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AvaII and BglI restriction maps of bacteriophage Mu
VIROLOGY 126, 563-575 (1983)
Avail and Bgll Restriction Maps of Bacteriophage Mu CARL F. MARRS 1 AND MARTHA M. HOWE 2 Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received August 23, 1982; accepted January 25, 1983 The AvaII a n d BglI r e s t r i c t i o n m a p s of b a c t e r i o p h a g e Mu were derived by r e s t r i c t i o n a n a l y s i s of a series of p l a s m i d clones c o n t a i n i n g s e g m e n t s of Mu D N A which, in combination, covered t h e e n t i r e Mu g e n o m e . T h e p l a s m i d s a n a l y z e d included pKN36, pKN54, pKN62, pKN50, pKN35, pKN27, pKN48, pKN82, a n d pKN56 f r o m t h e collection of W. S c h u m a n n a n d E. G. Bade, a n d pCM02, a newly c o n s t r u c t e d p l a s m i d c o n t a i n i n g t h e r i g h t m o s t i n t e r n a l EcoRI-PstI f r a g m e n t of Mu DNA. BglI cuts Mu D N A at 23 sites, p r o d u c i n g 24 f r a g m e n t s w h i c h r a n g e in size f r o m 0.05 kb up to t h e a p p r o x i m a t e l y 7-kb f r a g m e n t derived f r o m t h e r i g h t end. AvaII cuts Mu D N A at 17 s i t e s (including 2 w i t h i n t h e G s e g m e n t ) , p r o d u c i n g f r a g m e n t s w h i c h r a n g e in size f r o m 0.17 to 8.9 kb. The derived m a p s were confirmed by r e s u l t s of h y b r i d i z a t i o n of ~2P-labeled, n i c k - t r a n s l a t e d p l a s m i d D N A to AvaII- a n d BglI-digested Mu DNAs. E v i d e n c e for modification of one of t h e AvaII s i t e s in E. coli w a s obtained. INTRODUCTION
Mu is a temperate bacteriophage of Escherichia coli that can insert its DNA into the bacterial chromosome at many different sites during both lytic growth and the establishment of lysogeny (for reviews, see Howe and Bade, 1975; Bukhari, 1976). The DNA in the mature phage particle contains both conserved Mu sequences and variable host sequences consisting of 50 to 150 bp of host DNA at the left end (Bukhari et al., 1976) and 0.5 to 2.0 kb of host DNA at the right end (Daniell et al., 1973). The attached host sequences arise due to headful packaging of Mu DNA from an integrated form (Bukhari and Taylor, 1975) and appear to consist of randomly distributed segments of the host genome (Daniell et al., 1975). Mu DNA also contains a 3-kb invertible DNA segment, called the G segment, which is located near the right end of the molecule (Daniell et al., 1973) and which is involved in determining the host range of the phage (van de Putte et al., 1980; Kamp, 1981). RestricP r e s e n t a d d r e s s : C e t u s P a l o A l t o , P a l o A l t o , CA 94304. 2 A u t h o r to w h o m r e p r i n t r e q u e s t s s h o u l d be addressed. 563
tion enzyme cleavage maps of Mu DNA were obtained previously for enzymes EcoRI, HpaI, HindII, and HindIII (Allet and Bukhari, 1975; Magazin et al., 1977) and for Bali, BamHI, BgIII, EcoRI, HindIII, KpnI, PstI, and SalI (Kahmann et aL, 1977). With the exception of HindII and HpaI these enzymes cleave at only one or two sites within the conserved Mu sequences in Mu DNA. Mu DNA also possesses an unusual DNA modification (Hattman, 1979, 1980) which requires the morn function of Mu (Toussaint, 1976) and the dam function of the host (Toussaint, 1977; Khatoon and Bukhari, 1978) and which is expressed to high levels in phage grown by induction of a lysogen (Toussaint, 1976). This modification protects Mu from cleavage by a number of restriction enzymes, including HindII and PstI (Allet and Bukhari, 1975; R. Kahmann and D. Kamp, personal communication). We are interested in defining the physical location, kinetics of synthesis, and regulation of specific Mu transcripts made during phage development. This can be accomplished by determining the hybridization pattern of appropriate RNA samples to Mu DNA restriction fragments. Hybridization of E. coli RNA to the variable 0042-6822/83 $3.00 Copyright 9 1983 by AcademicPress, Inc. All rights of reproduction in any form reserved.
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MARRS AND HOWE
host sequences in Mu DNA can be prevented by using Mu DNA isolated from phage grown by heat induction of an RP4::Mu cts prophage in Proteus mirabilis, a strain with very little sequence homology to E. coli (Waggoner and Pato, 1978). To optimize resolution of the transcript locations it is preferable to use restriction enzymes which cleave Mu DNA into 15 to 25 fragments. Although HindII, which produces 18 fragments (Magazin et al., 1977), would have been suitable, our inability to construct an RP4:: Mu cts mom lysogen of P. mirabilis prevented its use. Therefore we chose to investigate the possibility of using the enzymes AvaII and BglI which had been found to cleave Mu mom+ DNA completely into approximately the right number of fragments (R. K a h m an n and D. Kamp, personal communication). To use these enzymes we first needed to know the locations of their cleavage sites in Mu DNA. In this paper we report the results of restriction endonuclease cleavage analysis of eight plasmids containing different cloned segments of Mu DNA and the AvaII and BglI restriction maps of mature Mu DNA which have been derived from these results. MATERIALS AND METHODS
Media LB broth and LB agar (Howe, 1973), NZ broth and TCMG agar (Schumm et al., 1980) and M9 medium (Miller, 1972) were described previously. LBtet and LBamp plates contained LB agar supplemented with 20 #g/ml tetracycline (Sigma) and 40 ttg/ml ampicillin (Sigma), respectively.
Bacteria, Bacteriophages, and Plasmids P. mirabilis strain AT3557 (RP4::Mu cts62/lac gal trp thy mal met nad ilv NalR; Waggoner and Pato, 1978) and E. coli K12 strain BU8305 (F'pro+lac::Mu cts62/Aprolac his met tyr Sm ~ MuR; Bukhari and A1let, 1975) were used for the preparation of Mu lysates by heat induction. E. coli C was used to grow r lysates (Pagano and Hutchison, 1971), and E. coli K12 strain K802 (supE hsdR gal met; Williams and
Blattner, 1979) was used to grow large lysates of }, phages. E. coli K12 strains MH812 (thr leu met lac supE hsdR hsdM; MacNeil et al., 1980) and WD5021 (gal lac rpsL; Howe, 1973) were used for Mu lysate titration. Strains WD5021 and EB105 (F-Alac~v Aara-leu498; Schumann et al., 1980) were used as hosts for plasmid DNA isolation for pCM02 or the pKN and vector plasmids, respectively. Bacteriophage strains included the following: Mu cts62, a heat-inducible derivative of Mu (Howe, 1973); hCharon 10 (hplac5 AH3 b/o256 KH54 sR14 ~ nin5 sHindIII6 ~ DK1; Williams and Blattner, 1979); )~KH100 nin5 (Daniels et al., 1980); a cts62 derivative of Mu A445-3 (Chow et aL, 1977) and r am3 (Hutchison and Sinsheimer, 1966). The Mu amber mutants used for marker rescue were from the collection of O'Day et aL, (1979). Plasmids used included the vector plasmids pBR322 (Bolivar et al., 1977; Sutcliffe, 1978), pBR325 (Bolivar, 1978) and pRSF2124 (So et al., 1975), and the hybrid plasmids pKN001 (Schumann et al., 1978), pKN54, pKN62 (Schumann, 1979), pKN56 (Schumann and Bade, 1979), pKN27, pKN48 (Schumann et al., 1979; Schumann et al., 1980), pKN35, pKN36, pKN50, pKN82 (Schumann et al., 1980), and pCM02. Plasmid pCM02 was made by cloning the region of Mu DNA from the right EcoRI site to the right PstI site into pBR322 using pKN48 as the Mu DNA source. After digestion of pKN48 and pBR322 DNA with both EcoRI and PstI and ligation with T4 ligase the DNA mixture was used to transform strain WD5021 (Cohen et al., 1972). Plasmid DNA from two Tet R Amp s transformants giving marker rescue (spot method of Engler et al., 1980) of amber mutations in Mu genes M, Y, and N, but not S or U, were screened by restriction analysis with EcoRI and PstI. Each produced the expected restriction p a t t e r n and one was designated pCM02.
