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Restriction mapping of the DNA of the Streptomyces temperate phage øC31 and its derivatives
C,ene, 14 (1981) 183-194 Elsevie~/Noxth-HollandBiomedicalPress
183
Restriction mapping of the DNA of the Streptomyces temperate phage ~C31 and its derivatives
(Streptomyces coelicolor, DNA cloning vector; deletion and insertion derivatives; lambda Sa/PI fragments)
Keith F. Chater, Celia J. Bruton and Juan E. Suarez *
John InnesInstitute, NorwichNR4 7UH(U.K.) (Received February27th, 1981) (Accepted March12th, 1981)
SUMMARY DNA of ¢C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHl, SalPI (=Pstl) and Xhol; one for Xbal; three for Hpal; five for Oal and Kpnl; six for EcoRl; about 13 for Hindlll; about 14 for 8cll; and more than 15 for FspAl, Hg/A1, Sacl, SalGI and Smal. A complete map of 20 sites (Xbal, Hapl, Clal, Kpnl and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.
INTRODUCTION The generation of a restriction map of a phage DNA or a plasmid immediately opens up two major routes of potentially profitable investigation: the genetic functions of physically defined segments of the molecule may be identified, and realistic assess. ment of the potential of the molecule as a DNA cloning vector cam be made. The Streptomyces temperate phage ¢C31 is of real interest from both standpoints. Genetically, it is by far the best characterised Of $treptomyces phages (for a review, see Lomovskaya et ~1., 1980); a linear recombination map of temperature-sensitive mutations belonging to 20 complemer,tation groups has Abbreviation:kb, kilobasepairs. * Present address: Departmentof Microb~.ology,Universityof Oviedo(Spain).
been def'med (Chinenova and Lomovskaya, 1975), and a variety of mutants altered in the regulation of lysogeny have been studied (Lomovskaya et al., 1979). The phage is able to form plaques on S. coelicolor A3(2) derivatives "'cured" of an uncharacterised endogenous resistance mechanism, and lysogenises S. coelicolor A3(2) efficiently. This raises the possibility of genetically studying the interactions between the phage and its host, since S. coelicolor A3(2) is genetically an extremely well-characterised organism (Chater and Hopwood, 1981; Hopwood et al., 1973). The ability of ,pc31 to infect and lysogenise not only this genetically interesting strain, but also a fairly high proportion of other Streptomyces species, gives it particular potential as a DNA cloning vector endogenous to Streptomyces (Chater et al., 1981). Such vectors are needed not only for "academic" studies but also in the applied context of the fermentation industry, which is dependent in a large measure on
0378-II 19/81/0000-0000/$02-50 © Ek,evier/Northq-loHand Biomedical l~ess
184
Streptomyces products, and where wide host range would be an especially useful feature of a DNA cloning vector. (For a di~ussion of possfole industrial applications of DNA cloning in Streptomyces, see Hopwood and Chater, 1980). We have therefore analysed 0C31 DNA with restriction enzymes, as described in this paper. As a result of this study we have already been able, taking advantage of an efficient trar.sfection system (Suarez and Chater, 1980a), to introduce DNA of the Escherichia coli plasmid pBR322 (Bolivar et al., 1977) into a 0(;31 deletion mutant, generating a chimaeric bifunctional rep!icon able to exist as a plasmid in E. coli or as a phage in Streptomyces (Suarez and Chater, 1980b).
MATERIALS ANq) METHODS (a) Bacteria, phages, and extraction of DNA The host used for phage propagation was S. lividans 66 (Lomovskaya et al., 1972). The 0(331 derivatives
used are listed in Table I. The presence of the c l or ctsl mutations did not affect the restriction patterns, so these mutations are not referred to in the text. Conditions for phage propagation, assay, pumqcation, and DNA extraction using phenol, were as previously described (Chater and Wilde, 1976) except that the divalent cation concentrations used during phage propagation were adjusted to 8 mM Ca 2÷ and 10 mM
Mg2÷. (b) Restriction enzymes Commercially produced enzymes were as follows: EcoRl (Miles Laboratories or Boehringer), Oal, Smal and Hpal (Boehringer); Aval, 8amHl, Kpnl, Xbal and Hindlll (Bethesda Research Laboratories); and 8cll and Xhol (New Et~gland Bio Labs). FspAI and HgiAl were gifts of Dr. N.L. Brown. Sa/PI (an isoschizomer of Pstl; Chater, 1977; Carter et al., 1981), SalGl and Sacl were purified from their producing Streptomyces strains essentially b y the procedures of Chater and Wilde 0 9 7 6 ) for S a d , Bickle et al. (1977) for SaIGl, and Greene et al. (1979) for SalPl. All enzymes were used at 37°C in
TABLE ! Derivatives of oC31 used for restriction mapping ~C31 genotype
Physical changes in DNA relevant to restriction mapping a
Origin
c I (the source of "wild-type" DNA throughout)
None (wild-type)
Provided by N.D. Lomovskaya ("Norwich stock"; Lomovskaya et al., 1980)
ctsl AM b
1.72 kb deletion (AM)
Provided by N.D. Lomovskaya ("Moscow stock"; Lomovskaya et al., 1980)
ctsl ctsl ctsl ctsl
1.17 kb deletion (AT) c Loss of an EcoRI site (d12) c 1.43 kb deletion (A23) c i.09 kb deletion (A31) C
Obtained during attempted in vitro generation of deletions using EcoRl on ctsl AM DNA (Chater, 1980; I.E. Suarez, unpublished results)
ctsl AM A23::pBR322
1.43 kb deletion (A23) c 4.362 kb insertion (pBR322)
In vitro insertion of pBR322 into an EcoRI site ofcts AM A23 (Suarez and Chater, 1980b)
plJ500
1.43 kb deletion (A23) C 4.362 kb insertion (pBR322) Cohesive ends joined to give covalently closed circular molecule
AM A7 AMdl2 AM A23 AM A31
Plasmid DNA isolated after transformation of
E coli 803 reeA with 0C31 etsl AM A23:: pBR322 DNA (Suarez and Cha"ter, 1980b)
a See Figs. 6 and 7 for additional data. b ctsl is a mutation in the repressor gene c rendering the product thermosensitive. c For AM see the second entry.
185 reaction mixtures containing lOmM Tris" HC1 pH 7.4, lOmM MgCI2, 10 mM 2-mercaptoethanol and 50 ~ NaC1, except for Smal (20°C in 30 mM TrisHCl pH 9.0, 8 mM MgC12 and 80 mM KC1), HpaI (37"C in 10raM Tris-HCI 7.4, 10raM MgCl2, l0 mM 2-mercaptoethanol, 20 mM KCI) and BclI .(60°C in 10ram Tris-HCl pH 7.9, 10raM MgCl2, 10mM 2-mereaptoethanol, 60 mM KCI).
of banding patterns on agarose gels, and are subject to errors of up to 5% with fragments of 1 to 15 kb, and higher errors for smaller or larger fragments.
RESULTS (a) Suscept~ility of 01231 DNA to various restriction enzymes
(¢) Agarose gel eleetrophoresis Horizontal 1% agarose gels were ruv., stained and photographed as in Carter et al. (1980).
(d) Estimation of moleen!nr sizes Standards were provided by ~,cI857Sam7 DNA digested with EcoRl, Hindlll (fragment sizes given by Daniels et al., 1980) and SalPl (fragment sizes recalibrated after those of Chater, 1977, by A.G. Hepburn, personal communication; TablelI). The reciprocals of the electrophoretic mobilities of the standards from 0.5 to 12 kb gave straight lines when plotted against molecular size as suggested by Southern (1979). Fragments ot ¢C31 DNA generated by restriction enzymes are designated by letter, in descending size order: e.g. EeoRI-C is the third largest fragment. obtained by EcoRl digestion. It should be borne in mind that all the fragment sizes are based on analysis
The numbers of target sites in ¢C31 DNA for various restriction enzymes were as follows: (o) BamHl, Xhol, SalPI (=estI); (1) XbaI; (3) HpaI; (5) C/al, KpnI; (6) EcoRl; (13)HindllI; (14) Bell; (>15) SalG I, Sac l, SmaI , A va I, Hg/AI,, Fsp Al. The digestion products after treatment with the enzymes cutting at six or fewer sites are shown in Fig. 1 and Table Ill. The molecular sizes of all the
X DNA ~
,.,.:
Fragment
kb
Fragment
kb
Ab B C D E F
11.600 4.980 4.645 4.362 2.713 2.440 .2.368
1 J L M N O P
2.063 1.917 1.113 1.045 0.885 0.564 0.533
G, H
{2.368
._
~
A-~ l--.
,...,
,.-
=:
_ A=B,L
-- E-
F'.-' E,-.-, F,-,
x
A-.. A-- A-.A+B'A.-. e.--'
B I~._. B-. B_.
TABLE II Molecular sizes of fragments obtained by cleavage of ~.c1857Sam7 D N A by SalPI a
~"C 31 DNA
B--
C.--. C.-. D.-.
B.~
D,... E,...F'-"
C~=
F..-. 5._. E..-.
..-.
F
t...~
_ _ ,
a Recalibrated by A.G. Hepburn (personal communication) from the originalestimates of Chater (1977). b A band correspondingto 14.04 kb is usually also present in 7, $a/Pl digests;it consists of fragments A and F joined by the 7, cohesiveends.
