Vol. 18, No. 2 Printed in U.SA.

JOURNAL OF VIROLOGY, May 1976, p. 793-798 Copyright D 1976 American Society for Microbiology

Restriction Endonuclease from Haemophilus gallinarum (HgaI) Cleaves Polyoma DNA at Four Locations KAZUO SHISHIDO AND PAUL BERG* Department of Biochemistry, Stanford University Medical Center, Stanford, California 94305 Received for publication 24 November 1975

A restriction endonuclease obtained from Haemophilus gallinarum (HgaI) cleaves polyoma DNA at four specific sites. Using the EcoRI, HindIII, and HpaII endonuclease restriction sites as reference, the four HgaI cleavage sites were mapped at 0.02, 0.14, 0.27, and 0.48 fractional lengths, clockwise, from the single EcoRI cleavage site. A restriction endonuclease (HgaI), isolated from Haemophilus gallinarum (8), makes multiple cleavages in a variety of bacteriophage DNAs (e.g., fdRF, T3, and X [8]), several bacterial plasmids (e.g., ColEl and pSC101 [our unpublished observations]), and adenovirus 2 DNA (R. J. Roberts, private communication). Our work has revealed that polyoma DNA is cleaved by HgaI endonuclease into four fragments and in this paper we report the physical map location of these cleavage sites. Polyoma DNA (large plaque strain [1]) was obtained from infected 3T6 mouse cells by standard methods for obtaining supercoiled DNA. HgaI endonuclease was isolated from Haemophilus gallinarum (provided by M. Takanami) according to a previously published method (8); EcoRI endonuclease, prepared according to Greene et al. (2), was a gift from M-T. Hsu; HindIII endonuclease was purified by a modification of Smith and Wilcox's procedure (7) and provided by S. Goff; HpaII endonuclease was isolated according to Sharp et al. (6) and given to us by T. Shenk. Enzyme digestions of polyoma DNA: (i) For digestion ofHgal, the DNA (0.4 ,ug) was incubated for 6 h at 37 C with 0.04 ,g of enzyme in 20 ,ul of buffer (10 mM Tris, pH 7.5, 7 mM MgCl2, 20 mM NaCl, 7 mM 2-mercaptoethanol). Partial digestions were carried out under identical conditions with less enzyme for shorter times at 25 C. (ii) EcoRI-DNA (0.4 ug) was digested for 30 min at 37 C with 0.10 jig of enzyme in 20 ,l of buffer (100 mM Tris, pH 7.5, 10 mM MgCl2). (ii) HindIII-DNA (0.4 ,ug) was digested for 2 h at 37 C with 0.05 ,ug of enzyme in 20 ,ul of buffer (10 mM Tris, pH 7.4, 60 mM NaCl, 6 mM MgCl2, and 1 mM dithiothreitol). (iv) HpaII-DNA (0.4 ,ug) was digested for a total of 6 h at 37 C with 0.01 4g of enzyme added at the beginning and 0.01 ,g after 3 h. The buffer (20 ,ul) was the same as for HgaI reactions except

with 2 ,ug of added gelatin. (v) After digestion of the DNA (0.4 jig) with HgaI (see above), 2.5 ,ul of 1 M Tris, pH 7.5, 2 ,ul of 50 mM MgCl2, and 0.10 ,g of EcoRI were added and the incubation continued for 30 min at 37 C. (vi) After digestion of the DNA (0.4 ,ug) for 4 h at 37 C with HgaI in 20 ,u of the HindIII digestion buffer containing 2 jig of gelatin, 0.05 ,g of HindIII enzyme was added and the reaction continued for 2 h more. Cleavage by HgaI in the HindIII digestion mixture containing gelatin was as efficient as with the conditions shown in (i). (vii) HgaI together with HpaIIDNA (0.4 ,ug) was digested with 0.04 ,ug of HgaI and 0.02 ,ug of HpaJ for 6 h at 37 C in 20 gl of the HgaI digestion buffer containing 2 ,ug of gelatin; the HpaII was added in half quantities at the start and after 3 h. Gel electrophoresis. Agarose gels (1.2% and/ or 2.5%, 6 mm in diameter, 150-mm and/or 250mm long) were prepared and electrophoresed in Tris-borate buffer (89 mM Tris-OH, 89 mM boric acid, 2.5 mM EDTA, pH 8.2 (6) (different conditions are described in the legends to the figures). The DNA bands were stained with ethidium bromide, visualized using a short wavelength UV light, and then photographed using a Vivitar orange (02) filter and Polaroid black and white Land Pack film type 105. HgaI cleaves polyoma DNA into four major fragments. A limit digest of polyoma DNA with HgaI restriction endonuclease contains four major fragments, HgaI-A to HgaI-D (Fig. la or b). With excess enzyme and longer incubation times the quantity of fragment HgaI-D occasionally decreases and a faster migrating fragment designated HgaI-D' appears (Fig. la). Whether HgaI-D is converted to HgaI-D' by the same enzyme that produces the other cleavages or by a contaminating activity needs to be examined further. Table 1 summarizes the size, expressed in