Preparation of Phages and D N A Large lysates of Mu and }, were prepared and titered as previously described (Schumm et al., 1980) except strain AT3557
AvaII AND BglI MAPS OF PHAGE MU was grown and induced in LB containing 50 # g / ml thymine and 10 ~g/ml nicotinic acid and the resulting lysate was titered on strain MH812. DNA was isolated from purified phage particles by phenol extraction and dialyzed as described by Williams et al. (1977). ~bX174am3 phage were isolated as described by Pagano and Hutchison (1971). The double-stranded RF form was isolated by infecting E. coli C grown to approximately 5 X 10s cells/ml in I liter of LB with q~X174am3 at a multiplicity of infection (m.o.i.) of 5-10. At 4 min postinfection chloramphenicol was added to a final concentration of 40 ttg/ml, and the flask was shaken at 37 ~ for 2 hr. The culture was then placed on ice, and the cells were harvested by centrifugation for 20 min at 10,000 g. After lysis of cells with lysozyme, Brij 58 and sodium deoxycholate the RF DNA molecules were isolated by polyethylene glycol precipitation and CsC1 ethidium bromide equilibrium gradient centrifugation (Humphreys et al., 1975). For plasmid DNA isolation strains containing plasmids were grown in 800 ml of M9 medium to a density of 5 X l0 s cells/ ml. Then, chloramphenicol was added to a final concentration of 175 ttg/ml, and the cultures were shaken for 15 to 24 hr. The cells were harvested and lysed, and the plasmid DNA was isolated by polyethylene glycol precipitation and CsC1 ethidium bromide equilibrium gradient centrifugation (Humphreys et al., 1975).
Restriction Endonuclease Digestions All restriction endonucleases were purchased from New England BioLabs, Inc., except for SinI which was obtained from Biotec. Digestions were carried out for 212 hr at 37 ~ in 30-#1 reaction vol containing 0.5-4 tLg of DNA with sufficient enzyme to give complete digestion. The buffer for
AvaII, BgII, BglII, EcoRI, HaeIII, HpaI, and MspI was 6 mM MgC12, 6 m M TrisHC1 (pH 7.9), and 6 m M mercaptoethanol. For BamHI, HincII, HindIII, and PstI the buffer was the same except for the addition of 50 mM NaC1; for SalI the addition was 150 mM NaC1. The buffer for SinI was 60 m M NaC1, 10 mM Tris-HC1 (pH 7.5),
565
and 6 mMMgC12. Digestion of a DNA sample with more than one enzyme was done either by simultaneous digestion or by sequential digestion, first with the enzyme requiring a low NaC1 condition and then with t h a t requiring the addition of salt. For most of this work cleavage at AvaII sites was accomplished with AvaII; however, in a few instances the more stable enzyme SinI was used.
Gel Electrophoresis Horizontal agarose gel electrophoresis (Shinnick et aL, 1975) was used for the separation of restriction fragm ent s 0.8 kb and larger. In general 1% agarose gels were used, and electrophoresis was carried out with the wicks 12 cm a p a r t at 8 V for 16 hr at room temperature. EcoRI digested hCharon 10 and hKH100 nin5 DNAs and HaeIII-digested r RF DNA were included to provide standard length m arker fragments of 19.8, 12.4, 9.6, 6.8, 5.7, 4.9, 3.6, 3.1, and 2.0 kb (Daniels et al., 1980) and 1.35, 1.08, 0.87, 0.60, 0.31, 0.28, 0.27, 0.23, 0.19, 0.12, and 0.07 kb (Sanger et al., 1977), respectively. Restriction fragments of 1.2 kb and smaller were separated by vertical 5% acrylamide gel electrophoresis (14 X 12 X 0.3 cm) using a Hoefer Scientific Instruments Model SE500 apparatus. TBE buffer was 0.09 M TrisBase, 0.09 M boric acid, and 0.0028 M NaEDTA, pH 8.3. Each gel was made by preparing a solution containing 4.75% acrylamide (BioRad), 0.25% bisacrylamide (N,N'-methylenebisacrylamide; BioRad), l xT BE buffer, 25% glycerol, 0.1% ammonium persulfate (freshly made; Mallinckrodt), and adding 0.025% TEMED (N,N,N',N'-tetramethylethylene diamine; Sigma) just prior to pouring the gel. After polymerization, samples containing 30 #l digested DNA and 12 #l of concentrated sample buffer (54% glycerol, 0.6xTBE, 0.2% SDS, 0.05 M NaEDTA (pH 8.0) and 0.2 m g/ ml bromophenol blue) were loaded into wells under 1X TBE buffer. Electrophoresis was carried out at 130 V for 4 to 4.5 hr. Samples containing HaeIII-digested q~X174 RF or MspI-digested pBR322 (sizes 0.62, 0.53, 0.40, 0.31, 0.24 (2), 0.22, 0.20, 0.19,
566
MARRS AND HOWE
0.18, 0.16 (2), 0.15 (2), 0.12, 0.11, 0.09, 0.076, 0.067, 0.034 (2), 0.026 (2), 0.015, and 0.004 (2) kb) (Sutcliffe, 1979) were routinely included to provide a set of standard length marker fragments. Both agarose and acrylamide gels were stained for 10 min in a solution of approximately 10 #g/ml ethidium bromide and then destained in distilled water for 30 to 45 min. The DNA bands were visualized and photographed as previously described (Shinnick et al., 1975). The relative mobilities of the standard fragments were plotted against their log molecular weights to produce a standard curve which was then used to determine the molecular weight of each experimental f r a g m e n t (Helling et al., 1974). In general multiple gels were run and measured for each fr ag men t and the molecular weights determined were averaged to give the final size reported.
End Labeling, Nick Translation, Fragment Transfer, and Hybridization DNA samples were labeled at the 5' ends using polynucleotide kinase (PL Biochemicals) and [~-32P]ATP (a gift of D. L. Daniels and F. Blattner) by the method of Maxam and Gilbert (1977). Introduction of a-32P-nucleotides (New England Nuclear Corp., Boston, Mass.) into plasmid and phage DNA was carried out by nick translation according to the method of Maniatis et al. (1975). DNA was t r a ns f e r r e d from agarose gels to nitrocellulose filters by the method of Southern (1975) and the filters were hybridized to the nick-translated DNA probes as described by Smithies et al. (1978). RESULTS AND DISCUSSION
The strategy used for mapping the many AvaII and BglI restriction sites in Mu DNA was to determine the locations of those sites in segments of Mu DNA cloned into plasmid vectors and then to use t hat information to derive the map for the intact Mu DNA molecule. The plasmids used were the previously isolated pKN plasmids of
W. Schumann, E. G. Bade, and their collaborators (Schumann et al., 1980) and pCM02 which was constructed from pKN48 as described in Materials and Methods. The Mu DNA segments contained in each plasmid and the locations and orientations of the inserts in the pBR322 and pBR325 vectors are depicted in Fig. 1. The plasmid restriction maps were derived primarily by analysis of the number and sizes of restriction fragments produced by digestion of plasmid DNA with single enzymes and with pairs of enzymes. Prior knowledge of the locations of most restriction sites in the vector DNAs (Bolivar, 1978; Sutcliffe, 1978, 1979) and several sites in the Mu DNA inserts (Magazin et al., 1977; K ahm ann et al., 1977) simplified the analysis. In general the digested DNAs were examined by both agarose and acrylamide gel electrophoresis with EcoRI digested },Charon 10 and ~,KH100 nin5 DNAs and HaeIII digested ~bX174 RF DNA and/or MspI digested pBR322 DNA electrophoresed in parallel to provide standards of known length. The results of these restriction analyses are given in Figs. 2-4 which present the derived maps of the AvaII and BglI sites for each plasmid and the sizes of the pertinent restriction fragments used to generate those maps. In a few cases additional information was required to derive the maps; this information is described below.
