sizes (5.9% and 5*80/,) were obtained, and HhuI sites were found in HaeIII fragments 6,7 and 14. For further information, the eight HpaII fragments were separated and cleaved with HaeIII. The data are summarised in Table 2. The data obtained in the experiments described above allowed all the HaeIII fragments between 69.5 and 28.2 map units (clockwise, see Fig. 3) on the physical map of polyoma DNA to be assigned unambiguously. For assignment of the 14 Hue111 fragments which came from 28.2 to 69.5 map units (clockwise) on the physical map, the following experiments were carried out.

FINE

STRUCTURE

OF POLYONR

VlRUS

.lJSA

451

Polyoma A3 DNA HpoIl+ \

HueIU

I

HuellI

a

(al

(b)

FIG. 1. (a) Electrophoretic separation of unlabelled Hue111 fragments of polyoma DNA on a 1.4% agarose cylindrical gel in Tris-acetate (pH 7.5) buffer; fragments were stained with ethidium bromide and visualised as described by Sharp et al. (1973). Fragments 1 and 2, easily separated on agarose gels, did not usually separate on polyacrylamide gels, see (b). The bromophenol blue dye marker, B +, co-migrated with HaeIII-7 and interfered with the visualisation of this fragment. Small fragments (< 3%) of the DNA length were not resolved by this procedure. (b) Autoradiogram of fragments produced from 32P-labelled polyoma DNA (A3 strain) by cleavage with (column A) H&I, (column B) HpaII + HaeIII, (column C) H&II. Reparation was carried out by electrophoresis on a 5% acrylamide slab gel (20 cm x 40 cm) in Tris-borat,n (pH 8.3) buffer. HpaII fragments are numbered from top to bottom on the left according to Griffin et al. (1974) and Hoe111 fragments from top to bottom on the right according to decreasing size (see Table 1). By a comparison of the products in columns B and C, HpaII sites can be seen to lie in Hoe111 fragments 1, 2, 6, 9, 17 (see Discussion) and 21. In DNA from the A2 and 208 strains, fragment 15’ (the slower of the 2 species with similar migrat,ions) was absent,, and fragment 14 was present in 2.molar yield (the second species being designated 14’. Fig. 3).

I

I I

II’IIIII

/III

I

FINE

STRUCTURE

OF

POLYOMA

TABLE

C’leavage of HpaII

fragments

VIRUS

453

DSA

2

of A2 polyoma

DNA

with HaeIII

H~xzIl Fragment ,111.

3

Size (% of full-length polyoma DNA)?

Hue111 subfragments

( y0 of full-length

27.3

10.0 3.4 2.7 1.9 1.6 1.4 1.0 0.9 0.8 0.2

(HueIII-4) (two-molar) (HneTII-10. (HueIIl- 11) (HaelII-13) (HpulI/Hue 1.6%) (HueIII-16) (HueIll-18) (HuelII-19) (HueIIl-20) (HueIIl-22)

21.4

10.2 6.0 3.9 1.2 0.1

(HueIII-3) (HueIIl-7) (HueIIl-8) (HpuII/HueIII (HpaII/HueIII

1.2%) 0.1%)

9.8 2.3 1.7 1.5 1.2 0.3

(H&II-5) (HueIII-12) (HaelIl-14) (HaeIII-15) (HpaIl/HueIII (H~ulI/HueIII

1.2%) 0.3 %)

16.8

4

13.2

6.8 (HpulI/HueIII 5.4 (HpuII/HaelII 1.0 (HaeIII-18’)

6.8%) 5.40/;,)

7.7

5.9 (HpuII/HnellI 1.7 (HaeIlI-14’)s 0.1 (HpuII/HaeIII

5.991,)

5 6

6.7

6.1 (HpalI/HueIII 0.6 (HpnII/HueIlI

6.1%) O.B”k,)

7

5.2

None

8

1.8

None

HprrIT/HtreIII

DNA)$

3.4O,,)

0.1%)

t Taken from Griffin et ccl. (1974). 1 Identities given in parentheses (see Table 3 and Fig. 3). $ In the A3 &rain of polyoma DNA this is 1.59/,.

(1) The restriction fragment HpaII-1 was cleaved with HindTIl, the resulting two This fragments (about 18.5% and 9.0%) isolated and further cleaved with HaeIII. allowed the assignment of HaeIII-10. 11, 20, 22 and most of HaeIII-4 to the region 26.6 and 45.0 map units. HaeIII-13, 16, 18, 19, part, of HaeIII-4 and a fragment similar in size to HaeIII-9 could be allocated to the region between 45.0 and 53.9 is known to have an HpaII cleavage map units (see Fig. 2 and Table 1). HaeIII-9 site, therefore it must lie at the junction of HpaII fragments 3 and 1. The endonuclease HphI (Kleid et al., 1976) cleaves polyoma DNA into nine fragments. Five of these &es have been found in HpaII-1 (Griffin, unpublished results) ; cleavage of HpalI-1 with HphI gives fragmenbs with sizes about 11.5%, 6*00/o,

B. E.

454

-3

I

Polymo

GRIFFIN

DNA ----l

SV40 DNA

30.7

J5*7

7-o 6.7 6-3 6.1 7

4.3

8 9 3.3

(0)

(b)

of 32P-labelled fragments obtained from polyoma A2 DN A and Frc. 2. (a) Autorediogram on a 6% acrylamide slab gel (20 cm x 40 cm) in Tris-borate (pNH 8.3) seperai ted by electrophoresis buffer. I A, products obtained by cleavage of the A2 DNA HpaII-2 restriction fragment ( GIriffin Colu ‘4) with HaeIII; the 3rd largest HaeIII fragment from polyoma DNA (see colu: ml,n D) et al., m to be one of the products. can be B, products obtained by cleavage of the A2 DNA HpaII-3 restriction fragmen .t with