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cleavage, two fragments (0.56 and 0.44) produced by HindIII cleavages, and seven fragments (0.27, 0.21, 0.17, 0.13, 0.08, 0.07, and 0.05) generated by HpaII endonuclease (3). When the DNA is only partially digested, there is produced, beside the four final products, six additional fragments designated HgaI1 to HgaI-6 (Fig. ic). Their sizes, expressed in polyoma fractional length, are also presented in -I Table 1. Each of the incompletely digested fragments can be accounted for as one containing two or A -i- 2 more of the fragments produced in the limit - 3 digest. For example, the length of HgaI-1 is A accounted for by the lengths HgaI-A+B+C ----4 best or D; HgaI-2 is very likely a partial digestion product containing fragments HgaI-A+B or 5 possibly HgaI-A+C+D; the length of HgaI-3 is best accounted for by the lengths of HgaI-A+C or D; HgaI-4 (though not obvious in this electro-6 phorogram was frequently detected in other partial digests) most likely contains fragments -B HgaI-B+C+D; HgaI-5 is either composed of BHgaI-B+C or HgaI-B+D; and HgaI-6 is very likely HgaI-C +D. From the occurrence of these fragments the most reasonable inference for the -c order of the HgaI endonuclease-generated fragments on the polyoma DNA map is a circular C-- D permutation of ABCD or ABDC. DLocation of the HgaI cleavage sites on the polyoma DNA map. Polyoma DNA contains a single EcoRI endonuclease cleavage site (3) which has been arbitrarily assigned the 0/1.0 coordinate of its physical map. To determine which of the HgaI fragments contains the EcoRI cleavage site, polyoma DNA was digested sequentially with HgaI and EcoRI endonucleases (compare the gel patterns Fig. 2A, a and b). Although the relative mobilities of HgaI-A to D (and D') were not appreciably changed, there was invariably evidence of a FIG. 1. Agarose gel electrophoresis of the frag- new fragment, about 0.02 fractional length, apments produced in limit and incomplete digests of pearing after EcoRI endonuclease digestion. By polyoma DNA with HgaI endonuclease. (a) DNA using electrophoresis conditions that better re(0.4 Mg) was completely digested as described. (b) I-

b'(I)

-L

Im

D'-

DNA (0.4 pg) was digested with 0.015 pg of HgaI for 6 h at 37 C. (c) DNA (0.8 ,ug) was digested with 0.005 pg of HgaI in 40 Al of buffer for 45 min at 25 C. In reaction a and b electrophoresis was carried out in 1.2% agarose gel at 25 V for 16 h. At the end of reaction c, electrophoresis was in 1.2% agarose gel at 150 V for 3 h.

TABLE 1. Molecular lengths offragments produced in limit and incomplete digests ofpolyoma DNA with

HgaI endonuclease Limit digest

Fragment

polyoma fractional lengths, of the four major (and the minor) fragments produced in a limit digest with HgaI. These were deduced from their electrophoretic mobilities using as molecular length standards full length linear polyoma DNA produced by EcoRI endonuclease

HgaI-A -B

-C -D -D'

Polyoma fractional length

0.54 0.21 0.13 0.12 0.10

Incomplete digest Fragment

HgaI-1 -2 -3 -4 -5 -6

Polyoma fractional length

0.89 0.75 0.66 0.46 0.34 0.25

VOL. 18, 1976

NOTES

A

B

(a) (b) (c)

795

() (b) (c)