pKN50 The evidence for a Mu-specific BglI fragment of approximately 0.23 kb in pKN50 in addition to the 0.23-kb vector fragm ent was (1) the presence of a broad band at 0.23 kb in BgII-digested pKN50 DNA, (2) the presence of an 0.24-kb band in BglI digests of pKN27 (which contains Mu DNA present in pKN50, pKN36, and pKN35), a plasmid whose vector region (RSF2124) did not have a BglI band near 0.23 kb, and (3) cleavage of the vector 0.23-kb band in pKN35 and pKN50 with AvaII to produce an 0.21-kb band but without the loss of the 0.23-kb band in pKN50 as would be expected if it were composed of two 0.23-kb fragments.
AvaII AND BglI MAPS OF PHAGE MU
567
A
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I
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)
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pKN48
pKNOO1
pKN27
pKN54
pKN50 pKN62
SU (
pKN82 pKN35
pKN56
pKN36
B
aCM02
C
FIG. 1. Locations of Mu-DNA segments present in plasmid clones. (A) The map at the top represents an approximate correlation of the genetic and physical maps of Mu (Magazin et al., 1977; Schumann et al., 1980; Schumm et al., 1980). The parentheses indicate the location of the invertible G segment; the open box at the right end represents the variable-end host DNA. The horizontal lines below the map indicate the Mu DNA present in each of the plasmids. The map is not drawn precisely to scale. The positions and orientations of Mu DNA inserts in (B) pBR322 and (C) pBR325 are given by the location and direction of arrows. The arrowheads point toward the right end of the Mu restriction map above. The maps are not drawn to scale, but the distances (in kb) between pertinent restriction sites are given.
T h e r e l a t i v e o r d e r s o f t h e t h r e e BglI f r a g m e n t s a n d f o u r A v a I I f r a g m e n t s loc a t e d b e t w e e n t h e EcoRI a n d H p a I s i t e s of pKN50 were determined from the res u l t s of d i g e s t i o n of p K N 5 0 w i t h b o t h A v a I I a n d BglI w h i c h r u l e d o u t a l l b u t 2 of t h e 144 p o s s i b l e o r d e r s . T h e s e t w o d i f f e r e d in t h e o r d e r o f t h e A v a I I f r a g m e n t s o f 0.90 a n d 0.17 kb. T h e o b s e r v a t i o n o f a 2.9-kb A v a I I p a r t i a l d i g e s t i o n p r o d u c t of p K N 5 0 s u g g e s t e d t h a t t h e 2.7-kb f r a g m e n t w a s a d j a c e n t to a f r a g m e n t o f a b o u t 0.2 kb, a n d t h u s s u p p o r t e d t h e o r d e r s h o w n in F i g . 3; however, the partial digestion pattern was complex and did not completely rule out the opposite order.
pKN35 To d e t e r m i n e t h e r e l a t i v e o r d e r of t b e 0.76- a n d 0.05-kb BglI f r a g m e n t s p K N 3 5 w a s d i g e s t e d w i t h b o t h BglI a n d A v a I I r e s u l t i n g in c l e a v a g e of t h e 0.76-kb BglI f r a g m e n t to p r o d u c e f r a g m e n t s of 0.59 a n d 0.17 kb. I f t h e 0.76-kb f r a g m e n t w e r e c l o s e r to t h e EcoRI s i t e , f r a g m e n t s of 0.61 a n d 0.15 k b w o u l d b e e x p e c t e d , w h i l e t h e opposite order would predict fragments of 0.66 a n d 0.10 kb. T h e o b s e r v e d f r a g m e n t sizes are more consistent with placement o f t h e 0.76-kb f r a g m e n t c l o s e r t o t h e EcoRI s i t e a s d e p i c t e d in F i g . 3; h o w e v e r , b e c a u s e D N A f r a g m e n t s do n o t a l w a y s m i g r a t e e x -
568
MARRS AND HOWE .81
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FIG. 2. Restriction Maps of pKN36, pKN54, and pKN62. The central horizontal line on each map represents the chimeric plasmid DNA linearized by opening at the unique EcoRI site of the vector and oriented such t h a t the Mu DNA s e g m e n t s are in their normal left to right orientation (as in Fig. 1). The line represents pBR322 vector DNA; the filled box r e p r e s e n t s the cloned Mu DNA; and the open boxes represent other DNA cloned with the Mu DNA f r a g m e n t . In the case of pKN54 this other DNA is primarily ColEl DNA adjacent to approximately 0.1-0.3 kb of host DNA; whereas in pKN62 the other DNA is composed of two separate f r a g m e n t s of ColE1 DNA. The solid vertical lines on each m a p indicate the locations of BglI (above the line) and A v a I I (below the line) restriction sites, and the n u m b e r s located between and above or below these sites indicate the sizes (in kb) of restriction f r a g m e n t s produced by digestion with these enzymes. The extents of t h e f r a g m e n t s s p a n n i n g the EcoRI site are indicated by the thin horizontal lines extending between the restriction sites; the sizes of these f r a g m e n t s are given at only one end of the map. The dashed vertical lines indicate the locations of previously known sites of cleavage by other enzymes as indicated on each map. These m a p s (drawn to scale) were derived by analysis of f r a g m e n t s produced by single and double digestion of plasmid DNA with a variety of enzymes. Below each m a p are given the observed sizes and interpreted locations of (a) new restriction f r a g m e n t s arising by digestion of BglI- or AvaIIdigested DNA with a second enzyme or (b) the unique restriction f r a g m e n t s generated by digestion with enzymes other t h a n BglI or AvaII. The locations of BglI and A v a I I sites within the vector
AvaII AND BglI MAPS OF PHAGE MU
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FIG. 3. Restriction maps of pKN50 and pKN35. See legend to Fig. 2.
actly according to their size in a c r y l a m i d e gels, the p o s i t i o n i n g of the 0.05-kb fragm e n t should be taken as t e n t a t i v e w i t h the
possibility t h a t it could be located b e t w e e n the 1.7- and 0.76-kb f r a g m e n t s or even bet w e e n the 4.1- and 1.3-kb f r a g m e n t s .
DNA were derived from the previously determined nucieotide sequence (Sutcliffe, 1979) except that the site dividing the 0.34-kb fragment into 0.30- and 0.04-kb fragments was left off the map because the fragment usually was not cleaved, presumably because the site was modified by the dcm methylase of E. coli (Backman, 1980).
