Unusual base sequence arrangement in phage φ29 DNA

Gene, 5(1979) 1--7 © Elsevier/North-Holland Biomedical Press--Printed in The Netherlands

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UNUSUAL BASE SEQUENCE ARRANGEMENT IN PHAGE ~29 DNA

(Endonucleolytic cleavage; recombinant DNA; Bacillus subtilis; plasmid~29 hybrids; ~29 HindIII fragments) JUNETSU ITO and RICHARD J. ROBERTS* Department of Cellular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 92037 and*Cold Spring Harbor Laboratories, Cold Spring Harbor, N Y 11724 (U.S.A.) (Received and accepted October 6th, 1978) SUMMARY

Susceptibility of Bacillus subtiUs phage ~29 DNA to 34 different restriction endonucleases was determined. Three enzymes, BglI, XbaI and BstEII, were found to cleave ~29 DNA only once at specific sites. The sites of these single cleavages have been mapped. Thirteen enzymes did not cut ~b29 DNA. ~29 HindHI DNA fragments inserted into pBR313 plasmid and propagated in Escherichia coU, were resistant to these restriction endonucleases. This result suggests that the insusceptibility is due to the absence of the nucleotide sequences on $29 recognized by the enzymes, and not to the presence of modified nucleotides. The genome of ~29, one of the smaller Bacillus phages, is a linear, nonpermuted duplex DNA molecule of about 18 000 base pairs (Anderson and Mosharrafa, 1968; Ito et al., 1976). A novel feature of the ~29 DNA is its tight association with protein (terminal protein), which binds at the ends of the DNA molecule (Ortin et at., 1971; Harding and Ito, 1976; Harding et al., 1978; Ito, 1978; Salas et al., 1978). The terminal protein appears to be the product of ~29 gene-3 which has been shown to be essential for DNA replication (Talavera et al., 1972; Yanofsky et al., 1976; Harding et al., 1978; Salas et al., 1978). However, at present little is known about the mechanism of replication of the ~29 DNA. Restriction endonucleases have proved to be extremely useful tools in studies, not only of gene organization and transcription, but also for determination of the location of initiation and termination of DNA replication (Nathans and Smith, 1975; Roberts, 1976). Several restriction endonucleases have already been used in various studies on ~ 29 (Inciarte et al., 1976; Ito and Kaw-mura, 1976; Ito et al., 1976). Recently m-ny more restriction endonucleases have become available. During the

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course of our study of susceptibility of ~29 DNA to 34 different restriction endonucleases, we found that many of them do not cleave this viral DNA~ Our results suggest that this insusceptibility of the DNA is due to the absence of the nucleotide sequence of ~29 DNA recognized by these enzymes and not due to the presence of some modified nucleotides in ¢29 DNA. The preparation of the ¢29 DNA has been described previously (Ito et al., 1976). The restriction endonucleases were prepared as described previously (quoted in Roberts, 1976) or obtained from either New England Biolabs or from Bethesda Research Laboratories. Digestion of the ¢29 DNA with these enzymes and analysis of the digestion products by electrophoresis in agarose gels were generally performed according to methods that were previously described (Ito et al., 1975; Roberts, 1976). The results of the cleavage of ~29 DNA with 34 restriction endonucleases are summarized in Table I. For comparison, the number of cleavage sites for the~e enzymes on ), DNA is also given in this table. The numbers given in Table I are a minimum estimate, because there may exiRt flragments which are too sm~l! to be detected on agarose gels, and bands may consist of more than one DNA fragment. Three enzymes, BglI, XbaI and BstEII, cut ¢29 DNA only once. The cleavage sites for these enzymes Were located in relation to EcoRI cleavage fragment of ¢29 which has been mapped previously (Ito et al., 1976). Fig. 1 demonstrates the method of mapping the cleavage sites of BglI and Xbal on ~29 DNA. It can be seen from the figure that both BglI and XbaI cleave the EcoRI-A fragments, that XbaI cut the BglI-A fragment and that BglI enzyme cleaves the XbaI-B fragment. It was also found that BstEII enzyme cleaves the Bg/I-A, XbaI-A and EcoRI-B fragments (Table II). From these results, it was possible to locate the cleavage sites of these enzymes as shown in Fig.2. Fragments generated by these restriction endonucieases should be extremely useful for marker rescue experiments to correlate the genetic and physical maps of ¢29. Restriction endonucleases, MboII, AluI, SaeI, MboI, Hinfl, HincII, HphI, and TaqI, produce many small fragments. Thus, these enzymes may be useful to construct a fine physical map of the ~29 genome and for DNA sequencing. To our suprise, many restriction endonucleases do not cleave ~29 DNA, although these enzymes are active on ), DNA (Table I). Enzyme HaeIH, for example, which cleaves the double-stranded DNA sequence at 5-GG ~C-3' 3-CC,GG'5' (Middleton et al., 1972; Roberts et al., 1975) digests ~, DNA more than 50 times, but does not cleave ¢29 DNA at all. ¢29 DNA is expected to contain such a sequence approx. 23 times by chance, since this DNA contains 18 000 base pairs and 38% GC (Rubio et al., 1974). Therefore, we considered the possibility that ¢29 DNA c~ntains modified bases, such as 5-methylcytosine. To examine this possibility, ~29 DNA fragments were inserted into an E. coli plasmid, pBR313 (Bolivar et al., 1977) and the resulting plasmid-~29 hybrid DNA was grown in K coli and tested for susceptibility to the enzymes that did not cleave $29 DNA prepared from phage particles. The procedure for