,,
Fig. 1. Diagrammatic rel~esentatlon of banding patterns of ¢C31 D N A cleavedby variousrestriction enzymes,in agarose gel electxophoresb. No attempt b made to indicate relative band intensities. Fragments smaller than I kb (i.e. Kpnl-E and -F and Hpal-D: Table lid are not shown.
186 TABLE Ill Molecular sizes of restriction enzyme fragments of ¢£~31 wild-t3,pe DNA a ""
A B
Xbal
HpaI
Clal
Kpnl
EcoRl
27.09b 14.10b
28.28 b 10.82
22.83b 10.87 2.11 2.11 1.77 1.50
30.72 b
13.73 9.55 6.57 3.43 3.15 2.97 1.79
C
1.91
D E F G
0.18 b
3.94 3.64 2.00 0.69 0.20 b
°
-
iI
z
a Sizes (kb) are mean values from at least two.determinations. b Determined indirectly: see RESULTS.
EcoRI fragments could be obtained relatively easily (the other enzymes all gave one particularly large fragment, the size of which could not be accurately determined) so that their sum, 41.19 kb, was taken to be the size of the entire ¢C31 DNA molecule for the h itial stages of restriction mapping. (b) Derivation of an EcoRl, Xbal and Hpai map o f ~C31 DNA The DNA of ~C31 has cohesive ends (Sladkova et al., 1977). The fragments EcoRI.B and -F carry these ends. This is shown by their tendency to associate to give a fragment of the predicted molecular size (Chater, 1980) and by their complete replacement by a fragment of the latter size during propagation of a ¢C31 ::pBR322 chimaeric molecule (¢C3 i AMA23:: pBR322) as a plasm!d (pIJ500) in Esche.richia cvli (Suarez and Chater, 1980b). In two deletions (A7, A31) EcoRI-F was fused to EcoRI.C, with no change in other fragrner.:ts (Fig. 2). This placed EcoRI-C next to EcoRI-F, giving the EcoRI fragment sequence: B
Fig. 2. Agarose-gel electrophoresis of the DNA" of 4~31 derivatives after complete or partial digestion with EcoRl, Xbal and Xbal + EcoRI. Sa/PI digests of 7,c1857Sam7 served as controls.
partial digestion fragment of 8.70kb was present only with Norwich stock DNA, and one of 6.75 kb was present only with Moscow stock DNA. This was compati01e with an arrangement in which (coRIC (6.57 kb) or 42zxu (4.85 kb) and EcoRI-G (1.79 kb) were adjacent, giving the EcoRI fragment sequence: B-(A,D,E)-G-C-F.
TABLE IV Fragments (kb) obtained by partial digestion of 0C31 DNA with EcoRl a DNA used
Partially digested fragments smaller than EcoRI-B (9.55kb) Observed
Predictedb
Composition
Wild type
8.70 5.06
8.36 4.94
C+G E+G
AM c
7.85 6.75 5.09
7.82 6.64 4.94
CAM + F CAM + G E+G
a Sizes (kb) ate the mean of t w o determinations. b Predictions based on the arrangement of fragments deduced :n RESULTS and given in Fig. 6, and on the fragment sizes given in Table III. c AM has a deletion of 1.72 kb from fragment C (see Table I). The resulting fragment CAM has a size of 4.85 kb.
187
EcoRI-G and -E were shown to be adjacent because they were fused by a mutation (all 2) obtained during attempts to eliminate EcoRI fragments in vitro by partial EcoRI digestion followed by DNA ligase treatment and transfection. The fusion of the fragments was not accompanied by any obvious deleti.on of DNA (Fig. 2), so it is possible that a point mutation or very smr~l deletion involving the EcoRI site separating fragments G and E was induced during the in vitro treatment. Analysis with EcoRI alone did not enable us to orient the remaining two fragments, A and D: thus a -partial EcoRl fragment map was established as ~follows:
op.4
°
•<
Of[
<:1
.~
,~
t
B-(A,D)-E-G-C-F
Xbal cleaved OC31 DNA at a single site to give fragments Xbal-A (size too large for direct determination) and Xbal-B (14.10kb) XbaI-B was the same size irrespective of the presence or absence of the ~dVl deletion (Fig. 2), and was therefore located at the left end of the partial restriction map given above. In an EcoRI/Xbal double digest (Fig. 2), EcoRI-A (13.73 kb) was cleaved by Xbal to give fragments of 12.40kb and 1.33kb. This implies that XbaI-B (14.10kb) consists of 1.33kb of EcoRI-A plus a further 12.77 kb of DNA at the left end of the molecule. The latter amount of DNA requires that EcoRI-D (3.43 kb) be interposed between the left end EcoRl fragment, EcoRI.B (9.55 kb), and EcoRI A. Thus the following complete EcoRI restriction fragment map of 0C31 DNA was obtained: B-D-A-E-G-C-F The sizes of fragments obtained after partial EcoRI digestion of Norwich or Moscow stock DNA (Table IV) were entirely in agreement with this sequence. Three DNA fragments were observed after digestion with HpaI (Fig. 3), initially suggesting that there were o~y two HpaI sites. In Hpal/EcoRI double digests, however, three EcoRl fragments- B, E and G - were reduced in size (Fig. 3), suggesting three target sites. The third site, undetected in digests with HpaI alone, was close to the left end of the OC31 DNA, since its cleavage resulted in: loss of the band of 12.52 kb made up by annealing of the cohesive ends of EcoRI-B and -F, and usually seen in EcoRl
Fig. 3. Agaxose-gel electrophoresis of the DNA of OC31 derivatives after cleavage with Hpal and HpaI + EcoRI (see Fig. 2 for ?~controls).
digests; the detection of a faint new band of 3.10 kb, corresponding to joining of EcoRI-F to the newly detected HpaI-D by annealing of the cohesive ends; and a very slight reduction in the size of EcoRI-B. Cleavage by HpaI of the adjacent fragments
188
EcoRI-F and 43 suggested that Hpal-C overlapped these two fragments. The sizes of the resulting four Hpal/EcoRl fragments were used to obtain accurate positioning of the Hpal sites within the EcoRl fragments. The largest (1.90 kb) was evidently the leftmc st, a. it was bigger than EcoR143 (1.79 kb), and too big to be part of HpaI.C (1.91 kb) when added to an:/ of the otaer three fragments. The two smallest fragments (1.12 kb and 0.82 kb) apparently made up EcoR143 (1.79 kb), and the remaining unassigned fragment (1.26 kb) therefore represented the HpalC/,:.coRI-E overlap. By subtracting this value (1.26 kb) from Hpal-C (1.91 kb), a predicted size of 0.65 kb for the Hpal-C/EcoR143 overlap was obtained. The 0.82 kb fragment observed was closest to this value. The p:edi:ted distance, based on the EcoRI map, from the ~'~*most of the Hpal sites to the right end of the o(?-I .L~A molecule (10.65 kb) was in good agreement ~_ the observed size of HpaI-B (10.82 kb). Moi:~ =~ HpaI-B was reduced to 9.19 kb in DNA that c~ .a~ned the AM deletion (1.73 kb), previously mapr in a region proposed to be within Hpal-B (Fig. 3) (c) Derivation o' , Kpnl restriction map of0C3i DNA Ideally it sh,,~Ad have been possible to recognise three bands pr~'.,~'~t on gels in less than equimolar amounts ruing @~31 DNA completely digested with KpnI, correspon ling to the t~o end fragments and their fusioI~ product formed t,y hydrogen bonding between the cohesive ends. In practice we could see only two such bands, the intensities of which varied inversely in relation to each other (Fig. 4, arrows). This was most easily explicable by assuming that the larger of the two (3.80 kb) was formed by hydrogen bonding between one of the phage DNA's cohesive ends carried by the smaller (KpnI-C, 3.64 kb) and the second cohesive end carried by a fragment (Kpnl-F) too small to be detected in our agarose gel system. In agreement with this, the 3.80 kb band was present in, and Kp~dC was absent from, Kpnl-digested plJ500, the plasmid form of ~C31Z~A23::pBR322, both fragments being present in the phage form. The internal fragment Kpnl-B (3.94kb) was shown to overlap EcoRIC and -F because deletions A7 and A31, which fused the latter fragments, eliminated Kpnl-B, with smaller fragments of 2.77 kb and 2.85 kb, respectively, being observed (Fig. 4).
G
cg t.,
,.;
,..; N
N
N
N
N
N
N
Fig. 4. Agarose-gcl electrophoresis of the DNA of 0C31 derivatives after cleavage with Kpnl or Kpnl + Xbai. Most tracks show traces of partial digest fragments: these tracks have been labelled as "partial" where they are particularly conspicuou~ (~e Fig. 2 for ~. control).