570
MARRS
AND
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BglI f r a g m e n t in pCM02 in addition to the 0.23-kb vector band was indicated by (1) the presence of a broad b r i g h t band at 0.230.24 kb in BglI digested pCM02 D N A and only a n a r r o w 0.23-kb band in BglI-digested pKN56 D N A and (2) the presence of an 0.24-kb band f r o m pKN48 (containing Mu D N A present in pCM02 and pKN56) but not f r o m its vector pRSF2124 w h i c h has no 0.23-kb vector band. The relative order of the 0.20-, 0.64-, a n d 1.6-kb AvaII f r a g m e n t s located between the EcoRI and HindIII sites was d e t e r m i n e d f r o m results of two types of experiments. First, t h r e e of the six possible orders were e l i m i n a t e d by the sizes of f r a g m e n t s resulting f r o m double digestion of pCM02 with BglI and AvaII since those orders would have gene r a t e d f r a g m e n t s of 1.3 or 1.5 kb which were not observed. The final order was then d e t e r m i n e d by partial AvaII digestion of the EcoRI-HindIII f r a g m e n t which was 5' end-labeled at the EcoRI site (Smith and Birnstiel, 1976) and produced a p a r t i a l digestion p r o d u c t of 0.35 kb and a complete digestion p r o d u c t of 0.20 kb. A s i m i l a r a p p r o a c h was used to define the relative order of the 0.24- and 0.10-kb BglI f r a g m e n t s , pCM02 was cleaved with either HindIII or HpaI, 5' end-labeled, subjected to no s e c o n d a r y digestion (HindIII) or digestion with PstI (HpaI) and then p a r t i a l l y digested with BglI. Besides f r a g m e n t s 0.11 kb or smaller and f r a g m e n t s 0.89 kb or larger produced by p a r t i a l or complete digestion in o t h e r regions of the plasmid, the a u t o r a d i o g r a m s showed partial digestion products in the size r a n g e 0.11 to 0.89 which could be used to o r d e r the f r a g m e n t s in the region of interest.
571
The a u t o r a d i o g r a m of pCM02 end-labeled at HindIII and p a r t i a l l y digested by BglI showed a complete digestion product at about 0.27 kb and a p a r t i a l digestion product at 0.53 kb, d e m o n s t r a t i n g t h a t the 0.24kb f r a g m e n t was closest to the HindIII site. The a u t o r a d i o g r a m of pCM02 end-labeled at HpaI and p a r t i a l l y digested by BglI showed b a n d s at 0.16, 0.37, a n d 0.61 kb. The only p a t t e r n which would a c c o m m o d a t e these d a t a was the location of two 0.10-kb f r a g m e n t s close to the HpaI site followed by the 0.24-kb f r a g m e n t close to the HindIII site. This was confirmed by the observed HindIII to HpaI distance of 0.91 kb as c o m p a r e d to predicted values of 0.77 kb for a single 0.10-kb f r a g m e n t and 0.87 kb for two 0.10-kb f r a g m e n t s .
pKN82 Cleavage of pKN82 by AvaII produced a series of f r a g m e n t s whose lengths s u m m e d to a size 8.6 kb l a r g e r t h a n the length of pKN82. This resulted f r o m the cleavage of molecules c o n t a i n i n g the G s e g m e n t in opposite o r i e n t a t i o n s t o produce different sized f r a g m e n t s . The i d e n t i t y and location of these f r a g m e n t s was d e t e r m i n e d f r o m the f r a g m e n t sizes r e s u l t i n g f r o m cleavage with AvaII and EcoRI, PstI, or SalI as shown in Fig. 4. D e t e r m i n a t i o n of the G(+) or G ( - ) orientation specificity of the f r a g m e n t s was made as follows: First, it was reasoned t h a t since G s e g m e n t inversion does not alter the total length of Mu DNA, the larger of the AvaII f r a g m e n t s s p a n n i n g the EcoRI site m u s t be derived f r o m the same G orientation as the smaller of the AvaII fragments s p a n n i n g the SalI site and vice versa. Thus, the 3.8- and 4.8-kb f r a g m e n t s m u s t be derived f r o m one o r i e n t a t i o n and the
FIG. 4. Restriction maps of pCM02, pKN82, and pKN56. See legend to Fig. 2. On the maps of pKN82 and pKN56 the large parentheses and the dashed arrow indicate the outer boundaries and the center, respectively, of the invertible G segment and the fragment sizes within brackets indicate sizes derived from the opposite orientation of the G segment. The grouping of the 0.90- and 0.08kb BglI fragments in parentheses with a small arrow below indicates that the order of these fragments, and therefore the precise location of the BglI site between them, is unknown. Not shown on these maps is the presence of a small amount (probably less than 0.2 kb) of host DNA between the ~ end of Mu and the pBR322 (pKN56) or pBR325 (pKN82) vector DNA.
572
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4.2- and 4.5-kb fragments from the other orientation. Identification of the f r a g m e n t pair present in G(+) DNA (the orientation of G allowing growth by infection of E. coli K12) was made by AvaII digestion of Mu cts62A445-3 DNA. This phage carries the 445-3 deletion which spans the G-/~ boundary and fixes the G segment in the G(+) orientation (Chow et al., 1977). Detection of the 4.8-kb fragment but not the 4.5-kb fragment defined the 4.8- and 3.8kb fragments as those arising from cleavage of G(+) DNA.
Construction and Confirmation of the BglI and A v a I I Maps of Mature Mu D N A The maps of BglI and AvaII restriction sites in Mu DNA (Fig. 5) were derived from the plasmid maps by placing the map of each individual cloned segment in its appropriate position in Mu DNA and determining the sizes of fragments spanning the cloning sites by summing the fragment sizes immediately flanking these sites. To test the size accuracy of these cloning junction fragments, the sizes of restriction fragments produced by AvaII or BglI cleavage of mature Mu DNA were compared to those predicted by the map. In addition, 32p-labeled probes prepared by nick translation of pKN001, pKN35, pKN50, pKN48, pCM02, pKN27, pKN82, pKN56, and Mu DNA (grown in Proteus mirabilis) were hybridized to AvaII and BglI restriction fragments of Mu DNA which had been grown in E. coli, cleaved, electrophoresed in agarose gels, and transferred to nitrocellulose paper by the method of Southern (1975). The latter experiment was designed to detect possible DNA rearrangements in the plasmids and potential comigrating fragments arising from different segments of the Mu genome. When the AvaII restriction pattern of Mu DNA grown in E. coli or P. mirabilis was compared to that predicted by the map, the majority of fragments were those predicted; however, there were two notable exceptions. Mu DNA grown in E. coli contained all the predicted fragments and two additional fragments of 2.9 and 1.1 kb. Mu DNA grown in P. mirabilis had only a small
573