FINE

STRUCTURE

OF

POLYOMA

VIRUS

DNA

455

4.00/,, 3.5% and 1.5% (two-molar yield). Cleavage of the HpaII/HindIII 18.5% fragment (see above) with HphI gave the 11.5% fragment and two 3.5% fragments. Cleavage of the HpaII/H&dIII 9.0% fragment gave the 4.0% and 1.574 fragments, and a new fragment 2.5”/b in size. The Hph 6.0% fragment contained the Hind111 site. Hph cleavage sites were found in HaeIII fragments 4, 11, 13 and 18, all fragments derived from HpaII-1. The cndonuclease PstI has been reported to cleave polyoma DNA at five sites, two of which have been located at 33.0 and 50.3 map units (Crawford & Robbins, 1976). Analysis with HaeIII showed PstI sites in HaeIII fragments 10 and 18. A comparison of the HaeIII endonuclease cleavage pattern of the temperaturesensitive mutant TsA with wild-type polyoma DNA showed HaeIII-6 and 20 to br absent in the mutant and a new fragment with a size corresponding to that expected from a fusion of HaeIIl-6 and 20 (about 8.5%) to be present (Miller et al., 1976). Tbe data presented above can all be explained by a HaeIII fragment alignment and order (smrting from the HpaII l-6 junction) : a 1.7% fragment (a subfragment of HaeIII-6). HaeIII fragments 20, 11, 10, 4, 16, 19 (or 19, IS), 13, 18 and a 3.4%, fragment (a subfragment of HaeIlI-9). The very small fragment, HaelII-22, must, lit hrtween HaeIII fragments 20 and 4, but its unambiguous assignment has not been made. Thus. most of the HaelI fragments between 23.2 and 54.2 map units can be assigned. (2) For assignment of HaeIII fragments to the region between 53.9 and 70.8 ‘map units, the following data were used. The endonuclease Barn1 cleaves polyoma DNA in restriction fragment HpaII-3 (Griffin & Fried, 1976). Analysis of the HaeIII cleavage pattern of the linear species produced by cleavage of polyoma DNA with BnmI, showed the Barn1 site to lie in fragment HaeIII-5 (Fig. 2(E)). Cleavage sites of t’he cndonuclease Bum1 (Table 1) were found in HaeIII-17 and HaeIII-15. These data: taken together with data on the size of the fragments produced, suggest that the alignment and order of the HaeIII fragments (starting from the HpaII 1-3 junction) are: a 0.3% fragment, and HaeIII fragments 12,5,15,14, and 17. The conconclusive assignment of HaeIlI fragments within HpaII-3 depended upon the isolation of partial digestion products by methods previously described (Griffin et al., HrrrIlI; the 5th largest, HtreIII fragment from polyoma DNA (see column U) can be seen t,o bcb 0111’ of the products. (‘olumn C, products obtained by cleavage of polyoma DNA with Hue111 and E’coRT. The EcoRl sitfl can be seen to lie in the 8th largest Hue111 fragment (see column D). Aft,er cleavage with &‘:coRI, the ninth largest fragment is present in S-molar yield. The very small product cleaved from HrreIII-8 with EcoRI cannot be seen on this gel. ( ‘olumn D, products obtained by cleavage of polyome DNA with HaeIII. For complete fragment numbering, see Fig. 1. (b) Autoradiogrem of 32P-labelled fragments obtained by cleavage of polyoma virus and SV40 I)SAs with rest,riction endonucleases. Products were separated essentially as described in (a). The ver)’ small products were all trapped in a 15% gel together with the dye marker, tB. (.‘olumn E, products obtained by cleavage of polyoma DNA with Hue111 and BamI. By comparison with the products in column F, the Bum1 site can be seen to lie in the 5th largest H&II fragment, which is a product of HpaII-3, see (a) column B. After cleavage with Barn1 the 6th largest HcteIII fragment was present in 2-molar yield. The small product cleaved from HaeILI-5 wit,h Bern1 could not be identified on this gel. (‘olumn F, products obtained by cleavage of polyoma DNA with HueIII. Only the 14 largest fragments can be clearly seen. (‘olumn G, products obtained by cleavage of SV40 DNA (A to J) with HaeIII. The fragment sizc%s given on the extreme right as percentages of the full-length genome are taken from Yang et ~(1. (1976).

B. E. GRIFFIN

456

1974). After limit digests on partially digested products HaeIII-14 was found to lie adjacent to the 1.2% HpaII 3-5 junction fragment (a subfragment of HaeIII-17) and to Ha&II-15; both partial digestion products were obtained and analysed. Similarly, HaeIII-5 was shown to lie adjacent to HaeIII-15 and HueIII-12. Confirmation for the assignment has also come from studies on defective polyoma species (Griffin t Fried, 1975; Lund et aE., 1977a). Much of the data given above are summarised in Tables 3 and 4. They lead to the Hue111 physical map for the A2 strain of polyoma DNA shown in Figure 3. Similar data on the polyoma A3 strain point to a difference in the two polyoma strains which can be mapped between 70.8 and 72.5 map units on the physical map. This was further confirmed when HpaII-5 was cleaved with HhaI : a change (reduction) in size of about 0.2 to 0.3% was observed when the small HhuI fragment from A3 HpaII-5 was compared with the small HhaI fragment from the A2 HpaIT-5 (see Discussion). The sizes given to Hue111 fragments (Table 1) were determined as described (see Materials and Methods). Many sizes were also deduced from comparisons with sizes of the HpaII fragments (Griffin et al., 1974). For an independent check, the HaeIII TABLE 3

Size and position (map units) of the HaeIII restriction endonuclease fragments from polyoma (A2 strain) DNA (clockwise) on the physical map (EcoRI site is zero map unit) Fragment

8 3 7 21 6 20 11 10

22 4

1’31 1% 13 18 9 12 5 15 14 17 14’1 2 18’ 1 8

no.