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TABLE I A CATALOGUE OF CLEAVAGES OF ~29 DNA BY RESTRICTION ENDONUCLEASES Microorganisms

Moraxella boris Arthrobacter luteus Moraxella boris Haemophilus influenzae Rf Haemophilus parahaemolytieus Haemophilus influenzae Rc ~ermus aquaticus YTI Haemophilus gallinarum Haemophilus influenzae Rd Haemophilus haemolyticus 8treptomyces aehromogenes Haemophilus aegyptie~s Ilaemophilus parainfluenzae Haemophilus parainfluenzae Eseheriehia eoli RY13 £seheriehia coil RY13 Proteus vulgaris Xanthomor~s badrii Bacillus globigii Bacillus stearothermophilus £seheriehia eoU R245 Haemophilus aegyptieus BaeiUus amyloUquefaciens H Bacillus globigii Brevibaeterium albidum Anabacna variabilis Klebsiella pneumoniae ok8 Providenvia stuartii 164 Streptomyees aehromogenes Streptomyees aibus G Serratia marceseens St) Xanthomonus maimeearum Proteus vu ar Xanthomonas holeieola

Enzyme

MboII AluI MboI HinfI HphI HineH Taq I HgaI HindIII HhaI 8aeII HaeII HpaII HpaI EeoRI EeoR 'a PvuI XbaI BglI BstEII EeoRII HaeIII Barn HI BglII Ball AvaI KpnI PstI SaeI SalI Sinai XmaI PvuII XhoI

Sequence

GA~GA-~8bp ~G'CT "~ATC G'ANTC GGT~A~8bp G~Py" PuAC T:CGA G~.ACGC A*A~CTT GCG "~ CCGC" GGb PuGCGC~Py C~CGG GTr+AAC (]" AATTC *PuPuA~TPyPy TC~cATCGb TAGA

Number of cleavage sites ~29 DNA

~ DNAa

>30 > 25 > 20 > 20 >20 > 15 >15 14 13 12 > 10 9 8 7 4 >15 3 1 1 1

>50 > 50 > 50 > 50 >50 34 >50 > 50 6 > 50 3 >30 > 50 11 5 >10 2 1 22 11

o

~" uc G~GATCC AG , "G#TCT T~G" CCA C" PyCG~uG GGTAC ~Cb CTGCA~G G#GVr' Cb G" T~GAC

o 0 0 0 0 0 0

>50

o

2

0

2

C C'GC

0

3

C" C~GGG b CA~" CTG CT" CGAG

0 0 0

3 15 1

5 5 15 3 2 18

a Taken from Roberts (1976, 1978) b Unpublished remits of R.J. Roberts (Sael and SaelI), J.E. Gingeras and R.J. Roberts (PVul and/~uII) and R. Wu and R.J. Roberts (KpnI)(see Roberts, 1978).

d o n i n g ~29 D N A fragments in K eoli plasmids will be described elsewhere. Fig. 3 shows s o m e o f t h e results o b t a i n e d with f o u r h y b r i d plasmids w h i c h c o n t a i n ~ 2 9 HindIII-D, E, F a n d G fragment, respectively. It is clear t h a t although t h e plasmid D N A is sensitive t o HaeIII, t h e c l o n e d ~29 D N A segm e n t s are insensitive t o t h e e n z y m e . Similar results were o b t a i n e d w i t h t h e