amount of the expected 3.9-kb fragment and had two extra fragments of 2.9 and 1.1 kb. Both the 2.9- and 1.1-kb fragments hybridized nick-translated probe from pKN35, the plasmid containing the 3.9-kb fragment. These data could be most simply explained if the 3.9-kb fragment contained an additional AvaII site (GGTACC) which partially overlapped with an E. coli dcm methylation site (CC~eAGG) (as shown for pBR322 by Backman [1980]) such t h a t plasmid DNA was completely modified and replicating Mu DNA was only partially modified, thus allowing for production of all three fragments. Presumably in P. mirabilis this site was modified more slowly, if at all, thus producing primarily the 2.9and 1.1-kb fragments. The fragment patterns produced by hybridization of the remaining plasmids to AvaII-digested Mu DNA and those produced by hybridization of the plasmids to BglI-digested Mu DNA were completely consistent with the patterns predicted by the maps, thus confirming the accuracy of the maps. These detailed restriction maps should be helpful in continued dissection of Mu genome organization, allowing for more precise localization of proteins, transcripts, specific mutations, and regulatory sites. ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, and by National Science Foundation Grants PCM 75-02465 and PCM 79-04055 to M.M.H. and by a National Institutes of Health Predoctoral Traineeship to C.F.M. on Training Grant GM 07133. M.M.H. is the recipient of Research Career Development Award AI 00274 from the National Institute of Allergy and Infectious Diseases. The authors gratefully acknowledge the assistance of D. Daniels and F. Blattner in the end-labeling experiments. REFERENCES ALLET, B., BLATTNER, F., HOWE, M., MAGAZIN, M., MOORE, D., O'DAY, K., SCHULTZ, D., and SCHUMM, J. (1977). Genetic and physical map of bacteriophage Mu. In "DNA Insertion Elements, Plasmids, and Episomes" (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 745-748. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
574
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ALLET, B., and BUKHARI,A. I. (1975). Analysis of bacteriophage Mu and },-ME hybrid DNAs by specific endonucleases. J. Mol. BioL 92, 529-540. BACKMAN, K. (1980). A cautionary note on the use of certain restriction endonucleases with methylated substrates. Gene II, 169-171. BOLIVAR, F. (1978). Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique E c o R I sites for selection of EcoRI generated recombinant DNA molecules. Gene 4, 121-136. BOLIVAR, F., RODRIGUEZ,R., GREENE, P. J., BETLACH, M., HEYNEKER, H. L., BOYER, H. W., CROSA,J., and FALKOW, S. (1977). Construction and characterization of new cloning vehicles. Gene 2, 95-113. BUKHARI, A. I. (1976). Bacteriophage Mu as a transposition element. Ann. Rev. Genet. 10, 389-412. BUKHARI, A. I., and ALLET, B. (1975). Plaque-forming k-ME hybrids. Virology 63, 30-39. BUKHARI, A. I., FROSHAUER, S., and BOTCHAN, M. (1976). Ends of bacteriophage Mu DNA. N a t u r e (London) 264, 580-583. BUKHARI, A. I., and TAYLOR, A. L. (1975). Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA. Proc. Nat. Acad. Sci. USA 72, 4399-4403. CHOW, L. W., KAHMANN,R., and KAMP,D. (1977). Electron microscopic characterization of DNAs of nondefective deletion mutants of bacteriophage ME. J. Mol. Biol. 113, 591-609. COHEN, S. N., CHANG,A. C. Y., and Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by Rfactor DNA. Proc. Nat. Acad. Sci. USA 69, 21102114. DANIELL, E., ABELSON, J., KIM, J. S., and DAVIDSON, N. (1973). Heteroduplex structures of bacteriophage Mu DNA. Virology 51, 237-239. DANIELL, E., KOHNE, D. E., and ABELSON, J. (1975). Characterization of the inhomogeneous DNA in virions of bacteriophage Mu by DNA reannealing kinetics. J. Virol. 15, 739-743. DANIELS, D. L., DEWET, J. R., and BLATTNER, F. R. (1980). New map of bacteriophage ~ DNA. J. Virol. 33, 390-400. ENGLER, J. A., FORGIE, R. A., and HOWE, M. M. (1980). Restriction endonuclease B a m H I cleaves bacteriophage Mu DNA within cistrons E and F. Gene 10, 79-83. HATTMAN, S. (1979). Unusual modification of bacteriophage Mu DNA. J. Virol. 32, 468-475. HATTMAN, S. (1980). Specificity of the bacteriophage Mu mom+-controlled DNA modification. J. ViroL 34, 277-279. HELLING, R. B., GOODMAN, H. M., and BOYER, H. W. (1974). Analysis of endonuclease R Eco R1 fragments of DNA from lambdoid bacteriophages and
other viruses by agarose-gel electrophoresis. J. Virol. 14, 1235-1244. HOWE, M. M. (1973). Prophage deletion mapping of bacteriophage ME-1. Virology 54, 93-101. HOWE, M. M., and BADE, E. G. (1975). Molecular biology of bacteriophage ME. Science 190, 624-632. HUMPHREYS, G. 0., WILLSHAW, G. A., and ANDERSON, E. S. (1975). A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 383, 457-463. HUTCHISON III, C. A., and SINSHEIMER, R. L. (1966). The process of infection with bacteriophage ~bX174. Mutations in a ~bX lysis gene. J. Mol. Biol. 18, 429447. KAHMANN, R., KAMP, D., and ZIPSER, D. (1977). Mapping of restriction sites in Mu DNA. In "DNA Insertion Elements, Plasmids, and Episomes" (A. I. Bukhari, J. Shapiro, and S. Adhya, eds.), pp. 335339. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. KAMP, D. (1981). Invertible deoxyribonucleic acid: The G segment of bacteriophage ME. I n "Microbiology1981" (D. Schlessinger, ed.), pp. 73-76. American Society for Microbiology, Washington, D. C. KHATOON, H., and BUKHARI, A. I. (1978). Bacteriophage ME-induced modification of DNA is dependent upon a host function. J. Bacteriol. 136, 423428. MACNEIL, D., HOWE, M. M., and BRILL, W. J. (1980). Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella p n e u m o n i a e n i f genes. J. Bacteriol. 141, 12641271. MAGAZIN, M., HOWE, M., and ALLET, B. (1977). Partial correlation of the genetic and physical maps of bacteriophage ME. Virology 77, 677-688. MANIATIS, T., JEFFREY, A., and KLEID, D. G. (1975). Nucleotide sequence of the rightward operator of phage h. Proc. Nat. Acad. Sci. USA 72, 1184-1188. MAXAM,A. M., and GILBERT, W. (1977). A new method for sequencing DNA. Proc. Nat. Acad. Sci. USA 74, 560-564. MILLER, J. H. (1972). I n "Experiments in Molecular Genetics," Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., p. 431. O'DAY, K., SCHULTZ,D., ERICSEN, W., RAWLUK,L., and HOWE, M. (1979). Correction and refinement of the genetic map of bacteriophage ME. Virology 93, 320328. PAGANO, J. S., and HUTCHISON III, C. A. (1971). Small, circular viral DNA: Preparation and analysis of SV40 and r DNA. Methods in Virology 5, 79123. SANGER, F., AIR, G. M., BARRELL, B. G., BROWN, N. L., COULSON, A. R., FIDDES, J. C., HUTCHISON, III, C. A., SLOCOMBE,P. M., and SMITH,M. (1977).
AvaII AND BglI MAPS OF PHAGE MU Nucleotide sequence of bacteriophage r DNA. Nature (London) 265, 687-695. SCHUMANN,W. (1979). Cloning and biological characterization of the immunity region of Escherichia coli phage Mu. Gene 5, 275-290. SCHUMANN, W., and BADE, E. G. (1979). In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions. Mol. Gen. Genet. 169, 97-105. SCHUMANN,W., BADE, a. G., DELIUS, S., and HUBERT, P. (1978). Cloning of a restriction fragment of phage Mu DNA coding for early functions. Mol. Gen. Genet. 160, 115-118. SCHUMANN,W., BADE, E. G., FORGIE, R. A., and HOWE, M. M. (1980). Cloning of DNA fragments of the right end of phage Mu and location of the HindIII, SaII, PstI, and BamHI restriction sites on the genetic map of Mu. Virology 104, 418-425. SCHUMANN,W., WESTPHAL, C., BADE, E. g., and HOLZER, L. (1979). Origin and binding specificity of proteins coded for by Mu prophages. Mol. Gen. Genet. 173, 189-196. SCHUMM, J. W., MOORE, D. D., BLATTNER, F. R., and HOWE, M. M. (1980). Correlation of the genetic and physical maps in the central region of the bacteriophage Mu genome. Virology 105, 185-195.