Size ( y0 of full-length polyoma DNA) 3.9 10.2 6.0 0.7 7.7 0.8 2.7 3.4 0.2 10.0 1.4 0.9 1.9 1.0 3.7 2.3 9.8 1.5 1.7 1.3 1.7 12.7 1.0 13.5 3.9

Location

(map units) ho.2

99.7- 3.6 3.6-13.8 13.8-19.8 19.8-20.5 20.5-28.2 28.2-29.0 29.0-31.7 31.7-35.1 35.1-35.3 35.3-45.3 45.3-46.7 46.7-47.6 47.6-49.5 49.5-60.6 50.6-64.2 54.2-56.6 56.6-66.3 66.3-67.8 67.8-69.5 69.5-70.8 70.8-72.5 72.6-85.2 85.2-86.2 86.2-99.7 99.7- 3.6

t In the A3 strain of polyoma DNA, this fragment is absent and a new fragment slightly larger than HaeIII-16 (1.6%) is present. f The position of these fragments has not yet been proved unambiguously.

with

size

FINE

STRUCTURE

OF POLYOMA

VIRUS

DNA

457

TABLE 4 Sites of cleavage of polyoma DNA (A2 strain) by restriction endonuclease from B. amyloliquefaciens (Baml), B. umbra (BumI), E. coli (EcoRI), H. aegyptius (Hae1s. and III), H. haemolyticus (Hhal), H. influenzae (Hind11 and III) and H. parainfluenzae (Hpall) Restriction enzyme

Cleavage site (map unit & 0.2)

Restriction enzyme

EcoRI Hind111 Hue111

0.0 1.4 3.6 8.8 13.8 14.0 19.8 19.9 20.5 26.2 26.4 26.6 28.2, 29.0, 31.7, 351, 35.3 35.6 45.0 45.3 (46.7, 48.6) 49.6, 50.5 53.9

HaeIlI Barn1 HaeIII Bum1 Hue111 Bum1 HpaII HaeIII HhaI HaeII HaeIII HpaII HaeIII HpaII Bum1 HpaII HaeIII

Bum1

HaeIII HhaI HaeIII HpaII Ha&I Hind11 HhaI HpuII Hue111 Hind11 Hind111 HaeIII HpaII

Cleavage site (map unit i: 0.2) 54.2, 58.5 66.3 67.4 6743, 70.2 70.7 70.X 72.4 72.4 72.5 78.4 85.2, 91.6 92.4 93.4, 99.7

56.5

69.5

86.2

98.5

restriction pattern of 32P-labelled SV40 DNA has been compared with the HaeIlI and2 (given restriction pattern of polyoma DNA (seeFig. 2 (FandG): polyomaHaeIII-1 values of 13.5% and 12.7%, respectively, of the full-length DNA) were found to migrate between the SV40 15*7°h and 10.2% fragments; polyoma fragments Hue111 3.4,5 and 6 (10.2%, 10*O”/& 9.8% and 7.7O/& respectively) all migrated between the SV40 10.2% and 7.0% fragments. Polyoma HaeIII-7 (6.0%) comigrated with the SV40 6.1% fragment. Polyoma HaeIII-8 and 9 (3.9% and 3.7:& respectively) migrated between the SV40 4.3% and 3.2% fragments. The migration of the SV40 3.376 fragment was slightly faster than polyoma H&I-IO (3.4%). The results for the larger fragments are therefore in good general agreement with SV40 Hue111 fragment sizes as determined by Yang et al. (1976). (Slight variations in sizes of SV40 DNA Hue111 fragments have been reported (Huang et al., 1973; Subramanian et al., 1974; Lebowitz et al., 1974; Yang et al., 1976. These are thought to reflect technical differences rather than a true difference in DNA (Yang et al., 1976).) (b) Analysis

of depurination

Jingerprints

The fingerprint made on the depurination products of 32P-labelled A2 polyoma virus DNA (see Fig. 4) showed several interesting features. (A fingerprint of the A3 strain of polyoma virus DNA (not shown) was indistinguishable from that of the A2 strain.) (1) Depurination

produced

over 50 discrete

(2) More than ten of the tracts one-molar yield.

in only

appeared

pyrimidine

to be unique,

tracts. that is apparently

present

B. E. GRIFFIN

458 A2

P~lpma

EcoRI

A2 DNA, HpaE

and HaeIII

H/ndlll physical

mops

FIG. 3. Concentric circles showing the physical maps of polyoma DNA (A2 strain) obtained by cleavage with restriction endonucleases H. parainfluenzae (HpaII, 8 sites, Griffin et al., 1974, inner ring) and H. aegyptius (HaeIII, 24 sites, outer ring). The map is divided into 100 units with the single EcoRI cleavage site at zero map unit. The origin of viral DNA replication (0) (Griffin et al., 1974) is shown. The location of the early and late transcription regions on the polyoma DNA HpaII physical map is taken from Kamen et al. (1974). Other restriction endonuclease sites have been mapped relative to the HpaII and Hoe111 physical maps. These include the endonucleases from E. coli (EcoRI), H. aegy&wr (HaeII), B. umyloliquefaciens (BarnI), H. influenzae (Hind11 and III), B. urnbra (BumI), and H. huemolyticus (Hhd). These sites are shown. Data for cleavage of polyoma DNA with an endonuclease from Providenciu stuurtii (P&I) and Klebsielh pneumoniae (KpnI) have been reported by Crawford & Robbins (1976) and for Huemophilw, gallinurum (HgaI) by Shishido t Berg (1976). Pet1 cleaves polyoma DNA at 5 sites which are reported to lie at 14.8, 16.5, 32.6, 60.3 and 80.0 map units; the cleavage site at 32.6 map units has been found to lie in fragment HaeIII-10 and that at 60.3 map units in HaeIII-18. KpnI cleaves polyoma DNA at two sites which are reported to lie at 11.6 and 59.2 map units. HgaI cleaves polyoma DNA at 4 sites which have been reported to lie at 2, 14, 27 and 48 map units.