This location for Kpnl-B eliminated the possibility that Kpnl-C (3.64 kb) could carry the right-hand end of the molecule (there would be insufficient room for this), and therefore indicated that Kpnl.C was located at the left an4 Kpnl.F (0.20 kb) at the right-hand end of the molecule. It was possible to deduce that Kpnl.D (2.00 kb) abutted onto KpnI.C. This followed from the finding from Kpnl +Xbal double digests (Fig. 4) that the single Xball site was located 8.59 kb from the left end of Kpnl-A, thus locating the latter Kpnl site about 5.51 kb from the left cohesive end carried by KpnI-C (3.64kb). The space intervening between KpnI-C and Kpnl-A (about 1.87 kb) could satisfactorily accommodate only Kpnl-D (2.00 kb) of the two remaining unmapped fragments (the other being KpnI-E: 0.69 kb). The question of whether KpnI-E was located to the left or to the right of Kpnl-B (the only possible
189 locations) was resolved by the finding that the deletion A23 removed KpnI-E (Fig. 4). Since Kpnl-B included the right hand end of EcoRI-C, and A23 was internal to EcoRI-C, Kpn~-E was evidently located to the left of Kpnl-B. The further finding that Kpnl-B was also absent from A23 DNA indicated that the whole of Kpnl-E was included within the deleted DNA, so that Kpnl-B had become fused to KpnI-A giving a very large fragment not resolved from the wild-type Kpnl-A in our conditions. This information was sufficient to complete the Kpnl fragment map as follows:
(d) Derivation of a C/al restriction map of qC31 DNA The ClaI restriction map was derived largely by the use of deletion and insertion mutants and the results with each of these (illustrated in Fig. 5) were as follows. In AM DNA, C/aI-D (one of two fragments of 2.11 kb found with wild-type DNA) was replaced by one of 0.38 kb. AM (a 1.72 kb deletion) had previously been located in the KpnI-A/EcoRI.C overlap, 31.7 to 36.5 kb from the left end of the whole q~C31 DNA molecule.
C-D-A-E-B-F This map was confirmed both by analysis of Kpnl + EcoRI double digests of DNA of wild-type ~C31 and its deletion and insertion derivatives AM, AMA23 and AMA23::pBR322 (Table VII) and by the analysis of fragments obtained after partial Kpnl digestion of the same set of DNAs (Table V): the observed sizes of all the fragments obtained were in close agreement with those predicted from the Kpnl and EcoRI maps.
¢x3
°I
"1
4-
•
<3
•
,<
TABLE V Sizes (kb) of partially digested fragments obtained by incomplete digestion of qC31 DNA with Kpnl a Partially digested fragments of less than 12 kb Observed
Predicted b
Composition
10.73 9.87
10.47 9.78
B+C+ D+ E + F B+ C + D + F
8.58
8.47
7.84
7.78
B+C + F
5.85
5.84
C+D+ F
B+C+E+ F
5.65
5.64
C+ D
4.78 4.56
4.83 4.63
B+E+ F B+E
4.10
4.14
B+F
a The same values were obtained with both wild-type and AM DNAs. b Prediction based on the arrangement of fragments deduced
in RESULTS and given in Fig. 6, and on the fragment sizes given in Table III.
Fig. 5. Agatose-gel electrophoresis o f the DNA of 0C31 derivatives after cleavage with C/aI, C/al + Xbal or Cla!+Hpal
(see Fig. 2 for 7~controls).
190 AM~23 DNA differed from AM DNA in the loss of C/aI-C (2.11 kb) and C/aI-F (1.50 kb) and the occurrence of a new fragment of 2.25 kb. This was consistent with the fusion of the two fragments by the A23 (1.43 kb) deletion, and located C/alE and -F to the fight of Oal-D. The combined length C/alC plus -F (3.61 kb) was too short to f'dl the shortest poss~le interval (about 4 ~ kb) between Oal-D and the right end of the full length molecule, so we concluded that at least one more fragment lay in this interval. Of the remaining three unmapped fragments only C/aI-E (1.77 kb) was small enough to be accommodated there. This gave the partial Oal fragment map:
(one still too large for accurate size determination and one of 6.32 kb). The fmding that pBR322 was inserted within C/aI-A resolved the orientation of C/aI-A and -13, giving the following partial C/al fragment map: B-A-D-(F,C)-E The relative orientation of C/al fragments F and C was obtained by Clal/EcoRI double digestion (Table VII) which resulted in the loss of C/aI-C but not of C/aI-F; and by Clal/Kpnl double digestion, as a result of which C/aI-F was lost but not C/aI-C. These observations were compatible only with the following C/al fragment map:
(B,A)-D-(F,C)-E. B-A-D-F--C-E AMA23::pBR322 DNA carries pBR322 inserted at a known EcoRI site (between EcoRI fragments C and G), with the single C~I site of pBR322 (Sutcliffe, 1979) 25 base pairs from the left end of the inserted plasmid. It differed from AMA23 DNA in the loss of C/aI-A and its replacement by two smaller fragments
TABLE VI Fragments (kb) obtained by partial digestion of 0C31 DNA with Clal DNA used
Wild type
~M
fragmentation patterns that were entkely consistent with this C/aI map. In addition, the two fragments C/aI-B and -E supposed to carry the cohesive ends of the phage were invariably present in less than equimolar amounts compared with the other fragments, and a fragment of 13.6 kb (the sum of C/aI-B and -E) increased in intensity as C/aI-B and -E decreased.
Partially digested fragments smaller than Clal-B (10.79 kb) DISCUSSION
Observed
Predicted a
4.03
7.78 5.89 5.67 4.02
3.62
3.78
D+ F +C+E D+ F +C F+C+E C+E .C+F ~D + F
-
6.06 5.67 4.16 4.02 2.06
l) AM + F + C + F+C+E DAM + F + C C+E D AM + F
4.64 4.25 2.74
DAM + ( F - C ) A 2 3 + (F - C)A23 + E DAM + (F - C)A23
-
4.03 AM A23
Analysis of C/al partial digests (Table VI) and of
CI~I + Kpnl, Oal + EcoRI, Oal + Xbal and Clal + Hpal double digests (Fig. 5 and Table VII) gave
4.16 -
Compositiona
The combined maps of cleavage sites for EcoRI, Clal, Kpnl, Hpal and Xbal for ¢C31 DNA are given in
E
E
a Ba.~d cn the arrangement and sizes of fragments and deletions given in Fig. 6 and Tables I and VIII.
Fig. 6, and maps of the DNAs of several deletion and insertion derivatives are given in Fig. 7. Table VIII gives our present "best estimates" for each interval of the map, used in drawing the combined map in Fig. 6. In a preliminary account of the EcoRI map of ~ 3 1 (Lomovskaya et al., 1980) the restriction map was oriented with respect to the denaturation and heteroduplex maps obtained by Sladkova et aL (1977; 1979). With the addition of the KpnI and C/aI maps it has become apparent that the middle of the molecule is relatively low in hexanucleotide restriction enzyme target sites containing four A:T and two G:C base pairs. This is consistent with the
191 TABLE VII Sizes (kb) of fragments obtained by digestion of 0C31 DNA or OC31 AM DNA with pairs of restriction enzymes
EcoRi + Kpnl
EcoRI + ClaI
Observed size
Presumed origin a
Predicted size b
Observed size
14.04 4.81 d"
EcoRI-A EcoRI-C.~ KpnI-A J
13.75
3.98 3.78 c " 3.57 3.42 3.13 3.00 e
EcoRI-B}
4.76
Clal + KpnI Presumed origin a
Predicted size b
Observed size
Presumed origin a
Predicted size b
13.89
EcoRI-A
11.60 c
13.75 11.49
16.75 f 5.24
C/aI-A C/aI-B .
23.63
EcoRI-B} C/aI-E
9.59
EeoRI-B
9.59
5.18
3.88 c
KpnI-A ) Kpnl-C K~nI-F y
3.82
KpnI-A
3.98
KpnI.C} KpnI-F
3.82
3.25
EcoRI-E
3.17
3.69
KpnI-C
3.61
KpnI-C
3.61 3.32 3.17
2.12 d
C/aI-D
2.11
2.13 d
C/aI-D
2.11
2.12
2.12
2.13
CIaI-C
2.12
EcoRI-D EcoRI-E
EcoRI-C AM } Kpnl-A EcoRI-F} KpnI-B
3.04
1.88
EcoRI-D} ClaI-A ClaI-E
2.75
1.80
EcoRI-G
1.79
. .
1.90
1.99
KpnI-D
2.00
2.00
KpnI-D
2.00
1.80
EcoRI-C} CIaI-A
1.80
0.85
1.80 1.18
EcoRI-G EcoRI-C.~ KpnI-B ~
1.79
1.49
C/aI-F
1.66
0.66
Clal-E KpnI-B }. ClaI-A KpnI-A } KpnI-E
1.18
1.20
C/aI-B
"
1.20
0.38 e
CIaI.DAM
0.39
O. lO
Kpni-E
0.69
1.06
EcoRI-C ~
1.06
not seen not seen
0.21
1.06
EcoRI-F } Clal.C
1.06
Kpnl-F CIaI-F } KpnI-B
0.38 e
C/aI-DAM
0.39
2.78
EcoRI-D)
C/aI-C
not seen
Kpnl.F
0.21
"
1.69
1.69 0.85 0.69
0.12
a Each fragment is either bounded by homologous sites, in which case it is identical to the single digest fragment listed, or by heterologous sites, in which case it forms the length of DNA in common between the two fragments listed. b Based cn the map co-ordinates given in Fig. 6 and Table VIII. c Contains both ends of the 0C31 molecule joined by their cohesive ends. d Seen only with wild-type DNA. e Seen only with AM DNA. f Size estimates are very inaccurate with such large fragments.
occurrence in the homologous part of the denaturation map of the longest high melting point segment of the molecule - i.e. that richest in G:C base pairs. All viable deletion mutants so far examined lack segments from EcoRI.C, often extending into EcoRI. F (except where the deletion is accompanied by clearplaque phenotype, when the segment lost is close to the centre of the linear molecule, within EcoRI-A; Lomovskaya et al., 1980). Fortunately, the EcoRI C-F region is well marked with restriction sites, which has facilitated the physical mapping of further dele-
tions (W. Springer, K.F. Chater and C J . Bruton, unpublished data). The absence o f deletions from o:her regions o f the map might reflect any of several cir.: ,mstances: in particular, these other regions may virtually entirely comprise genetic information essential for plaque formation (as would be consistent with the finding that in 19 out o f 20 complementation groups found for tehaperature-sensitive ¢C31 mutants the mutation mapped to the left o f the c gene, which is almost in the centre of the physical map); or the mechanisms(s) b y which deletions are formed in 0C31
192
1
3
I 1
4
. I,, ,i
56
78
II
I I
10 11 13 1/,
9
I
II
I 12
2
/i
8
D
9-590 13 10-790
r. . . . L
i
15 171920 21
I
Iii
I
II 1618
t,1.195kb
I 22
A
! ,
,
A 22.625 A 30.765
EcoRI D F 12;110[g~E 2.1C20[1~95
........