SHINNICK,T. M., LUND, E., SMITHIES,0., and BLATTNER, F. R. (1975). Hybridization of labeled RNA to DNA in agarose gels. Nucleic Acids Res. 2, 19111929. SMITH, H. 0., and BIRNSTIEL, M. L. (1976). A simple method for DNA restriction site mapping. Nucleic Acids Res. 3, 2387-2399. SMITHIES, O., BLECHL, A. E., DENNISTON-THOMPSON, K., NEWELL, N., RICHARDS, J. E., SLIGHTOM, J. L., TUCKER, P. W., and BLATTNER, F. R. (1978). Cloning
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human fetal -y globin and mouse a-type globin DNA: Characterization and partial sequencing. Science 202, 1284-1289. So, M., GILL, R., and FALKOW, S. (1975). The generation of a Col E1-Apr cloning vehicle which allows detection of inserted DNA. MoL Geva Genet. 142, 239-249. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioL 98, 503-517. SUTCLIFFE,J. G. (1978). pBR322 restriction map derived from the DNA sequence: Accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 5, 2721-2728. SUTCLIFFE,J. G. (1979). Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43, 77-90. TOUSSAINT, A. (1976). The DNA modification function of temperate phage Mu-1. Virology 70, 17-27. TOUSSAINT, A. (1977). DNA modification of bacteriophage Mu-1 requires both host and bacteriophage functions. J. Virol. 23, 825-826. VANDE PUTTE, P., CRAMER,S., and GIPHART-GASSLER, M. (1980). Invertible DNA determines host specificity of bacteriophage Mu. Nature (London) 286, 218-222. WAGGONER, B. T., and PATO, M. L. (1978). Early events in the replication of Mu prophage DNA. J. Virol. 27, 587-594. WILLIAMS, B. G., and BLATTNER, F. R. (1979). Construction and characterization of the hybrid bacteriophage lambda Charon vectors for DNA cloning. J. Virol. 29, 555-575. WILLIAMS, B. G., BLATTNER, F. R., JASKUNAS, S. R., and NOMURA,M. (1977). Insertion of DNA carrying ribosomal protein genes of Escherichia coli into Charon vector phages. J. BioL Chem. 252, 7344-7354.
Avail and Bgll Restriction Maps of Bacteriophage Mu CARL F. MARRS 1 AND MARTHA M. HOWE 2 Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received August 23, 1982; accepted January 25, 1983 The AvaII a n d BglI r e s t r i c t i o n m a p s of b a c t e r i o p h a g e Mu were derived by r e s t r i c t i o n a n a l y s i s of a series of p l a s m i d clones c o n t a i n i n g s e g m e n t s of Mu D N A which, in combination, covered t h e e n t i r e Mu g e n o m e . T h e p l a s m i d s a n a l y z e d included pKN36, pKN54, pKN62, pKN50, pKN35, pKN27, pKN48, pKN82, a n d pKN56 f r o m t h e collection of W. S c h u m a n n a n d E. G. Bade, a n d pCM02, a newly c o n s t r u c t e d p l a s m i d c o n t a i n i n g t h e r i g h t m o s t i n t e r n a l EcoRI-PstI f r a g m e n t of Mu DNA. BglI cuts Mu D N A at 23 sites, p r o d u c i n g 24 f r a g m e n t s w h i c h r a n g e in size f r o m 0.05 kb up to t h e a p p r o x i m a t e l y 7-kb f r a g m e n t derived f r o m t h e r i g h t end. AvaII cuts Mu D N A at 17 s i t e s (including 2 w i t h i n t h e G s e g m e n t ) , p r o d u c i n g f r a g m e n t s w h i c h r a n g e in size f r o m 0.17 to 8.9 kb. The derived m a p s were confirmed by r e s u l t s of h y b r i d i z a t i o n of ~2P-labeled, n i c k - t r a n s l a t e d p l a s m i d D N A to AvaII- a n d BglI-digested Mu DNAs. E v i d e n c e for modification of one of t h e AvaII s i t e s in E. coli w a s obtained. INTRODUCTION
Mu is a temperate bacteriophage of Escherichia coli that can insert its DNA into the bacterial chromosome at many different sites during both lytic growth and the establishment of lysogeny (for reviews, see Howe and Bade, 1975; Bukhari, 1976). The DNA in the mature phage particle contains both conserved Mu sequences and variable host sequences consisting of 50 to 150 bp of host DNA at the left end (Bukhari et al., 1976) and 0.5 to 2.0 kb of host DNA at the right end (Daniell et al., 1973). The attached host sequences arise due to headful packaging of Mu DNA from an integrated form (Bukhari and Taylor, 1975) and appear to consist of randomly distributed segments of the host genome (Daniell et al., 1975). Mu DNA also contains a 3-kb invertible DNA segment, called the G segment, which is located near the right end of the molecule (Daniell et al., 1973) and which is involved in determining the host range of the phage (van de Putte et al., 1980; Kamp, 1981). RestricP r e s e n t a d d r e s s : C e t u s P a l o A l t o , P a l o A l t o , CA 94304. 2 A u t h o r to w h o m r e p r i n t r e q u e s t s s h o u l d be addressed. 563
tion enzyme cleavage maps of Mu DNA were obtained previously for enzymes EcoRI, HpaI, HindII, and HindIII (Allet and Bukhari, 1975; Magazin et al., 1977) and for Bali, BamHI, BgIII, EcoRI, HindIII, KpnI, PstI, and SalI (Kahmann et aL, 1977). With the exception of HindII and HpaI these enzymes cleave at only one or two sites within the conserved Mu sequences in Mu DNA. Mu DNA also possesses an unusual DNA modification (Hattman, 1979, 1980) which requires the morn function of Mu (Toussaint, 1976) and the dam function of the host (Toussaint, 1977; Khatoon and Bukhari, 1978) and which is expressed to high levels in phage grown by induction of a lysogen (Toussaint, 1976). This modification protects Mu from cleavage by a number of restriction enzymes, including HindII and PstI (Allet and Bukhari, 1975; R. Kahmann and D. Kamp, personal communication). We are interested in defining the physical location, kinetics of synthesis, and regulation of specific Mu transcripts made during phage development. This can be accomplished by determining the hybridization pattern of appropriate RNA samples to Mu DNA restriction fragments. Hybridization of E. coli RNA to the variable 0042-6822/83 $3.00 Copyright 9 1983 by AcademicPress, Inc. All rights of reproduction in any form reserved.
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host sequences in Mu DNA can be prevented by using Mu DNA isolated from phage grown by heat induction of an RP4::Mu cts prophage in Proteus mirabilis, a strain with very little sequence homology to E. coli (Waggoner and Pato, 1978). To optimize resolution of the transcript locations it is preferable to use restriction enzymes which cleave Mu DNA into 15 to 25 fragments. Although HindII, which produces 18 fragments (Magazin et al., 1977), would have been suitable, our inability to construct an RP4:: Mu cts mom lysogen of P. mirabilis prevented its use. Therefore we chose to investigate the possibility of using the enzymes AvaII and BglI which had been found to cleave Mu mom+ DNA completely into approximately the right number of fragments (R. K a h m an n and D. Kamp, personal communication). To use these enzymes we first needed to know the locations of their cleavage sites in Mu DNA. In this paper we report the results of restriction endonuclease cleavage analysis of eight plasmids containing different cloned segments of Mu DNA and the AvaII and BglI restriction maps of mature Mu DNA which have been derived from these results. MATERIALS AND METHODS
Media LB broth and LB agar (Howe, 1973), NZ broth and TCMG agar (Schumm et al., 1980) and M9 medium (Miller, 1972) were described previously. LBtet and LBamp plates contained LB agar supplemented with 20 #g/ml tetracycline (Sigma) and 40 ttg/ml ampicillin (Sigma), respectively.