(3) Two exceptionally large oligopyrimidines were present, which by consideration of their position on the polyoma DNA fingerprint (Fig. 4) and by comparison with the fingerprint of fd DNA (Ling, 1972) must be between 15 and 20 nucleotides long. (The largest oligopyrimidine in fd DNA was 20 nucleotides long ; in SV40 DNA the largest appears to be only 11 or 12 nucleotides long (see Fig. 5).) (4) Among the unique species, one very large oligodeoxycytidine could be seen, which by its position on the fingerprint appeared to be an octamer. (5) One very large oligothymidine was present, which by its position appeared also to be an octamer. An autoradiograph of the depurination products of 32P-labelled A2 polyoma virus DNA and a schematic diagram are shown in Figure 4. The assessment of size, composition and yield is based on the work described by Ling (1972) for fd DNA. To complete the comparison of polyoma virus DNA with SV40 DNA, an autoradiogram of the depurination products of SV40 DNA (small-plaque strain) was also made (see Fig. 5(a)). In order to assign the unique oligopyrimidines to a specific region on the polyoma

Polyomo wrus DNA

first-dimension (01

(electrophorews,

pH 36) (b)

polyoma virus A2 PIG. 4. (a) An autoradiogram of the depurination products of 32P-labelled DNA. The oligopyrimidines were separated in the 1st dimensions by electrophoresis on cellulose acetate strips at pH 3,5, and in the 2nd dimension by homochromatography at 60°C on DEAEcellulose thin-layer sheets (20 cm x 40 cm). (See Materials and Methods for procedures and references.) (b) A schematic diagram of the depurination fingerprint of polyoma virus DNA shown in (a). The identification of the products in terms of composition (deoxycytidines, C, and thymidines, T) follows from the results of Ling (1972). Although nothing can be said about the sequence of the various oligomers, the compositions can be directly read off, at least to the decamer level. The nine oligomers which are larger than decamers have been assigned to the HpuII restriction fragment from which they are derived, four from HpaII-1 (see Fig. 5(b)) and one each from HpaII fragments 2. 5, 6, 7 and 8 (see Figs 6, 8 and 9). The single deoxycytidine-rich species has been assigned to Up~rI1-8 (Fig. 8) and the single thymidine-rich species to HpalI-3 (Fig. 6). Numbers inside the circles are used to designate the HpaII fragment from which the oligopyrimidinn is derived.

460

B. E. GRIFFIN

Polyoma virus DNA- HpaII-

Electmphoresis, (al

I

pH 3.5 (b)

FIQ. 5. Autoradiogram showing a comparison of the depurination products of (a) 3aP-labelled SV40 DNA (small-plaque strain) and (b) fragment, HpaII-1 from polyoma DNA (A2 strain). The oligopyrimidines were separated by the 2-dimensional procedure described in Fig. 4. This comparison is made as shown, because although more complex, the overall pattern of SV40 DNA appears to resemble polyoma H&I-l more than polyoma virus DNA (see Fig. 4). The species designated (*) (9 and 11.long oligomers) appear in both fingerprints, clearly in multi-molar yield in SV40 DNA, and the g-long (and possibly the 11-long oligomer also) in multi-molar yield in the polyoma fragment. Polyoma, virus and SV40 DNAs have been found (Ferguson & Davis, 1976) to have a region of limited homology which from the data available can be mapped in polyoma fragment HpaII-1 (Fried et aE., 1974). Whether the similarities found above do in fact represent this homology region must await further investigation.

FINE

STRUCTURE

OF

POLYOMA

VIRUS

DKA

4til

DNA, the DNAs from the eight HpaII restriction fragments were depurinated and fingerprints made in the same way as for whole A2 DNA. These are shown in Figures 6. 8 and 9. This analysis showed that: (1) The largest oligopyrimidine (see above) came from HpaII-8 (Fig. 8) and the second largest from HpaII-5 (Fig. 6). Hparr-5

Hpo!J-3

d

Elecirophoresls,

pH 3.5

HpaII restriction endoFIG. 6. Autoradiograms of the depurination products of 32P-labelled nuclease fragments (HpaII-3 and HpaII-6) from the region around the origin of replication in polyoma DNA (see Griffin et al., 1974, and Fig. 3). The fingerprint of HpaII-3 (AZ strain) can be seen (*) to have the largest oligothymidylic acid (probably T,) found in polyoma virus DNA. By analysis of HaeIII fragments from HpaII-3, t,his has b-en found to lie near the HpaII 3-5 junction and the origin of DNA replication (see Fig. 7). For other characteristics of HpaII-3, see Lund et al. (1977a,b). The fingerprint of HpaII-6 (A2 strain) shows (*) the very large oligomer (about 17.nucleotides long) found to be the 2nd largest in polyoma DNA (see Fig. 4). By analysis of the H&II fragments from HpaII-6, this has been found to lie near the HpaII 3-6 junction and the origin of DNA replication (see Fig. 7).