A 28-39.0
0-180
I
0-690-'E B 10~5
0
B
ClaI 05
39B25 [
Hpal
A
l~ZtO,
KpnI
XbaI
26.955
Fig. 6. Restriction map of OC31 wild-type DNA. The combined sequence of restriction enzyme target sites obtained by superimposing the separate maps was entkely confirmed by electrophoretic analysis following digestion by pairwise combinations of the enzymes {Table VII). The map intervals are the "best estimates" given in Table VIII.
cos witdtype I I
K I
K I..
E C I 1
E Xb J I
Hp
~H
cos ! i
tc 1
K 1
E C Ii
E Xb I [
'
E HpE I I I
cos
E HpE ,i I I
K
K
E C
E Xb
E HpE
I
I
I I
I I
I
K
K
E C
E Xb
[
[
I [
I I
E HpE I I [
|
AHA23: "pBR~22 |
Hp
I
I
K
E C
EXb
I
I
I I
I I
E HpE
I
cos
CC K E C K II I I I l 39.~75kb Ii I. KC cos
E
(C EC
K
Ilil
]1 38050kb cos
£ I
Hp
K
II
£ I
I I!1. Hp
Hp
cos
C K EC K I I I I I r.l.19Skb KC
Hp
Hp
tos AM?a7~
I
C I
Hp
Hp
~H~za.I
[ ,
EB
I,_ I I , II !1 Hp C
CC K C K II I,~L.J, 3e.305kb / I
K
cos
PE il
E EC IIII
K
1142.412kb cos
Fig. 7. Restriction maps of ~C31 deaivativea. Restriction enzyme target sites are abbreviated as follows: B = B a m m , C = C1al, E = EcoRI, Hp = Hpal; K = KpnI;P = Patl/Sa/Pl; Xb = Xbal. The cohesive ends of the molecular are labelled "cos".
193 TABLE VIII
REFERENCES
Best estimates for intervals between restriction enzyme target sites in 0C31 DNA a Restriction fragment b
HpaI-D KpnI-C-HpoI-A KpnI-D EcoRI-B-KpnI-A ClaI-B-EcoRI.D EcoRI-D-CIaI-A XbaI-B-EcoRI-A EcoRI-A-XbaI-A HpaI-A-EcoRI-E EcoRI-E-HpaI-C HpaI-C-EcoRI-G EcoRI-G-HpaI.B ClaI-A-EcoRI-C C/aI-D Kpnl-A-ClaI-F Kpnl-E ClaI-F-KpnI-B EcoRI-C-CiaI-C ClaI.C-EcoRI-F CluI-E-KpnI-F Kpnl-F
Bracketing target sites c
Best estimate
1, 2 2, 3 3, 4 4, 5 5, 6 6, 7 7, 8 8, 9 9, 10 10, 11 11, 12 12, 13 1.,, 14 14, 15 15, 16 16, 17 17, 18 18, 19 19, 20 20, 21 21, 22
0.180 3.430 2.000 3.980 1.200 2.120 1.330 12.420 1.910 1.260 0.720 1.065 1.800 2.110 0.850 0.690 O.115 d 1.060 1.060 1.690 0.205 d
(kb)
Sum 41.195 a Determined arbitrarily by averaging and accomodating independent estimates of map intervals. b Overlap between fragments indicated by giving left-most fragment first. c Numbering as in Fig. 6. d Obtained by subtraction.
DNA may be such that certain regions of the DNA are preferred substrates for deletion events. It is not yet clear whether the single Xbal site is in an essential region of the DNA, though this seems probable considering the high density of essential genes in the left half of the map.
ACKNOWLEDGMENTS We thank David Hopwood for critically reading the manuscript. J.E.S., on leave from the University of Oviedo, Spain, was the recipient of a grant from the Vicente Canada Blanch Foundation.
Bickle; T.A., Pirotta, V. and Imber, R.: A simple, genert.1 procedure for purifying restriction endonucleases. Nucl. Acids Res. 4 (1977) 2561-2572. Bolivar, F., Rodriguez, R., Greene, PJ., Betlach, M., Heyneker, H.L., Boyer, H.W., Crosa, J. and Falkow, S- Constmction and characterisation of new cloning vehicles. A multipurpose cloning system. Gene 2 (1977) 95-113. Carter, J.A., (:hater, K.F., Bruton, C.J. and Brown, N.L: A comly~ison of DNA cleavage by the restriction enzymes SalPl and Ps~I. Nucl. Acids Res. 8 (1980) 4943-4954. Chater, K.F.: A site-specific endodeoxyribonuclease from Streptornyces albus CMI 52766 sharing site-specificity with P~ovidencia stuartii endonuclease PstI. Nucl. Acids Res. 4 (1977) 1989-1998. Chater, K.F.: Actinophage DNA. Dev. Ind. Micmbiol. 21 (1980) 65-74. Chater, K.F., Bruton, C.J., Suarez, J.E. and Springer, W.: Streptomyces phages and their applications in DNA cloning, in Schlessinger, D. (Ed.), Microbiology - 1981. American Society for Microbiology, Washington DC, in press. Chater, K.F. and Hopwood, D.A.: Streptomyces genetics, in Goodfellow, M., Mordarski, M. and Williams, S.T. (Eds.), Biology of the Actinomycetes. Academic Press, London, 1981, in press. Chater, K.F. and Wilde, L.C.: Restriction of a bacteriophage of Streptomyces albus G involving endonuclease Sail. J. Bacteriol. 128 (1976) 644-650. Chinenova, T.A. and Lomovskaya, N.D.: Temperaturesensitive mutants of a~inophage ~ 3 1 of Streptomyces coellcolor A3(2). Genelika 11 (1975) 132-141. Daniels, D.L,, De Wet, J.R. and Blattner, F.R.: New map of bacteriophage lambda DNA. J. Vtrol. 33 (1980) 390-400. Greene, P.J., Heyneker, H.L., Bolivar, F:, Rodriguez, R.L., Betlach, M.C., Covarrubias, A.A., Backman, K., Russell, DJ., Tait, R. and Boyer, H.: A general method for the purification of restriction enzymes. Nucl. Acids Res. 5 (1978) 2373-2380. Hopwood, D.A., Chater, K.F., Dowding, J.E. and Vivlan, A.: Recent advances in 5treptomyces coelicolor genetics. Bacteriol. Rev. 37 (1973) 371-405. Hopwood, D. ~,. and Chater, K.F.: Fresh approaches to antibiotic production. Phil. Trans. Roy. Soc. Lond. B 290 0980) 313-328. Lomovskaya, N.D., Chater, K.F. and Mkrtumlan, N.M.: Genetics and molecular biology of Streptomyces bacteriophages. Microbiel. Rev. 44 (1980) 206-229. Lomovskaya, ~.D., Mk~tumian, N~., Gostimskaya, N.L. and Danilenko, V.N.: Characterisation of temperate actinophage 0C31 isolated from Streptomyces coelico/or A3(2). J. Virol. 9 (1972) 258-262. Lomovskaya, N.D., Voeykova, T.A., Sladkova, I.A., Chinenova, T.A., Mkrtumlan, N.M. and Slavinskaya, EN.: Genetic relationship between actinomycetes and antinophages, in Sebek, O.K. and Laskin, A.I. (Eds.), Genetics of Industrial Microorganisms. Proceedings of the Third International Symposium on Genetics of Industrial Micro-
194 organisms. American Society for Microbiology, Washington, DC, 1979, pp. 141-146. Sladkova, I.A., Lomow,k,aya, N.D. and Chinenova, T.A.: The structure and size of the genome of actinopbage 0C31 of Szreptomyces coelicolor A3(2). Genetika 13 (1977) 342-344. Sladkova, l,S,., Chinenova, T.A., Lomovskaya, N.D. and $ikx~mian, N.M.: Genetic cimmcteristics and genome struct~e of Streptomycex codicolor A3(2) actinophages. Genet~a 11 (1979) 1953-1962. Southern, E.M.: Measurement of DNA length by gel electro° phozesis..Mmlyt. Biochem. 100 (1979) 319-323.
Suarez, J.E. and Chater, K.F.: Polyethylene glycol-assisted transfection of Streptomyces protoplasts. J. Bacteriol. 142 (1980a) 8-14. Sua~ez, J.E. and Chater, K.F.: DNA cloning in Streptomyces: a bifunctional replicon comprising pBR322 inserted into a Streptomyces phage. Nature (London) 286 (1980b) 527-529. Sutcliffe, J.G.: Complete nucleotide sequence of the EscheW. ¢hia ¢oU plasmkl pBR322. Cold Spring Harbor Syrup. Quant. Biol. 43 (1979) 77-90. Communicated by Z. H~ade~-nd.