Bacteria, Bacteriophages, and Plasmids P. mirabilis strain AT3557 (RP4::Mu cts62/lac gal trp thy mal met nad ilv NalR; Waggoner and Pato, 1978) and E. coli K12 strain BU8305 (F'pro+lac::Mu cts62/Aprolac his met tyr Sm ~ MuR; Bukhari and A1let, 1975) were used for the preparation of Mu lysates by heat induction. E. coli C was used to grow r lysates (Pagano and Hutchison, 1971), and E. coli K12 strain K802 (supE hsdR gal met; Williams and
Blattner, 1979) was used to grow large lysates of }, phages. E. coli K12 strains MH812 (thr leu met lac supE hsdR hsdM; MacNeil et al., 1980) and WD5021 (gal lac rpsL; Howe, 1973) were used for Mu lysate titration. Strains WD5021 and EB105 (F-Alac~v Aara-leu498; Schumann et al., 1980) were used as hosts for plasmid DNA isolation for pCM02 or the pKN and vector plasmids, respectively. Bacteriophage strains included the following: Mu cts62, a heat-inducible derivative of Mu (Howe, 1973); hCharon 10 (hplac5 AH3 b/o256 KH54 sR14 ~ nin5 sHindIII6 ~ DK1; Williams and Blattner, 1979); )~KH100 nin5 (Daniels et al., 1980); a cts62 derivative of Mu A445-3 (Chow et aL, 1977) and r am3 (Hutchison and Sinsheimer, 1966). The Mu amber mutants used for marker rescue were from the collection of O'Day et aL, (1979). Plasmids used included the vector plasmids pBR322 (Bolivar et al., 1977; Sutcliffe, 1978), pBR325 (Bolivar, 1978) and pRSF2124 (So et al., 1975), and the hybrid plasmids pKN001 (Schumann et al., 1978), pKN54, pKN62 (Schumann, 1979), pKN56 (Schumann and Bade, 1979), pKN27, pKN48 (Schumann et al., 1979; Schumann et al., 1980), pKN35, pKN36, pKN50, pKN82 (Schumann et al., 1980), and pCM02. Plasmid pCM02 was made by cloning the region of Mu DNA from the right EcoRI site to the right PstI site into pBR322 using pKN48 as the Mu DNA source. After digestion of pKN48 and pBR322 DNA with both EcoRI and PstI and ligation with T4 ligase the DNA mixture was used to transform strain WD5021 (Cohen et al., 1972). Plasmid DNA from two Tet R Amp s transformants giving marker rescue (spot method of Engler et al., 1980) of amber mutations in Mu genes M, Y, and N, but not S or U, were screened by restriction analysis with EcoRI and PstI. Each produced the expected restriction p a t t e r n and one was designated pCM02.
Preparation of Phages and D N A Large lysates of Mu and }, were prepared and titered as previously described (Schumm et al., 1980) except strain AT3557
AvaII AND BglI MAPS OF PHAGE MU was grown and induced in LB containing 50 # g / ml thymine and 10 ~g/ml nicotinic acid and the resulting lysate was titered on strain MH812. DNA was isolated from purified phage particles by phenol extraction and dialyzed as described by Williams et al. (1977). ~bX174am3 phage were isolated as described by Pagano and Hutchison (1971). The double-stranded RF form was isolated by infecting E. coli C grown to approximately 5 X 10s cells/ml in I liter of LB with q~X174am3 at a multiplicity of infection (m.o.i.) of 5-10. At 4 min postinfection chloramphenicol was added to a final concentration of 40 ttg/ml, and the flask was shaken at 37 ~ for 2 hr. The culture was then placed on ice, and the cells were harvested by centrifugation for 20 min at 10,000 g. After lysis of cells with lysozyme, Brij 58 and sodium deoxycholate the RF DNA molecules were isolated by polyethylene glycol precipitation and CsC1 ethidium bromide equilibrium gradient centrifugation (Humphreys et al., 1975). For plasmid DNA isolation strains containing plasmids were grown in 800 ml of M9 medium to a density of 5 X l0 s cells/ ml. Then, chloramphenicol was added to a final concentration of 175 ttg/ml, and the cultures were shaken for 15 to 24 hr. The cells were harvested and lysed, and the plasmid DNA was isolated by polyethylene glycol precipitation and CsC1 ethidium bromide equilibrium gradient centrifugation (Humphreys et al., 1975).
Restriction Endonuclease Digestions All restriction endonucleases were purchased from New England BioLabs, Inc., except for SinI which was obtained from Biotec. Digestions were carried out for 212 hr at 37 ~ in 30-#1 reaction vol containing 0.5-4 tLg of DNA with sufficient enzyme to give complete digestion. The buffer for
AvaII, BgII, BglII, EcoRI, HaeIII, HpaI, and MspI was 6 mM MgC12, 6 m M TrisHC1 (pH 7.9), and 6 m M mercaptoethanol. For BamHI, HincII, HindIII, and PstI the buffer was the same except for the addition of 50 mM NaC1; for SalI the addition was 150 mM NaC1. The buffer for SinI was 60 m M NaC1, 10 mM Tris-HC1 (pH 7.5),
565
and 6 mMMgC12. Digestion of a DNA sample with more than one enzyme was done either by simultaneous digestion or by sequential digestion, first with the enzyme requiring a low NaC1 condition and then with t h a t requiring the addition of salt. For most of this work cleavage at AvaII sites was accomplished with AvaII; however, in a few instances the more stable enzyme SinI was used.
Gel Electrophoresis Horizontal agarose gel electrophoresis (Shinnick et aL, 1975) was used for the separation of restriction fragm ent s 0.8 kb and larger. In general 1% agarose gels were used, and electrophoresis was carried out with the wicks 12 cm a p a r t at 8 V for 16 hr at room temperature. EcoRI digested hCharon 10 and hKH100 nin5 DNAs and HaeIII-digested r RF DNA were included to provide standard length m arker fragments of 19.8, 12.4, 9.6, 6.8, 5.7, 4.9, 3.6, 3.1, and 2.0 kb (Daniels et al., 1980) and 1.35, 1.08, 0.87, 0.60, 0.31, 0.28, 0.27, 0.23, 0.19, 0.12, and 0.07 kb (Sanger et al., 1977), respectively. Restriction fragments of 1.2 kb and smaller were separated by vertical 5% acrylamide gel electrophoresis (14 X 12 X 0.3 cm) using a Hoefer Scientific Instruments Model SE500 apparatus. TBE buffer was 0.09 M TrisBase, 0.09 M boric acid, and 0.0028 M NaEDTA, pH 8.3. Each gel was made by preparing a solution containing 4.75% acrylamide (BioRad), 0.25% bisacrylamide (N,N'-methylenebisacrylamide; BioRad), l xT BE buffer, 25% glycerol, 0.1% ammonium persulfate (freshly made; Mallinckrodt), and adding 0.025% TEMED (N,N,N',N'-tetramethylethylene diamine; Sigma) just prior to pouring the gel. After polymerization, samples containing 30 #l digested DNA and 12 #l of concentrated sample buffer (54% glycerol, 0.6xTBE, 0.2% SDS, 0.05 M NaEDTA (pH 8.0) and 0.2 m g/ ml bromophenol blue) were loaded into wells under 1X TBE buffer. Electrophoresis was carried out at 130 V for 4 to 4.5 hr. Samples containing HaeIII-digested q~X174 RF or MspI-digested pBR322 (sizes 0.62, 0.53, 0.40, 0.31, 0.24 (2), 0.22, 0.20, 0.19,
566
MARRS AND HOWE
0.18, 0.16 (2), 0.15 (2), 0.12, 0.11, 0.09, 0.076, 0.067, 0.034 (2), 0.026 (2), 0.015, and 0.004 (2) kb) (Sutcliffe, 1979) were routinely included to provide a set of standard length marker fragments. Both agarose and acrylamide gels were stained for 10 min in a solution of approximately 10 #g/ml ethidium bromide and then destained in distilled water for 30 to 45 min. The DNA bands were visualized and photographed as previously described (Shinnick et al., 1975). The relative mobilities of the standard fragments were plotted against their log molecular weights to produce a standard curve which was then used to determine the molecular weight of each experimental f r a g m e n t (Helling et al., 1974). In general multiple gels were run and measured for each fr ag men t and the molecular weights determined were averaged to give the final size reported.