B. E.

462

GRIFFIN

Oligomers from origin of replication

69.5 to 70.7 map units

70.8 to 72.4 I ‘IQ. 7. Autoradiograms from “UC :lease fragments

mop untts

region in polyoma DNA

70.7 to 70.0

map units

72.4 to 78.4 map units

Hue111 of the depurination products of 32P-labelled ((a) to (0)) the region of the DNA around the origin

restriction em IOof replication in

FINE

STRUCTURE

OF POLYOMA

(2) The single long run of thymidines

(probably

(3) The single long run of deoxycytidines

VIRUS

DN-\

T,) came from H@II-3

(probably

C,) came from HpaII-8

463

(Fig. 6). (Fig. 8).

(4) Of the other large unique oligopyrimidines, four came from HpaII-1 (Fig. 5(b)) and one each from HpaII-2, 6 and 7 (Figs 8 and 9). The results described above are shown on the schematic diagram in Figure 4(b). (5) Fingerprints of the HpaII-5 fragments smaller t,han A2, see above) were qualitatively

from A2 DNA or A3 DNA (0.2 to 0.30,, indistinguishable (data not shown).

When the DNA being examined was further diminished in size, and depurination fingerprints made of some of the individual Hue111 fragments, it was possible to assign some of the smaller oligopyrimidines to specific areas of polyoma DNA (see Lund ~‘kal., 1977a,b). Depurination fingerprints of the small fragments which lie in the region where the origin of DNA replication has been mapped (at 7153 map units. Grifhn et al.. 1974) are shown (Fig. 7). The fragments were obtained by Hue111 cleavage of HpaII-3 and 5; (a) fragment from 69.5 to 70.7 map units on the polyomn virus physical map (from a subfragment of HaeIII-17); (b) fragment from 70.7 to 70.8 map units (from the other subfragment of HaeIII-17); and (c) fragment from Hnelll-14’ (70.8 to 72.5 map units) (see Table 3 and Fig. 3). In (a) the fragment can be seen to contain the single long run of thymidines (T,) found in HpaIT-3 (see above). In (c) the fragment can be seen to contain the second largest (about 17 nucleotides long) oligopyrimidine present in wild-type polyoma DNA and found in HpaII-5 (SW ahovr), The depurination fingerprint of the fragment (b) suggests that this fragment, is 5 to 10 base-pairs in length ; the single characterist,ic oligopyrimidine appears to be either C,T or C&T,. Cleavage of the DNA from the A3 strain of virus gives a HaeIII fragment (HaeTII15’) from HpaII-5 which differs in size from A2 HaeIII-14’ (see above) by 10 to 15 base-pairs (Haelll-15’ is smaller). The depurination fingerprints of HaeIIl-14’ and 15 (not, shown) were qualitatively indistinguishable.

4. Discussion In order to understand in molecular terms the interactions of a virus (polyoma virus) and its host and to attempt to explain lytic or (viral) transformation events. it seemed reasonable to begin with defining the viral DNA. The establishment of a physical map of polyoma DNA and the localisation of some biological markers on this map (Griffin et al., 1974) was a first step in this direction. This paper attempts to define further the polyoma genome in terms of DNA segments of decreasing size (restriction endonuclease fragments) and the primary sequences (depurination products) found within these segments. This approach has not only been useful in polyoma virus (see Griffin et al., 1974, and Fig. 3) and (d) the rest of H&I-5. (a) Fingerprint of the Hue111 fragment from HpnII-3 which lies at the HpaII 3-5 junction (69.5 to 70.7 map units on the physical map, see Fig. 3). This fragment can be seen (*) to have th(b oligothymidine-rich tract (probably Ts) found in HpaII-3 (see Fig. 6). (h) Fingerprint of the Hue111 fragment from HpaII-5 which lies at the H@I 3-6 junction (70.7 to 70.8 map units on the physical map, Fig. 3). (c) Fingerprint of the Hue111 fragment from HpaII-5 which lies at 70.8 to 72.5 map units on the polyoma physical map (see Fig. 3). This fragment can be seen (*) to have the very long and characteristic pyrimidine tract (about 17 nucleotides long) from HpaII-5 (see Fig. 6). (tl) Fingerprint of the HaeIII fragment from HpaII-5 which lies at 72.5 to 78.4 map units on the polyoma physical map (Fig. 3). The fingerprints ((b) to (d)) on superimposition can be seen to give the fingerprint of the entire HpaII-5 fragment (Fig. 6).

If , *, I a

22

. B

FINE

STRUCTURE

OF POLYOMA

VIRUS

1)X);;\

465

defining the DNA of one wild-type polyoma strain but has also been useful in comparing strains and analysing variant and defective polyoma species (Griffin & Fried. 1975; Fried & Griffin, 1977 ; Lund et al., 1977aJ). (a) Analysis