183
Restriction mapping of the DNA of the Streptomyces temperate phage ~C31 and its derivatives
(Streptomyces coelicolor, DNA cloning vector; deletion and insertion derivatives; lambda Sa/PI fragments)
Keith F. Chater, Celia J. Bruton and Juan E. Suarez *
John InnesInstitute, NorwichNR4 7UH(U.K.) (Received February27th, 1981) (Accepted March12th, 1981)
SUMMARY DNA of ¢C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHl, SalPI (=Pstl) and Xhol; one for Xbal; three for Hpal; five for Oal and Kpnl; six for EcoRl; about 13 for Hindlll; about 14 for 8cll; and more than 15 for FspAl, Hg/A1, Sacl, SalGI and Smal. A complete map of 20 sites (Xbal, Hapl, Clal, Kpnl and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.
INTRODUCTION The generation of a restriction map of a phage DNA or a plasmid immediately opens up two major routes of potentially profitable investigation: the genetic functions of physically defined segments of the molecule may be identified, and realistic assess. ment of the potential of the molecule as a DNA cloning vector cam be made. The Streptomyces temperate phage ¢C31 is of real interest from both standpoints. Genetically, it is by far the best characterised Of $treptomyces phages (for a review, see Lomovskaya et ~1., 1980); a linear recombination map of temperature-sensitive mutations belonging to 20 complemer,tation groups has Abbreviation:kb, kilobasepairs. * Present address: Departmentof Microb~.ology,Universityof Oviedo(Spain).
been def'med (Chinenova and Lomovskaya, 1975), and a variety of mutants altered in the regulation of lysogeny have been studied (Lomovskaya et al., 1979). The phage is able to form plaques on S. coelicolor A3(2) derivatives "'cured" of an uncharacterised endogenous resistance mechanism, and lysogenises S. coelicolor A3(2) efficiently. This raises the possibility of genetically studying the interactions between the phage and its host, since S. coelicolor A3(2) is genetically an extremely well-characterised organism (Chater and Hopwood, 1981; Hopwood et al., 1973). The ability of ,pc31 to infect and lysogenise not only this genetically interesting strain, but also a fairly high proportion of other Streptomyces species, gives it particular potential as a DNA cloning vector endogenous to Streptomyces (Chater et al., 1981). Such vectors are needed not only for "academic" studies but also in the applied context of the fermentation industry, which is dependent in a large measure on
0378-II 19/81/0000-0000/$02-50 © Ek,evier/Northq-loHand Biomedical l~ess
184
Streptomyces products, and where wide host range would be an especially useful feature of a DNA cloning vector. (For a di~ussion of possfole industrial applications of DNA cloning in Streptomyces, see Hopwood and Chater, 1980). We have therefore analysed 0C31 DNA with restriction enzymes, as described in this paper. As a result of this study we have already been able, taking advantage of an efficient trar.sfection system (Suarez and Chater, 1980a), to introduce DNA of the Escherichia coli plasmid pBR322 (Bolivar et al., 1977) into a 0(;31 deletion mutant, generating a chimaeric bifunctional rep!icon able to exist as a plasmid in E. coli or as a phage in Streptomyces (Suarez and Chater, 1980b).
MATERIALS ANq) METHODS (a) Bacteria, phages, and extraction of DNA The host used for phage propagation was S. lividans 66 (Lomovskaya et al., 1972). The 0(331 derivatives
used are listed in Table I. The presence of the c l or ctsl mutations did not affect the restriction patterns, so these mutations are not referred to in the text. Conditions for phage propagation, assay, pumqcation, and DNA extraction using phenol, were as previously described (Chater and Wilde, 1976) except that the divalent cation concentrations used during phage propagation were adjusted to 8 mM Ca 2÷ and 10 mM
Mg2÷. (b) Restriction enzymes Commercially produced enzymes were as follows: EcoRl (Miles Laboratories or Boehringer), Oal, Smal and Hpal (Boehringer); Aval, 8amHl, Kpnl, Xbal and Hindlll (Bethesda Research Laboratories); and 8cll and Xhol (New Et~gland Bio Labs). FspAI and HgiAl were gifts of Dr. N.L. Brown. Sa/PI (an isoschizomer of Pstl; Chater, 1977; Carter et al., 1981), SalGl and Sacl were purified from their producing Streptomyces strains essentially b y the procedures of Chater and Wilde 0 9 7 6 ) for S a d , Bickle et al. (1977) for SaIGl, and Greene et al. (1979) for SalPl. All enzymes were used at 37°C in
TABLE ! Derivatives of oC31 used for restriction mapping ~C31 genotype
Physical changes in DNA relevant to restriction mapping a
Origin
c I (the source of "wild-type" DNA throughout)
None (wild-type)
Provided by N.D. Lomovskaya ("Norwich stock"; Lomovskaya et al., 1980)
ctsl AM b
1.72 kb deletion (AM)
Provided by N.D. Lomovskaya ("Moscow stock"; Lomovskaya et al., 1980)
ctsl ctsl ctsl ctsl
1.17 kb deletion (AT) c Loss of an EcoRI site (d12) c 1.43 kb deletion (A23) c i.09 kb deletion (A31) C
Obtained during attempted in vitro generation of deletions using EcoRl on ctsl AM DNA (Chater, 1980; I.E. Suarez, unpublished results)
ctsl AM A23::pBR322
1.43 kb deletion (A23) c 4.362 kb insertion (pBR322)
In vitro insertion of pBR322 into an EcoRI site ofcts AM A23 (Suarez and Chater, 1980b)
plJ500
1.43 kb deletion (A23) C 4.362 kb insertion (pBR322) Cohesive ends joined to give covalently closed circular molecule
AM A7 AMdl2 AM A23 AM A31
Plasmid DNA isolated after transformation of
E coli 803 reeA with 0C31 etsl AM A23:: pBR322 DNA (Suarez and Cha"ter, 1980b)
a See Figs. 6 and 7 for additional data. b ctsl is a mutation in the repressor gene c rendering the product thermosensitive. c For AM see the second entry.
185 reaction mixtures containing lOmM Tris" HC1 pH 7.4, lOmM MgCI2, 10 mM 2-mercaptoethanol and 50 ~ NaC1, except for Smal (20°C in 30 mM TrisHCl pH 9.0, 8 mM MgC12 and 80 mM KC1), HpaI (37"C in 10raM Tris-HCI 7.4, 10raM MgCl2, l0 mM 2-mercaptoethanol, 20 mM KCI) and BclI .(60°C in 10ram Tris-HCl pH 7.9, 10raM MgCl2, 10mM 2-mereaptoethanol, 60 mM KCI).
of banding patterns on agarose gels, and are subject to errors of up to 5% with fragments of 1 to 15 kb, and higher errors for smaller or larger fragments.
RESULTS (a) Suscept~ility of 01231 DNA to various restriction enzymes
(¢) Agarose gel eleetrophoresis Horizontal 1% agarose gels were ruv., stained and photographed as in Carter et al. (1980).
(d) Estimation of moleen!nr sizes Standards were provided by ~,cI857Sam7 DNA digested with EcoRl, Hindlll (fragment sizes given by Daniels et al., 1980) and SalPl (fragment sizes recalibrated after those of Chater, 1977, by A.G. Hepburn, personal communication; TablelI). The reciprocals of the electrophoretic mobilities of the standards from 0.5 to 12 kb gave straight lines when plotted against molecular size as suggested by Southern (1979). Fragments ot ¢C31 DNA generated by restriction enzymes are designated by letter, in descending size order: e.g. EeoRI-C is the third largest fragment. obtained by EcoRl digestion. It should be borne in mind that all the fragment sizes are based on analysis
The numbers of target sites in ¢C31 DNA for various restriction enzymes were as follows: (o) BamHl, Xhol, SalPI (=estI); (1) XbaI; (3) HpaI; (5) C/al, KpnI; (6) EcoRl; (13)HindllI; (14) Bell; (>15) SalG I, Sac l, SmaI , A va I, Hg/AI,, Fsp Al. The digestion products after treatment with the enzymes cutting at six or fewer sites are shown in Fig. 1 and Table Ill. The molecular sizes of all the
X DNA ~
,.,.:
Fragment
kb
Fragment
kb
Ab B C D E F
11.600 4.980 4.645 4.362 2.713 2.440 .2.368
1 J L M N O P
2.063 1.917 1.113 1.045 0.885 0.564 0.533
G, H
{2.368
._
~
A-~ l--.
,...,
,.-
=:
_ A=B,L
-- E-
F'.-' E,-.-, F,-,
x
A-.. A-- A-.A+B'A.-. e.--'
B I~._. B-. B_.
TABLE II Molecular sizes of fragments obtained by cleavage of ~.c1857Sam7 D N A by SalPI a
~"C 31 DNA
B--
C.--. C.-. D.-.
B.~
D,... E,...F'-"
C~=
F..-. 5._. E..-.
..-.
F
t...~
_ _ ,
a Recalibrated by A.G. Hepburn (personal communication) from the originalestimates of Chater (1977). b A band correspondingto 14.04 kb is usually also present in 7, $a/Pl digests;it consists of fragments A and F joined by the 7, cohesiveends.
,,
Fig. 1. Diagrammatic rel~esentatlon of banding patterns of ¢C31 D N A cleavedby variousrestriction enzymes,in agarose gel electxophoresb. No attempt b made to indicate relative band intensities. Fragments smaller than I kb (i.e. Kpnl-E and -F and Hpal-D: Table lid are not shown.