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Fig.1. Electrophoresk of fragmant4 produced by the digestion of 429 DNA with restriction endonueleases. The digested DNAs (0.6--1,0.g) were applied to a 0.8% agarose slab gel (12 × 18 × 0.8 era). Electrophoresis was carried out as described previously (Ito et al., 1076). (1) ~, DNA digested with EcoRI; (2) ~29 DNA digested with Bg/I; (3) ~29 DNA digested with BglI and XbaI; (4) ~29 DNA digested with XbaI; (5) 429 DNA digested with Xbal and EeoRI; (6) 429 DNA digested with Bg/I and EeoRI; (7) ~29 DNA digested with EcoRI; (8) ;~ DNA digested with EeoRI. TABLE HA

SIZE OF FRAGMENTS GENERATED FROM 429 DNA BY XbaI, BglI, BstEII AND BY DOUBLE ENZYME D~GESTION Molecular weights wer~ estimated by comparing the relative eleetrophoretic mobilities of DNA fragments with mobilities of EeoRI fragments of the ~, DNA and 429 DNA. Fragments

XbaI

Bg/I

BstEII

XbaI +

XbaI +

BgU +

BglI

BstEH

BstEII

~.70 5.20

5.50 3.95

5.10 3.95

0.35

2.50

3.00

Molecular weight (10 s) A B C

6.40 5.50

7.00 5.20

8.05 3.95

T A B L E IIB R E D I G E S T I O N O F ~29 EcoRI D N A F R A G M E N T S

W I T H XbaI, 8gII,A N D BstEII

The figures represent molecular weight (.106) of each DNA fragment. Fragments

EeoRI

EeoRI + XbaI

EcoRI + BglI

EcoRI + BstEII

A B C D E F

6.10 3.60 1.30 0.62 0.40

5.50 3.60 1.30 0.62 0.60 0.40

5.20 3.60 1.30 0.94 0.60 0.40

6.20 2.00 1.65 1.30 0.60 0.40

ECORI JECORI

,"con1:

A

I I EcoR~

1

s

tt

!

!

I

4,500

I

+

t

SglZ Xbolr

Sole polrs

++1 °1

I

9,000

Sit I:IT

I

I

13,500

I

I

le, OOO

Fig.2. Location of the single cleavage sites of XbaI, BglI and BstEII in ~29 DNA. Distance in nudeotide pairs was computed from the molecular weights of each DNA fragment obtained from their electrophoretic mobflitias. The £¢oRI cleavage map is taken from Ito et al., (1976).

other restriction enzymes (BamHI, PstI, S a l I a n d S m a I ) , suggesting that insensitivity of the 029 DNA to many restriction endonucleases is not due to the presence of the modified nucleotides in the DNA. Instead, it appears t h a t the 029 DNA lacks the nucleotide sequences recognized by these enzymes. ACKNOWLEDGEMENTS The authors thanks Miss Phyllis Myers and Mrs. Gayle Mildner for their invaluable technical assistance and one author (J.I.) wishes to thank the generous hospitality of Drs. Thomas R. Broker and Louise T. Chow during his visits to the Cold Spring Harbor Laboratories. This research was supported by N.I.H. grants GM 25081 (J.I.), CA 13106 (R.J.R.), National Science Foundation Grant GB 43912 (R.J.R.) and the American Cancer Society grant NP-203A (J.I.). J.L is the recipient of a Faculty Research Award of the American Cancer Society.