of polyoma

DNA

with restriction

endonucbases

The endonuclease from H. aegyptim, HueIII, which recognises and cleaves at a site whose sequence is 5’ -G-G-C-C- 3’ (Murray & Old, 1974) cleaved A2 polyoma EDNA (a large-plaque strain) into 24 fragments. The sizes of the Hue111 fragments of A2 polyoma DNA and the position of each cleavage site are summa.rised in Table 3. and correlated with the HpaII physical map and some biological markers in Figure 3. (Table 4 summarises the data on some other restriction endonuclease cleavage sites u.hich have been determined with respect to the Hue111 sites.) The DNAs from another large-plaque strain (A3), a small-plaque strain (P16) and a minute-plaque strain (208) (Fried et al., 1974) were also cleaved with HaeIII (Griffin, unpublished results). The restriction endonuclease cleavage patterns of 208 and A2 DNAs were indistinguishable. By this analysis, DNA from the A3 strain has been shown to differ from A2 (or 208) DNA in one respect only, it is missing about’ 10 t,o 15 base-pairs from the region of the DNA which lies between 70.8 and 72.4 map units (in HaeI 11-14’ in A2 DNA, see Figs l(b) and 3). It was not clear from further studies on this region from the A2 and A3 strains. either by cleavage with other restriction endonucleases or by depurination analysis (see below), whether th(a deletion represents a loss of non-essential sequences or whether the sequences are so important to the virus that they are duplicated in the A2 strain. As an argument for these sequences from this region being essential to the virus (and therefore duplicated), it, should be noted that in certain polyoma defective species (Griffin & Fried, 1975: Lund pf al., 1977aJ) this region, which contains (or is near) the origin of DNA replicat(ion, is always preserved and even duplicated. On the other hand, non-rssent,ial rrgions have been found in SV40 DNA on either side of the origin of DNA replicat,ion (Shenk et al., 1976) and similar results have been found for polyoma DNA (Lund PI al.. 1977a.h; Fried & Griffin, 1977). This point will probably be solved only when primary sequence data are available for this region of A2 and A3 DNA. (In addition to sequence changes found in different strains around the origin of viral DNA replication, or near the 5’ ends of the messenger RNAs, see Fig. 3. some variation has been observed in the region of the DNA near where the 3’ ends of thts polyoma mRNAs have been mapped (Kamen et al., 1974; Tiirler et al., 1976: seal Fig. 3). The size of the H~aII/HaeIII product from 26.6 to 28.2 map units (see Fig. 3

of 321’-labellod Fra. X. Aut,oradiograms of the depurination products HpcrII restriction endonuclcasv fragments (HprcII-4, 8 and 7) from a part of the ‘early region’ (SW Fig. 3) of polyoma DNA. All fragments are derived from the A2 strain. In t,ht! fingerprint of HpaII-4, the most notable characteristic is the relative abundance of th(s hvxamer, C,T,(*). There are no large characteristic oligomers present,. The fingerprint, of H@I-8 shows (*) the 2 very characteristic oligomers present in this fragment, One is the largest (about 20.nucleotides long) depurination product present in polyoma virus DSA. and the ot,her is t,he largest oligodeoxycytidine species (probably C,) present in the viral DNA (see Fig. 4). HpnII-8 was found to be the most G + G-rich (52.306) of thr HpnII fragments from polyoma virus DNA (Griffin et al., 1974). The fingerprint of HpuII-7 shows (*) a large (about 13-nucleotides long) unique, deoxycytitlincSrich species. HpaII-6 (Fig. 9) and 7 are similar in size (6.776 and 5.2%, resprctirely) and girl, fingerprints similar in complexity.

B. E. GRIFFIN

466 HpaD-2

t

HpaU- 6

Electrophoresis,

pH 3.5

restriction Pm. 9. Autoradiograms of the depurination products of two 32P-labelled H@I endonuclease fragments (HpaII-2 and 6) which are derived mainly from ‘early region’ of polyoma DNA (see Fig. 3). Both fragments come from the A2 strain. The fingerprint of H&I-2 can be seen (*) to have one of the large (about 13.nucleotides long) oligomers unique in polyoma virus DNA; this oligomer is deoxycytidine-rich. For other characteristics of HpaII-2, see Lund et al. (1977a,b). The fingerprint of HpaII-6 shows (*) a large (12 to 13-nucleotides long) unique, thymidine-rich oligomer.

and Table 4) has been found to vary between strains also by about 10 to 15 base-pairs. This has not yet been further studied.) By a similar analysis with HaeIII, the polyoma small-plaque strain Pl6 has been found to differ markedly from the A2 and A3 strains in the region from 67.8 to 72.4 (around the HpaII 3-5 junctions, see Fig. 3) and 26.6 to 45.3 (in H~aII-1) map units (data not shown). All the apparent changes appear to occur in the regions containing (or adjacent) to the 5’ and 3’ ends of polyoma messenger RNAs (Kamen et al., 1974). P16 is presumably the same as (or related to) the small-plaque strain studied by Ziff

FINE

STRUCTURE

OF

YOLYOMA

VJRlTS

DNA

467

(Crawford et al., 1974) and the results reported here roughly correspond to his results. A partial HaeIII map of polyoma virus DNA has been previously described 1)~ Summers (1975). This map, however, was not correlated with the known physical map of polyoma DNA (Griffin et aE., 1974) and differs somewhat from the Haell i physical map shown for A2 polyoma DNA (Fig. 3). The latter has been obtained by a combinabion of methods, in part by partial digestion with HaeIlI but in the main 1)~ digestion with one or more additional restriction endonucleases. From a comparison of the HaeIII maps of A2 and A3 DNA, it seems clear that Summers was working \q,ith a strain which was similar to the A3 strain. Two interesting technical observations emerged from the cleavage of polyoma DNA with these restriction endonucleases. (1) When cleaving with two or more restriction endonucleaxes, there appears t,o 1~ban order of preference for using the enzymes. Thus, whereas all the HpalI frapmerits which contain HaeIIl cleavage sites were cleaved by HaeIII, to give a “typical” HpaII/HaeIII double digestion pattern, not all the HaelII fragments which cont,ain HpaII sites were cleaved by HpalI. That is, the Hpall/HaeIIl double digestion pattern differs from the HaeIlI/HpaIl double digestion pattern (data’ not shown). The result remains the same regardless of whether the second enzyme was added in t,he presence of the first enzyme, or to a solution from which the first enzyme ha,d been removed. It seems reasonable to conclude that when the HpaII cleavage site lies near the end of a fragment, cleavage may not occur or may occur w&h difficulty. HaeIII-17 is only partly cleaved with HpaII, and HaeIII-21 is not, cleaved at all. The converse is not true. HaeIlI can apparently cleave near the end of a fragment. One explanation for this is that site recognition for the Hpall endonuclease requires more than just the cleavage site sequences 5’ C-C-G-G 3’ (Garfin & Goodman, 1974). With HaellI endonuclease this is either not the case, or all the required site recognition sequences are present in the HpaIl fragments. (2) When non-denaturing gels are used and fragment sizes are small, base composition may affect the mobility during the electrophoretic separation of fragments. That is, a fragment may not migrate entirely according to its size. This fact has already been observed by Mertz & Berg (1974) for SV40 DNA and by Thomas & Davis (1975) for phage h DNA, and is discussed in detail by Ziegler et al. (1972). In the case of polyoma DNA this is especially dramatic. The l.SqG HpalI/HaeIII fragment which comes from 69.5 to 70.7 map units on the polyoma A2 physical map migrates more slowly than the 1.3% HaeIlI fragment (data not shown) which comes from 69.5 to 70.8 map units (see Figs 1 and 3). The former is a HaeIII subfragment of HlJalI-3. Analysis of the base composition of HpaII-3 showed the A-+T content to be 55*2:/i (Griffin et al., 1974). A similar analysis on each of the HaelII subfragments from Hpall-3 (Griffin, unpublished results) has shown that whereas most fragments differ only slightly in composition from HpaII-3, the A+T content of the fragment which comes from 69.5 to 70.7 map units is considerably higher (approx. 62*5!4). Ziegler et al. (1972), in studying the effect of base composition on electrophoretic mobility of DNA on acryla.mide gels, found that A+T-rich species were retarded relative t’o G + C-rich species and postulated that A.T base-pairs occupy a volume wit,h a larger cross-sectional radius than G*C base-pairs. If the short set of sequences removed from the region 70.7 to 70.8 (about 5 base-pairs) is composed mainly of G.C base-pairs, the removal of five such pairs would make HueIll-17, a fragment ::I