-A -B

-C -D -D

FIG. 2. (Right) Fragments ofpolyoma DNA generated by co-digestion with HgaI and EcoRI and /or HgaI and HindIlf endonucleases. (a) Fragments generated by sequential digestion with HgaI and EcoRI endonucleases. (b) Fragments generated by digestion with HgaI endonuclease alone. (c) Fragments generated by sequential digestion with HgaI and HindIll endonucleases. After the digestions (see text, Methods), electrophoresis was carried out in 2.5% agarose gels (6-mm diameter and 150-mm length) at 150 V for3 h. (left) Size ofpolyoma DNA HgaI-A fragment after digestion with EcoRI orHindIlf endonucleases. (a) Digest with HgaI followed by EcoRI endonuclease. (b) Digest with HgaI endonuclease only. (c) Digest with HgaI followed by HindIlf endonuclease. After each digestion, a HindIII digest of polyoma DNA was added to provide internal length standards and then the electrophoresis was carried out in 1.2% agarose gels at 50 V for 15 h.

solve the larger fragments, HgaI-A was judged to be the source of the additional small fragment (compare the gel patterns of Fig. 2B, a and b); the increased mobility of fragment HgaI-A afterEcoRI endonuclease digestion corresponds to a shortening of about 0.02 fractional length. Thus, one end of the HgaI-A fragment contains the EcoRI cleavage site. HindIII cleaves polyoma DNA twice, at map

positions 0.01 and 0.45, to yield two fragments 0.44 and 0.56 polyoma fractional length (3). Sequential cleavage of polyoma DNA by HgaI and HindIII yields a new fragment of about 0.02 to 0.03 fractional length and fragment HgaI-B is shortened by an equivalent amount (Fig. 2A, c). Thus, the EcoRI restriction site is at one end of HgaI-A and the HindIII restriction site at

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NOTES

0.46 occurs at one end ofHgaI-B. This, and the should be cleaved nearly in half to yield new partial digestion results, suggest an arrange- fragments 0.06 to 0.07 fractional length; bement of HgaI fragments shown in the inner cause of the comigration ofHpaI-C and HpaII-4 ring of Fig. 3 with the order of fragments HgaI- this is difficult to discern directly, but the unusually large yield of fragments of 0.06 to 0.07 C and HgaI-D being as indicated or reversed. Which of the two arrangements was correct fractional length is consistent with that assigncould be resolved by examining the products ment. Fragment HpaII-1 should disappear and formed by co-digestion of the DNA with HgaI be replaced by a fragment having about the and HpaII endonuclease. HpaII endonuclease same length of HgaI-B and one of 0.06 fracmakes eight cleavages in polyoma DNA at the tional length. HpaII-2 is the precursor of fragmap locations indicated in the outer ring of Fig. ments having 0.03 and 0.06 fractional length 3 (3). If the order of HgaI fragments is as shown and, of course, one of 0.12, the length of HgaIin Fig. 3, fragments HpaII-3, 4, 5, 7, and 8 and D. Thus, all fragments can be accounted for on HgaI-D would be expected to remain intact the basis of the map positions assigned to the after co-digestion with the two enzymes; this cleavage sites for both enzymes. Does HgaI endonuclease cleavage produce expectation is confirmed by the gel pattern of the doubly digested DNA (Fig. 4b compared cohesive ends? The existence of complementary with the mixture of singly digested DNA shown or "cohesive" ends on restriction endonucleasein Fig. 4a). Similarly, HpaII-1, 2 and possibly 6 generated fragments can often be detected by as well as HgaI-A, C and possibly B should the formation of covalently closed circles after disappear from the double digest and be re- incubation with DNA ligase (5). Digestion of placed by smaller fragments. For example the polyoma DNA with HgaI endonuclease (to proHgaI-A segment is cleaved to HpaII-3, 4, 5, 7 duce a limit digest as described above) followed and 8, plus new fragments of about 0.03 and by incubation for 48 h with DNA ligase at 10 C, 0.06 fractional length. But fragment HgaI-C yielded no circular molecules (

Restriction endonuclease from Haemophilus gallinarum (HgaI) cleaves polyoma DNA at four locations.

Vol. 18, No. 2 Printed in U.SA. JOURNAL OF VIROLOGY, May 1976, p. 793-798 Copyright D 1976 American Society for Microbiology Restriction Endonucleas...
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