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Fig.3. Susceptibility of the cloned ¢29 HindIII fragments to HaeIII endonuclease. Hybrid plesmids were constructed by ligating HindIH cleaved pBR313 DNA and ¢29 DNA. The ligated DNA was introduced into K yoU 8F8(C600 rk- inK- reeBC') by transformation. Since the single HindHI site on pBR313 DNA is within the tetracycline resistance gene, insertion of DNA fragments into the HindIII site results is loss of the tetracycline resistent phenotype (Bolivar et al., 1977). The apRtc s clones were selected (the detailed procedure for cloning ¢29 DNA fragments will be described elsewhere). These experiments were performed using standard procedures which conform to the NIH Guidelines. The hybrid plasmid DNAs were isolated according to the procedure of Bolivar ct al., (1977). (1) ¢29 DNA digested with HindIII; (2) pJI14 DNA digested with HindIII; (3) pJI15 DNA digested with HindIH; (4) pJI16 DNA digested with HindIH; (5) pJI17 DNA digested with HindIII; (6) pBR313 DNA digested with HindIII; (7) pBR313 DNA digested with HindIII and HaeHI; (8) pJI14 DNA digested with HindIII and HaelII; (9) pJI15 digested with HindIH and HaeHI; (10) pJ16 DNA digested with HindIII and HaeIII; (11) pJI17 DNA digested with HindIII ~nd HaeHI; (12) ¢29 DNA digested with HindIII.

REFERENCES

Anderson, D.L. and Mosharrafa, E.T., Physical and biological properties of phage ¢29 deoxyribonucleic acid, J. Viroi., ~?(1968) 1185--1190. Bolivar,~F., Rodriguez, R.L., Betlach, M.C. and Boyer, H.W., Construction and characterization of new cloning vehicles, I. Ampicillin-resistant derivatives of the plasmid pMB9, Gene, 2 (1977) 75--93.

Harding, N.E. and Ito, J., DNA replication of bacteriophage ~29: Isolation of a DNAprotein complex from Bacillus subtilis cells infected with wild type and with a suppressor-sensitive mutant, Virology, 73(1976) 389--401. Harding, N.E., Ito, J. and David, G.S., Identification of the protein firmly bound to the ends of bacteriophage ~29 DNA, Virology, 84 (1976) 279--292. Inciarte, M.R., Lazaro, J.M., Salas, M. and Vinuela, E., Physical map of bacteriophage ~29 DNA, Virology, 74 (1976) 314--323. Ito, J., Kawamura, F. and Yanofsky, S., Analysis of ~29 and ~15 genomes by bacterial restriction endonucleases, EcoRI and HpaI, Virology, 70 (1976) 37--51. Ito, J. and Kawamura, F., Use of restriction endonucleases in analyzing the genome of bacteriophages ~29 and ~15, in D. Schlessinger, (Ed.), Microbiology 1976, American Society for Microbiology, Washington, D.C., 1976, pp. 367--379. Ito, J., Bacteriophage ¢29 terminal protein: its association with the 5'-termini of the ~29 genome, J. Virol., in press. Middleton, J.H., Edgell, M.H. and Hutchison, C.A., III, Specific fragments of ~X174 deoxyribonucleic acid produced by a restriction enzyme from Haemophilus aegyptius, endonuclease Z, J. Virol., 10 (1972) 42--50. Nathans, D. and Smith, H.O., Restriction endonucleases in the analysis and restructuring of DNA molecules, Annu. Rev. Biochem., 44 (1975) 273--293. Ort~n, J., Vifiuela, E., Salas, M. and V~squez, C., DNA-protein complex in circular DNA from phage ~29, Nature New Biol., 234 (1971) 275--277. Roberts, R.J., Restriction endonucleases, CRC Crit. Rev. Biochem., 4 (1976) 123--164. Roberts, R.J., Restriction and modification enzymes and their recognition sequences, Gene, 4(1978) 183--193. Roberts, R.J., Breitmeyer, J.B., Tabachnik, N.F. and Myers, P.A., A second specific endonuclease from Haemophilus aegyptius, J. Mol. Biol., 91 (1975) 121--123. Rubio, V., Salas, M., Vifiue]a, E., Usobiaga, P., Saiz, J.L. and Llopis, J.F., Biophysical properties of bacteriophage ~ 29, Virology, 57 (1974) 112--121. Salas, M., Mellado, R.P., Vifiuela, E. and Sogo, J.M., Characterization of a protein covalently linked to the 5'-termini of the DNA of Bacillus subtilis phage ~29, J. Mol. Biol., 119 (1978) 269--291. Talavera, A., Salas, M., and Vifiuela, E., Temperature-sensitive mutants affected in DNA synthesis in phage ~29 of Bacillus sub#ilis., Eur. J. Biochem., 31 (1972) 367--371. YaL:ofsky, S., Kawamura, F. and Ito, J., Thermolabile transfecting DNA from temperature. sel.~sitive mutant of phage ~29, Nature, 259 (1976) 60--63. Communicated by F.E. Young.