468

B. E. GRIFFIN

with a total of only about 65 base-pairs, even more A+T-rich. Making this assumption, and assuming that the depurinationproduct (Fig. 7(b)) is CIT, it can be postulated that the sequence between 70.7 and 70.8 map units is C-C(C,T)-C (if it is indeed only 5 base-pairs), since it would need to accommodate both an HpaII and an Hue111 cleavage site as well as the depurination product. The decanucleotide sequence, G-G 4 C-C-(C,T)-C 4 C-G-G fulfils these requirements. From depurination studies (see Results and Fig. 7) HaeIII-17 has been found to contain a long run of thymidines (probably 8). Should these be located near the HpaII cleavage site, it is possible that cleavage with HpaII results in the exposure of a region of the DNA that can easily become denatured; the presence of single-stranded regions in a DNA would certainly be expected to affect the mobility of a fragment during electrophoresis. Whatever the explanation for the unusual electrophoretic migration of HueIII-17, this phenomenon has been generally observed, not only with the fragment from this region in wild-type DNAs but also with similar fragments from polyoma defective (Griffin & Fried, 1975; Lund et al., 1977a,b) and variant (Fried & Griffin, unpublished results) species. It has proved to be a useful marker for the presence of viral sequences from 69.5 to 70.7 map units in polyoma variant and defective species. Moreover, it suggests the possibility of an easily denaturable site near the origin of DNA replication (see Fig. 3) which may serve a function in replication. Although clearly the restriction fragment HaeIII-17 has at least one A+T-rich region (since it contains a sequence of about eight thymidines) this is not a region in which T4 gene 32 protein has been found to bind in polyoma. DNA (Monjardino & James, 1975; Yaniv et al., 1975). (b) Analysis

of depurinution products of polyoma DNA

In addition to the restriction endonuclease cleavage analyses, depurination analyses of polyoma DNA and restriction endonuclease fragments have been carried out, some of which are shown (Figs 4 to 9). The depurination fingerprint of the whole viral (A2) DNA is shown in Figure 4. (It should be noted that the resulting fingerprints of oligomers represent the sequences of the entire molecule, since every pyrimidine tract from one strand of DNA has its complementary purine tract on the other strand.) It can be seen at a glance that polyoma DNA has many large unique (uni-molar yield) oligopyrimidines. These large oligomers have been placed into specific regions on the physical map of polyoma DNA by comparing fingerprints of the individual HpuII restriction fragments (Figs 4, 5, 7, 8 and 9) with that of the full-length viral DNA (Fig. 4). The presence of unique oligoppimidines allows depurination to be used as a diagnostic probe of sequences present in polyoma variant and defective species (Griffin & Fried, 1975; Lund et al., 1977a,b). As the areas of the DNA being examined become ever smaller (e.g. see fingerprints of Hue111 fragments in Fig. 7), the fingerprints become increasingly more useful as diagnostic tools, since even smaller oligomers become significant markers. For example, a depurination study has been made on fragments obtained from the region around the origin of DNA replication (mapped at 71f3 map units, Griffin et al., 1974). Figure 7(a) to (c) shows the fingerprints of fragments from 69.5 to 72.5 map units; this includes the HpuII 3-5 junction. (In order to show that this method is a reliable one, a fingerprint of the region 72.5 to 78-4 is also shown (Fig. 7(d)).)