186 TABLE Ill Molecular sizes of restriction enzyme fragments of ¢£~31 wild-t3,pe DNA a ""
A B
Xbal
HpaI
Clal
Kpnl
EcoRl
27.09b 14.10b
28.28 b 10.82
22.83b 10.87 2.11 2.11 1.77 1.50
30.72 b
13.73 9.55 6.57 3.43 3.15 2.97 1.79
C
1.91
D E F G
0.18 b
3.94 3.64 2.00 0.69 0.20 b
°
-
iI
z
a Sizes (kb) are mean values from at least two.determinations. b Determined indirectly: see RESULTS.
EcoRI fragments could be obtained relatively easily (the other enzymes all gave one particularly large fragment, the size of which could not be accurately determined) so that their sum, 41.19 kb, was taken to be the size of the entire ¢C31 DNA molecule for the h itial stages of restriction mapping. (b) Derivation of an EcoRl, Xbal and Hpai map o f ~C31 DNA The DNA of ~C31 has cohesive ends (Sladkova et al., 1977). The fragments EcoRI.B and -F carry these ends. This is shown by their tendency to associate to give a fragment of the predicted molecular size (Chater, 1980) and by their complete replacement by a fragment of the latter size during propagation of a ¢C31 ::pBR322 chimaeric molecule (¢C3 i AMA23:: pBR322) as a plasm!d (pIJ500) in Esche.richia cvli (Suarez and Chater, 1980b). In two deletions (A7, A31) EcoRI-F was fused to EcoRI.C, with no change in other fragrner.:ts (Fig. 2). This placed EcoRI-C next to EcoRI-F, giving the EcoRI fragment sequence: B
Fig. 2. Agarose-gel electrophoresis of the DNA" of 4~31 derivatives after complete or partial digestion with EcoRl, Xbal and Xbal + EcoRI. Sa/PI digests of 7,c1857Sam7 served as controls.
partial digestion fragment of 8.70kb was present only with Norwich stock DNA, and one of 6.75 kb was present only with Moscow stock DNA. This was compati01e with an arrangement in which (coRIC (6.57 kb) or 42zxu (4.85 kb) and EcoRI-G (1.79 kb) were adjacent, giving the EcoRI fragment sequence: B-(A,D,E)-G-C-F.
TABLE IV Fragments (kb) obtained by partial digestion of 0C31 DNA with EcoRl a DNA used
Partially digested fragments smaller than EcoRI-B (9.55kb) Observed
Predictedb
Composition
Wild type
8.70 5.06
8.36 4.94
C+G E+G
AM c
7.85 6.75 5.09
7.82 6.64 4.94
CAM + F CAM + G E+G
a Sizes (kb) ate the mean of t w o determinations. b Predictions based on the arrangement of fragments deduced :n RESULTS and given in Fig. 6, and on the fragment sizes given in Table III. c AM has a deletion of 1.72 kb from fragment C (see Table I). The resulting fragment CAM has a size of 4.85 kb.
187
EcoRI-G and -E were shown to be adjacent because they were fused by a mutation (all 2) obtained during attempts to eliminate EcoRI fragments in vitro by partial EcoRI digestion followed by DNA ligase treatment and transfection. The fusion of the fragments was not accompanied by any obvious deleti.on of DNA (Fig. 2), so it is possible that a point mutation or very smr~l deletion involving the EcoRI site separating fragments G and E was induced during the in vitro treatment. Analysis with EcoRI alone did not enable us to orient the remaining two fragments, A and D: thus a -partial EcoRl fragment map was established as ~follows:
op.4
°
•<
Of[
<:1
.~
,~
t
B-(A,D)-E-G-C-F
Xbal cleaved OC31 DNA at a single site to give fragments Xbal-A (size too large for direct determination) and Xbal-B (14.10kb) XbaI-B was the same size irrespective of the presence or absence of the ~dVl deletion (Fig. 2), and was therefore located at the left end of the partial restriction map given above. In an EcoRI/Xbal double digest (Fig. 2), EcoRI-A (13.73 kb) was cleaved by Xbal to give fragments of 12.40kb and 1.33kb. This implies that XbaI-B (14.10kb) consists of 1.33kb of EcoRI-A plus a further 12.77 kb of DNA at the left end of the molecule. The latter amount of DNA requires that EcoRI-D (3.43 kb) be interposed between the left end EcoRl fragment, EcoRI.B (9.55 kb), and EcoRI A. Thus the following complete EcoRI restriction fragment map of 0C31 DNA was obtained: B-D-A-E-G-C-F The sizes of fragments obtained after partial EcoRI digestion of Norwich or Moscow stock DNA (Table IV) were entirely in agreement with this sequence. Three DNA fragments were observed after digestion with HpaI (Fig. 3), initially suggesting that there were o~y two HpaI sites. In Hpal/EcoRI double digests, however, three EcoRl fragments- B, E and G - were reduced in size (Fig. 3), suggesting three target sites. The third site, undetected in digests with HpaI alone, was close to the left end of the OC31 DNA, since its cleavage resulted in: loss of the band of 12.52 kb made up by annealing of the cohesive ends of EcoRI-B and -F, and usually seen in EcoRl
Fig. 3. Agaxose-gel electrophoresis of the DNA of OC31 derivatives after cleavage with Hpal and HpaI + EcoRI (see Fig. 2 for ?~controls).
digests; the detection of a faint new band of 3.10 kb, corresponding to joining of EcoRI-F to the newly detected HpaI-D by annealing of the cohesive ends; and a very slight reduction in the size of EcoRI-B. Cleavage by HpaI of the adjacent fragments
188
EcoRI-F and 43 suggested that Hpal-C overlapped these two fragments. The sizes of the resulting four Hpal/EcoRl fragments were used to obtain accurate positioning of the Hpal sites within the EcoRl fragments. The largest (1.90 kb) was evidently the leftmc st, a. it was bigger than EcoR143 (1.79 kb), and too big to be part of HpaI.C (1.91 kb) when added to an:/ of the otaer three fragments. The two smallest fragments (1.12 kb and 0.82 kb) apparently made up EcoR143 (1.79 kb), and the remaining unassigned fragment (1.26 kb) therefore represented the HpalC/,:.coRI-E overlap. By subtracting this value (1.26 kb) from Hpal-C (1.91 kb), a predicted size of 0.65 kb for the Hpal-C/EcoR143 overlap was obtained. The 0.82 kb fragment observed was closest to this value. The p:edi:ted distance, based on the EcoRI map, from the ~'~*most of the Hpal sites to the right end of the o(?-I .L~A molecule (10.65 kb) was in good agreement ~_ the observed size of HpaI-B (10.82 kb). Moi:~ =~ HpaI-B was reduced to 9.19 kb in DNA that c~ .a~ned the AM deletion (1.73 kb), previously mapr in a region proposed to be within Hpal-B (Fig. 3) (c) Derivation o' , Kpnl restriction map of0C3i DNA Ideally it sh,,~Ad have been possible to recognise three bands pr~'.,~'~t on gels in less than equimolar amounts ruing @~31 DNA completely digested with KpnI, correspon ling to the t~o end fragments and their fusioI~ product formed t,y hydrogen bonding between the cohesive ends. In practice we could see only two such bands, the intensities of which varied inversely in relation to each other (Fig. 4, arrows). This was most easily explicable by assuming that the larger of the two (3.80 kb) was formed by hydrogen bonding between one of the phage DNA's cohesive ends carried by the smaller (KpnI-C, 3.64 kb) and the second cohesive end carried by a fragment (Kpnl-F) too small to be detected in our agarose gel system. In agreement with this, the 3.80 kb band was present in, and Kp~dC was absent from, Kpnl-digested plJ500, the plasmid form of ~C31Z~A23::pBR322, both fragments being present in the phage form. The internal fragment Kpnl-B (3.94kb) was shown to overlap EcoRIC and -F because deletions A7 and A31, which fused the latter fragments, eliminated Kpnl-B, with smaller fragments of 2.77 kb and 2.85 kb, respectively, being observed (Fig. 4).
G
cg t.,
,.;
,..; N
N
N
N
N
N
N
Fig. 4. Agarose-gcl electrophoresis of the DNA of 0C31 derivatives after cleavage with Kpnl or Kpnl + Xbai. Most tracks show traces of partial digest fragments: these tracks have been labelled as "partial" where they are particularly conspicuou~ (~e Fig. 2 for ~. control).