FINE

Figure

STRUCTURE

OF POLYOMA

VIRUS

DNA

469

7(b) to (d) should be capable of superimposition to give the fingerprint of HpaII-6 (Fig. 6) ; this can indeed be seen to be the case. Fingerprints of two of the Hue111 fragments which precede 69.5 map units (HueIII-14 and 15, Fig. 3) are given by Lund et al. (1977aJ). Besides the unique oligomers referred to above (one in Fig. 7(a) and one in Fig. 7(c)). there appears to be a pentamer with composition T,C!, which is present in two (a,nti possibly all three) of the fragments which cover the region from 69.5 to 72.4 map units (T,C, is missing from a number of polyoma restriction fragments of comparable size). Whether t#his represents a sequence repeat or is entirely a matter of coincidence has not yet been determined. It seems worth noting that a 27 nucleotide long palindrome has been found by Subramanian et al. (19763) in the region that precedes and includes the 5’ end of the early messenger RNA of SV40. This palindromic sequence contains three HaeIII restriction sites (the polyoma DNA 69.5 to 72.5 map units contains two HaeIII sites) and if depurinated would give two copies of the pentamer T,C,. The palindrome is found in SV40 DNA in the region bet,ween a sequence which contains eight, consecutive thymidines (discussed below) and the start of the coding sequences for the early mRNA. From a rough consideration of the depurination fingerprints of polyoma virus and SV40 DPiAs, it would appear that these two viruses which show so much similarity in the organi&ion of their genomes (Fried & Griffin, 1977) show very little similarity at the molecular level. Heteroduplex studies by Ferguson & Davis (1975) showed only limited homology between the two viral DNAs. The fingerprints of polyoma virus and SV40 DNAs (Figs 4 and 5(a)) also indicate little homology. What homology there is apparently resides in polyoma DNA within the HpaII-1 restriction fragment (that region of the genome which has been shown at least in part to code for VPI, the majol capsid protein of polyoma virus (Smith et al., 1975). When the fingerprint of SV40 is can in fact be compared with that of polyoma HpaII-1 (Fig. 5(b)) certain similarities seen. Whether they are of significance remains to be seen. The results from the depurination studies on SV40 (Fig. 5(a)) are in agreement with some of the sequence studies on that DNA. Subramanian et al. (1976a) by studying t’ranscript,ion products of XV40 DNA found 12 stretches of six or more deoxyadenylic acids. The depurination fingerprint shows SV40 DNA to contain several copies of the complementary hexa- and heptathymidylic acids, and at least one copy of octathymidylic acid. B,y contrast, depurination of polyoma DNA (Fig. 4) appears to generate at most two copies of hexathymidylic acid, none of hepta- and only one of octathymidylic acid. (All studies suggest that SV40 DNA is more A+T-rich than polyoma DNA; see Tooze (1973).) The most interesting point is that in XV40 the octathymidylic acid has been found to occur within sequences immediately preceding those which speci@ the 5’ end of the early messenger RNA and in the region which conta.ins the origin of DNA replication. In polyoma DNA, the octathymidylic acid has been found in HaeIII-17 (see Fig. 7(a)), the Ha&I fragment which maps at the HpdI 3-5 junction (see Fig. 3). The data of Kamen & Shure (1976) suggest that the 5’ end of ear.ly messenger RNA is coded for by sequences in HpaII-5 near the HpalI 3-S junction. Therefore, the octathymidylic acid sequence in both polyoma virus and S\‘40 DNAs comes from the same region of the genome. In conclusion, the fine structure mapping of polyoma DNA as presented here has been very useful for probing variability among polyoma strains and analysing variantIs. and defective species (see Lund et al., 1977a,h: Griffin $ Fried. 1975;

470

B. E.

GRIFFIN

Fried & Griffin, 1977). It has allowed tentative correlations to be made between certain regions of the DNAs of both polyoma virus and SV40. It clearly indicates that the next stage for analysis and comparison must go beyond sequence patterns to the actual primary sequences. I thank Dr Sherman Weissman for allowing me to see his manuscripts of the sequences of SV40 fragment EcoRII-G before publication. Without these, some of the analogies drawn between polyoma virus and SV40 DNAs could not have been made. I also thank Miss Christine Barry for excellent technical assistance, and numerous friends and colleagues including Drs Rich Roberts, John Arrand, Bob Kamen, Bob Old, Nigel Brown and Mr Alan Robbins for gifts of restriction enzymes. I am grateful to Dr Jesse Summers for a discussion before publication of his results on restricting polyoma DNA with HueHI. Finally, I acknowledge the helpful interest shown in this work by my colleagues Dr Mike Fried and Dr Elsebet Lund. REFERENCES Barrell, B. G. (1971). Proc. Nucleic Acid Res. 2, 751-779. Brownlee, G. G. & Sanger, F. (1969). Eur. J. Biochem. 11, 395-399. Burton, K. & Petersen, G. B. (1960). Biochem. J. 75, 17-27. Burton, K., Lunt, M. R., Petersen, G. B. & Siebke, J. C. (1963). Cold Spring Harbor Sym. Quant. Biol. 28, 27-34. Crawford, L. V. & Robbins, A. K. (1976). J. Gen. l’irol. 31, 315-321. Crawford, L. V., Robbins, A. K. & Nicklin, P. M. (1974). J. Gen. F’iroZ. 25, 133-142. Danna, K. J. & Nathans, D. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 2913-2917. Danna, K. J., Sack, G. H. & Nathans, D. (1973). J. Mol. BioZ. 78, 363-376. Dhar, R., Subramanian, K. N., Pan, J. & Weissman, S. M. (1977). Proc. Nat. Acad. Sci., U.S.A. 14, 827-831. Dingman, C. W. & Peacock, A. C. (1968). Biochemistry, 7, 668-674. Eagle, H. (1959). Science, 130, 432-437. Ferguson, J. & Davis, R. W. (1975). J. Mol. Biol. 94, 135-149. Folk, W. R., Fishel, B. R. & Anderson, D. M. (1975). Virology, 64, 277-280. Fried, M. (1974). J. l’irol. 13, 9399946. Fried, M. & Griffin, B. E. (1977). In Advances in Cancer Research (Klein, G. & Weinhouse, S., eds), vol. 24, pp. 67-113, Academic Press, New York. Fried, M., Griffin, B. E., Lund, E. & Robberson, D. L. (1974). Cold Spring Harbor Symp. Quant.

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