This location for Kpnl-B eliminated the possibility that Kpnl-C (3.64 kb) could carry the right-hand end of the molecule (there would be insufficient room for this), and therefore indicated that Kpnl.C was located at the left an4 Kpnl.F (0.20 kb) at the right-hand end of the molecule. It was possible to deduce that Kpnl.D (2.00 kb) abutted onto KpnI.C. This followed from the finding from Kpnl +Xbal double digests (Fig. 4) that the single Xball site was located 8.59 kb from the left end of Kpnl-A, thus locating the latter Kpnl site about 5.51 kb from the left cohesive end carried by KpnI-C (3.64kb). The space intervening between KpnI-C and Kpnl-A (about 1.87 kb) could satisfactorily accommodate only Kpnl-D (2.00 kb) of the two remaining unmapped fragments (the other being KpnI-E: 0.69 kb). The question of whether KpnI-E was located to the left or to the right of Kpnl-B (the only possible
189 locations) was resolved by the finding that the deletion A23 removed KpnI-E (Fig. 4). Since Kpnl-B included the right hand end of EcoRI-C, and A23 was internal to EcoRI-C, Kpn~-E was evidently located to the left of Kpnl-B. The further finding that Kpnl-B was also absent from A23 DNA indicated that the whole of Kpnl-E was included within the deleted DNA, so that Kpnl-B had become fused to KpnI-A giving a very large fragment not resolved from the wild-type Kpnl-A in our conditions. This information was sufficient to complete the Kpnl fragment map as follows:
(d) Derivation of a C/al restriction map of qC31 DNA The ClaI restriction map was derived largely by the use of deletion and insertion mutants and the results with each of these (illustrated in Fig. 5) were as follows. In AM DNA, C/aI-D (one of two fragments of 2.11 kb found with wild-type DNA) was replaced by one of 0.38 kb. AM (a 1.72 kb deletion) had previously been located in the KpnI-A/EcoRI.C overlap, 31.7 to 36.5 kb from the left end of the whole q~C31 DNA molecule.
C-D-A-E-B-F This map was confirmed both by analysis of Kpnl + EcoRI double digests of DNA of wild-type ~C31 and its deletion and insertion derivatives AM, AMA23 and AMA23::pBR322 (Table VII) and by the analysis of fragments obtained after partial Kpnl digestion of the same set of DNAs (Table V): the observed sizes of all the fragments obtained were in close agreement with those predicted from the Kpnl and EcoRI maps.
¢x3
°I
"1
4-
•
<3
•
,<
TABLE V Sizes (kb) of partially digested fragments obtained by incomplete digestion of qC31 DNA with Kpnl a Partially digested fragments of less than 12 kb Observed
Predicted b
Composition
10.73 9.87
10.47 9.78
B+C+ D+ E + F B+ C + D + F
8.58
8.47
7.84
7.78
B+C + F
5.85
5.84
C+D+ F
B+C+E+ F
5.65
5.64
C+ D
4.78 4.56
4.83 4.63
B+E+ F B+E
4.10
4.14
B+F
a The same values were obtained with both wild-type and AM DNAs. b Prediction based on the arrangement of fragments deduced
in RESULTS and given in Fig. 6, and on the fragment sizes given in Table III.
Fig. 5. Agatose-gel electrophoresis o f the DNA of 0C31 derivatives after cleavage with C/aI, C/al + Xbal or Cla!+Hpal
(see Fig. 2 for 7~controls).
190 AM~23 DNA differed from AM DNA in the loss of C/aI-C (2.11 kb) and C/aI-F (1.50 kb) and the occurrence of a new fragment of 2.25 kb. This was consistent with the fusion of the two fragments by the A23 (1.43 kb) deletion, and located C/alE and -F to the fight of Oal-D. The combined length C/alC plus -F (3.61 kb) was too short to f'dl the shortest poss~le interval (about 4 ~ kb) between Oal-D and the right end of the full length molecule, so we concluded that at least one more fragment lay in this interval. Of the remaining three unmapped fragments only C/aI-E (1.77 kb) was small enough to be accommodated there. This gave the partial Oal fragment map:
(one still too large for accurate size determination and one of 6.32 kb). The fmding that pBR322 was inserted within C/aI-A resolved the orientation of C/aI-A and -13, giving the following partial C/al fragment map: B-A-D-(F,C)-E The relative orientation of C/al fragments F and C was obtained by Clal/EcoRI double digestion (Table VII) which resulted in the loss of C/aI-C but not of C/aI-F; and by Clal/Kpnl double digestion, as a result of which C/aI-F was lost but not C/aI-C. These observations were compatible only with the following C/al fragment map:
(B,A)-D-(F,C)-E. B-A-D-F--C-E AMA23::pBR322 DNA carries pBR322 inserted at a known EcoRI site (between EcoRI fragments C and G), with the single C~I site of pBR322 (Sutcliffe, 1979) 25 base pairs from the left end of the inserted plasmid. It differed from AMA23 DNA in the loss of C/aI-A and its replacement by two smaller fragments
TABLE VI Fragments (kb) obtained by partial digestion of 0C31 DNA with Clal DNA used
Wild type
~M
fragmentation patterns that were entkely consistent with this C/aI map. In addition, the two fragments C/aI-B and -E supposed to carry the cohesive ends of the phage were invariably present in less than equimolar amounts compared with the other fragments, and a fragment of 13.6 kb (the sum of C/aI-B and -E) increased in intensity as C/aI-B and -E decreased.
Partially digested fragments smaller than Clal-B (10.79 kb) DISCUSSION
Observed
Predicted a
4.03
7.78 5.89 5.67 4.02
3.62
3.78
D+ F +C+E D+ F +C F+C+E C+E .C+F ~D + F
-
6.06 5.67 4.16 4.02 2.06
l) AM + F + C + F+C+E DAM + F + C C+E D AM + F
4.64 4.25 2.74
DAM + ( F - C ) A 2 3 + (F - C)A23 + E DAM + (F - C)A23
-
4.03 AM A23
Analysis of C/al partial digests (Table VI) and of
CI~I + Kpnl, Oal + EcoRI, Oal + Xbal and Clal + Hpal double digests (Fig. 5 and Table VII) gave
4.16 -
Compositiona
The combined maps of cleavage sites for EcoRI, Clal, Kpnl, Hpal and Xbal for ¢C31 DNA are given in
E
E
a Ba.~d cn the arrangement and sizes of fragments and deletions given in Fig. 6 and Tables I and VIII.
Fig. 6, and maps of the DNAs of several deletion and insertion derivatives are given in Fig. 7. Table VIII gives our present "best estimates" for each interval of the map, used in drawing the combined map in Fig. 6. In a preliminary account of the EcoRI map of ~ 3 1 (Lomovskaya et al., 1980) the restriction map was oriented with respect to the denaturation and heteroduplex maps obtained by Sladkova et aL (1977; 1979). With the addition of the KpnI and C/aI maps it has become apparent that the middle of the molecule is relatively low in hexanucleotide restriction enzyme target sites containing four A:T and two G:C base pairs. This is consistent with the
191 TABLE VII Sizes (kb) of fragments obtained by digestion of 0C31 DNA or OC31 AM DNA with pairs of restriction enzymes
EcoRi + Kpnl
EcoRI + ClaI
Observed size
Presumed origin a
Predicted size b
Observed size
14.04 4.81 d"
EcoRI-A EcoRI-C.~ KpnI-A J
13.75
3.98 3.78 c " 3.57 3.42 3.13 3.00 e
EcoRI-B}
4.76
Clal + KpnI Presumed origin a
Predicted size b
Observed size
Presumed origin a
Predicted size b
13.89
EcoRI-A
11.60 c
13.75 11.49
16.75 f 5.24
C/aI-A C/aI-B .
23.63
EcoRI-B} C/aI-E
9.59
EeoRI-B
9.59
5.18
3.88 c
KpnI-A ) Kpnl-C K~nI-F y
3.82
KpnI-A
3.98
KpnI.C} KpnI-F
3.82
3.25
EcoRI-E
3.17
3.69
KpnI-C
3.61
KpnI-C
3.61 3.32 3.17
2.12 d
C/aI-D
2.11
2.13 d
C/aI-D
2.11
2.12
2.12
2.13
CIaI-C
2.12
EcoRI-D EcoRI-E
EcoRI-C AM } Kpnl-A EcoRI-F} KpnI-B
3.04
1.88
EcoRI-D} ClaI-A ClaI-E
2.75
1.80
EcoRI-G
1.79
. .
1.90
1.99
KpnI-D
2.00
2.00
KpnI-D
2.00
1.80
EcoRI-C} CIaI-A
1.80
0.85
1.80 1.18
EcoRI-G EcoRI-C.~ KpnI-B ~
1.79
1.49
C/aI-F
1.66
0.66
Clal-E KpnI-B }. ClaI-A KpnI-A } KpnI-E
1.18
1.20
C/aI-B
"
1.20
0.38 e
CIaI.DAM
0.39
O. lO
Kpni-E
0.69
1.06
EcoRI-C ~
1.06
not seen not seen
0.21
1.06
EcoRI-F } Clal.C
1.06
Kpnl-F CIaI-F } KpnI-B
0.38 e
C/aI-DAM
0.39
2.78
EcoRI-D)
C/aI-C
not seen
Kpnl.F
0.21
"
1.69
1.69 0.85 0.69
0.12
a Each fragment is either bounded by homologous sites, in which case it is identical to the single digest fragment listed, or by heterologous sites, in which case it forms the length of DNA in common between the two fragments listed. b Based cn the map co-ordinates given in Fig. 6 and Table VIII. c Contains both ends of the 0C31 molecule joined by their cohesive ends. d Seen only with wild-type DNA. e Seen only with AM DNA. f Size estimates are very inaccurate with such large fragments.
occurrence in the homologous part of the denaturation map of the longest high melting point segment of the molecule - i.e. that richest in G:C base pairs. All viable deletion mutants so far examined lack segments from EcoRI.C, often extending into EcoRI. F (except where the deletion is accompanied by clearplaque phenotype, when the segment lost is close to the centre of the linear molecule, within EcoRI-A; Lomovskaya et al., 1980). Fortunately, the EcoRI C-F region is well marked with restriction sites, which has facilitated the physical mapping of further dele-
tions (W. Springer, K.F. Chater and C J . Bruton, unpublished data). The absence o f deletions from o:her regions o f the map might reflect any of several cir.: ,mstances: in particular, these other regions may virtually entirely comprise genetic information essential for plaque formation (as would be consistent with the finding that in 19 out o f 20 complementation groups found for tehaperature-sensitive ¢C31 mutants the mutation mapped to the left o f the c gene, which is almost in the centre of the physical map); or the mechanisms(s) b y which deletions are formed in 0C31
192
1
3
I 1
4
. I,, ,i
56
78
II
I I
10 11 13 1/,
9
I
II
I 12
2
/i
8
D
9-590 13 10-790
r. . . . L
i
15 171920 21
I
Iii
I
II 1618
t,1.195kb
I 22
A
! ,
,
A 22.625 A 30.765
EcoRI D F 12;110[g~E 2.1C20[1~95
........
A 28-39.0
0-180
I
0-690-'E B 10~5
0
B
ClaI 05
39B25 [
Hpal
A
l~ZtO,
KpnI
XbaI
26.955
Fig. 6. Restriction map of OC31 wild-type DNA. The combined sequence of restriction enzyme target sites obtained by superimposing the separate maps was entkely confirmed by electrophoretic analysis following digestion by pairwise combinations of the enzymes {Table VII). The map intervals are the "best estimates" given in Table VIII.
cos witdtype I I
K I
K I..
E C I 1
E Xb J I
Hp
~H
cos ! i
tc 1
K 1
E C Ii
E Xb I [
'
E HpE I I I
cos
E HpE ,i I I
K
K
E C
E Xb
E HpE
I
I
I I
I I
I
K
K
E C
E Xb
[
[
I [
I I
E HpE I I [
|
AHA23: "pBR~22 |
Hp
I
I
K
E C
EXb
I
I
I I
I I
E HpE
I
cos
CC K E C K II I I I l 39.~75kb Ii I. KC cos
E
(C EC
K
Ilil
]1 38050kb cos
£ I
Hp
K
II
£ I
I I!1. Hp
Hp
cos
C K EC K I I I I I r.l.19Skb KC
Hp
Hp
tos AM?a7~
I
C I
Hp
Hp
~H~za.I
[ ,
EB
I,_ I I , II !1 Hp C
CC K C K II I,~L.J, 3e.305kb / I
K
cos
PE il
E EC IIII
K
1142.412kb cos
Fig. 7. Restriction maps of ~C31 deaivativea. Restriction enzyme target sites are abbreviated as follows: B = B a m m , C = C1al, E = EcoRI, Hp = Hpal; K = KpnI;P = Patl/Sa/Pl; Xb = Xbal. The cohesive ends of the molecular are labelled "cos".
193 TABLE VIII
REFERENCES
Best estimates for intervals between restriction enzyme target sites in 0C31 DNA a Restriction fragment b
HpaI-D KpnI-C-HpoI-A KpnI-D EcoRI-B-KpnI-A ClaI-B-EcoRI.D EcoRI-D-CIaI-A XbaI-B-EcoRI-A EcoRI-A-XbaI-A HpaI-A-EcoRI-E EcoRI-E-HpaI-C HpaI-C-EcoRI-G EcoRI-G-HpaI.B ClaI-A-EcoRI-C C/aI-D Kpnl-A-ClaI-F Kpnl-E ClaI-F-KpnI-B EcoRI-C-CiaI-C ClaI.C-EcoRI-F CluI-E-KpnI-F Kpnl-F
Bracketing target sites c
Best estimate
1, 2 2, 3 3, 4 4, 5 5, 6 6, 7 7, 8 8, 9 9, 10 10, 11 11, 12 12, 13 1.,, 14 14, 15 15, 16 16, 17 17, 18 18, 19 19, 20 20, 21 21, 22
0.180 3.430 2.000 3.980 1.200 2.120 1.330 12.420 1.910 1.260 0.720 1.065 1.800 2.110 0.850 0.690 O.115 d 1.060 1.060 1.690 0.205 d
(kb)
Sum 41.195 a Determined arbitrarily by averaging and accomodating independent estimates of map intervals. b Overlap between fragments indicated by giving left-most fragment first. c Numbering as in Fig. 6. d Obtained by subtraction.
DNA may be such that certain regions of the DNA are preferred substrates for deletion events. It is not yet clear whether the single Xbal site is in an essential region of the DNA, though this seems probable considering the high density of essential genes in the left half of the map.
ACKNOWLEDGMENTS We thank David Hopwood for critically reading the manuscript. J.E.S., on leave from the University of Oviedo, Spain, was the recipient of a grant from the Vicente Canada Blanch Foundation.
Bickle; T.A., Pirotta, V. and Imber, R.: A simple, genert.1 procedure for purifying restriction endonucleases. Nucl. Acids Res. 4 (1977) 2561-2572. Bolivar, F., Rodriguez, R., Greene, PJ., Betlach, M., Heyneker, H.L., Boyer, H.W., Crosa, J. and Falkow, S- Constmction and characterisation of new cloning vehicles. A multipurpose cloning system. Gene 2 (1977) 95-113. Carter, J.A., (:hater, K.F., Bruton, C.J. and Brown, N.L: A comly~ison of DNA cleavage by the restriction enzymes SalPl and Ps~I. Nucl. Acids Res. 8 (1980) 4943-4954. Chater, K.F.: A site-specific endodeoxyribonuclease from Streptornyces albus CMI 52766 sharing site-specificity with P~ovidencia stuartii endonuclease PstI. Nucl. Acids Res. 4 (1977) 1989-1998. Chater, K.F.: Actinophage DNA. Dev. Ind. Micmbiol. 21 (1980) 65-74. Chater, K.F., Bruton, C.J., Suarez, J.E. and Springer, W.: Streptomyces phages and their applications in DNA cloning, in Schlessinger, D. (Ed.), Microbiology - 1981. American Society for Microbiology, Washington DC, in press. Chater, K.F. and Hopwood, D.A.: Streptomyces genetics, in Goodfellow, M., Mordarski, M. and Williams, S.T. (Eds.), Biology of the Actinomycetes. Academic Press, London, 1981, in press. Chater, K.F. and Wilde, L.C.: Restriction of a bacteriophage of Streptomyces albus G involving endonuclease Sail. J. Bacteriol. 128 (1976) 644-650. Chinenova, T.A. and Lomovskaya, N.D.: Temperaturesensitive mutants of a~inophage ~ 3 1 of Streptomyces coellcolor A3(2). Genelika 11 (1975) 132-141. Daniels, D.L,, De Wet, J.R. and Blattner, F.R.: New map of bacteriophage lambda DNA. J. Vtrol. 33 (1980) 390-400. Greene, P.J., Heyneker, H.L., Bolivar, F:, Rodriguez, R.L., Betlach, M.C., Covarrubias, A.A., Backman, K., Russell, DJ., Tait, R. and Boyer, H.: A general method for the purification of restriction enzymes. Nucl. Acids Res. 5 (1978) 2373-2380. Hopwood, D.A., Chater, K.F., Dowding, J.E. and Vivlan, A.: Recent advances in 5treptomyces coelicolor genetics. Bacteriol. Rev. 37 (1973) 371-405. Hopwood, D. ~,. and Chater, K.F.: Fresh approaches to antibiotic production. Phil. Trans. Roy. Soc. Lond. B 290 0980) 313-328. Lomovskaya, N.D., Chater, K.F. and Mkrtumlan, N.M.: Genetics and molecular biology of Streptomyces bacteriophages. Microbiel. Rev. 44 (1980) 206-229. Lomovskaya, ~.D., Mk~tumian, N~., Gostimskaya, N.L. and Danilenko, V.N.: Characterisation of temperate actinophage 0C31 isolated from Streptomyces coelico/or A3(2). J. Virol. 9 (1972) 258-262. Lomovskaya, N.D., Voeykova, T.A., Sladkova, I.A., Chinenova, T.A., Mkrtumlan, N.M. and Slavinskaya, EN.: Genetic relationship between actinomycetes and antinophages, in Sebek, O.K. and Laskin, A.I. (Eds.), Genetics of Industrial Microorganisms. Proceedings of the Third International Symposium on Genetics of Industrial Micro-
194 organisms. American Society for Microbiology, Washington, DC, 1979, pp. 141-146. Sladkova, I.A., Lomow,k,aya, N.D. and Chinenova, T.A.: The structure and size of the genome of actinopbage 0C31 of Szreptomyces coelicolor A3(2). Genetika 13 (1977) 342-344. Sladkova, l,S,., Chinenova, T.A., Lomovskaya, N.D. and $ikx~mian, N.M.: Genetic cimmcteristics and genome struct~e of Streptomycex codicolor A3(2) actinophages. Genet~a 11 (1979) 1953-1962. Southern, E.M.: Measurement of DNA length by gel electro° phozesis..Mmlyt. Biochem. 100 (1979) 319-323.
Suarez, J.E. and Chater, K.F.: Polyethylene glycol-assisted transfection of Streptomyces protoplasts. J. Bacteriol. 142 (1980a) 8-14. Sua~ez, J.E. and Chater, K.F.: DNA cloning in Streptomyces: a bifunctional replicon comprising pBR322 inserted into a Streptomyces phage. Nature (London) 286 (1980b) 527-529. Sutcliffe, J.G.: Complete nucleotide sequence of the EscheW. ¢hia ¢oU plasmkl pBR322. Cold Spring Harbor Syrup. Quant. Biol. 43 (1979) 77-90. Communicated by Z. H~ade~-nd.