End Labeling, Nick Translation, Fragment Transfer, and Hybridization DNA samples were labeled at the 5' ends using polynucleotide kinase (PL Biochemicals) and [~-32P]ATP (a gift of D. L. Daniels and F. Blattner) by the method of Maxam and Gilbert (1977). Introduction of a-32P-nucleotides (New England Nuclear Corp., Boston, Mass.) into plasmid and phage DNA was carried out by nick translation according to the method of Maniatis et al. (1975). DNA was t r a ns f e r r e d from agarose gels to nitrocellulose filters by the method of Southern (1975) and the filters were hybridized to the nick-translated DNA probes as described by Smithies et al. (1978). RESULTS AND DISCUSSION
The strategy used for mapping the many AvaII and BglI restriction sites in Mu DNA was to determine the locations of those sites in segments of Mu DNA cloned into plasmid vectors and then to use t hat information to derive the map for the intact Mu DNA molecule. The plasmids used were the previously isolated pKN plasmids of
W. Schumann, E. G. Bade, and their collaborators (Schumann et al., 1980) and pCM02 which was constructed from pKN48 as described in Materials and Methods. The Mu DNA segments contained in each plasmid and the locations and orientations of the inserts in the pBR322 and pBR325 vectors are depicted in Fig. 1. The plasmid restriction maps were derived primarily by analysis of the number and sizes of restriction fragments produced by digestion of plasmid DNA with single enzymes and with pairs of enzymes. Prior knowledge of the locations of most restriction sites in the vector DNAs (Bolivar, 1978; Sutcliffe, 1978, 1979) and several sites in the Mu DNA inserts (Magazin et al., 1977; K ahm ann et al., 1977) simplified the analysis. In general the digested DNAs were examined by both agarose and acrylamide gel electrophoresis with EcoRI digested },Charon 10 and ~,KH100 nin5 DNAs and HaeIII digested ~bX174 RF DNA and/or MspI digested pBR322 DNA electrophoresed in parallel to provide standards of known length. The results of these restriction analyses are given in Figs. 2-4 which present the derived maps of the AvaII and BglI sites for each plasmid and the sizes of the pertinent restriction fragments used to generate those maps. In a few cases additional information was required to derive the maps; this information is described below.
pKN50 The evidence for a Mu-specific BglI fragment of approximately 0.23 kb in pKN50 in addition to the 0.23-kb vector fragm ent was (1) the presence of a broad band at 0.23 kb in BgII-digested pKN50 DNA, (2) the presence of an 0.24-kb band in BglI digests of pKN27 (which contains Mu DNA present in pKN50, pKN36, and pKN35), a plasmid whose vector region (RSF2124) did not have a BglI band near 0.23 kb, and (3) cleavage of the vector 0.23-kb band in pKN35 and pKN50 with AvaII to produce an 0.21-kb band but without the loss of the 0.23-kb band in pKN50 as would be expected if it were composed of two 0.23-kb fragments.
AvaII AND BglI MAPS OF PHAGE MU
567
A
c
AB
I
Clys D ! H 113 ITJKL MY
)
I
J
pKN48
pKNOO1
pKN27
pKN54
pKN50 pKN62
SU (
pKN82 pKN35
pKN56
pKN36
B
aCM02
C
FIG. 1. Locations of Mu-DNA segments present in plasmid clones. (A) The map at the top represents an approximate correlation of the genetic and physical maps of Mu (Magazin et al., 1977; Schumann et al., 1980; Schumm et al., 1980). The parentheses indicate the location of the invertible G segment; the open box at the right end represents the variable-end host DNA. The horizontal lines below the map indicate the Mu DNA present in each of the plasmids. The map is not drawn precisely to scale. The positions and orientations of Mu DNA inserts in (B) pBR322 and (C) pBR325 are given by the location and direction of arrows. The arrowheads point toward the right end of the Mu restriction map above. The maps are not drawn to scale, but the distances (in kb) between pertinent restriction sites are given.
T h e r e l a t i v e o r d e r s o f t h e t h r e e BglI f r a g m e n t s a n d f o u r A v a I I f r a g m e n t s loc a t e d b e t w e e n t h e EcoRI a n d H p a I s i t e s of pKN50 were determined from the res u l t s of d i g e s t i o n of p K N 5 0 w i t h b o t h A v a I I a n d BglI w h i c h r u l e d o u t a l l b u t 2 of t h e 144 p o s s i b l e o r d e r s . T h e s e t w o d i f f e r e d in t h e o r d e r o f t h e A v a I I f r a g m e n t s o f 0.90 a n d 0.17 kb. T h e o b s e r v a t i o n o f a 2.9-kb A v a I I p a r t i a l d i g e s t i o n p r o d u c t of p K N 5 0 s u g g e s t e d t h a t t h e 2.7-kb f r a g m e n t w a s a d j a c e n t to a f r a g m e n t o f a b o u t 0.2 kb, a n d t h u s s u p p o r t e d t h e o r d e r s h o w n in F i g . 3; however, the partial digestion pattern was complex and did not completely rule out the opposite order.
pKN35 To d e t e r m i n e t h e r e l a t i v e o r d e r of t b e 0.76- a n d 0.05-kb BglI f r a g m e n t s p K N 3 5 w a s d i g e s t e d w i t h b o t h BglI a n d A v a I I r e s u l t i n g in c l e a v a g e of t h e 0.76-kb BglI f r a g m e n t to p r o d u c e f r a g m e n t s of 0.59 a n d 0.17 kb. I f t h e 0.76-kb f r a g m e n t w e r e c l o s e r to t h e EcoRI s i t e , f r a g m e n t s of 0.61 a n d 0.15 k b w o u l d b e e x p e c t e d , w h i l e t h e opposite order would predict fragments of 0.66 a n d 0.10 kb. T h e o b s e r v e d f r a g m e n t sizes are more consistent with placement o f t h e 0.76-kb f r a g m e n t c l o s e r t o t h e EcoRI s i t e a s d e p i c t e d in F i g . 3; h o w e v e r , b e c a u s e D N A f r a g m e n t s do n o t a l w a y s m i g r a t e e x -
568
MARRS AND HOWE .81
2.O
.23
2.3
1.7
pKN36
i i Eco Bam RI HI
~ .09.25 .34,28 HI
--d.481 k.88-1 I
8gl I + Barn HI Bgl I =f- Eco RI Ava II + Bam HI
i Eco Rt
Bam
p-,.4~.s41
I~1.2I-.ss-I 1-1.~
2.7---~.421
2.3
.23
3.7
pKN54
,', / \ \ \ ~
/ \ .09 . 2 5 . 3 4 . 2 8 Eco Hind RI III
"'
)k\ ~.o II II ' .' I I
/ Pst
Eco Hind Pst
RI
Ill
I
Eco RI
Bgl I + ECO RI
I-.~2-1
I
Ava I1+ Eco RI
I--.81~
~1.8 ----1.6,I
I
H i n d III
4.8
2.s~ I
i.s-
8gl I + Hind III
I--.89~
}--1.2 I
Ava II -b Hind III
F-.~91 - - , . , --I
I--,.2-pJ9~.67I 1.9 I II ,.~---I-1.8II ~.9---II
Pst I
Bgl I -t- Pst I Ava II + Pst I
.23
2.3
2.7
1.5--
1,1
2,8
pKN62
//\\
,09,25.34.28
ECO Hind RI III Bgll+
ECO RI
I
I
J \
I
I
I
Pst
Pst
Eco
I
I
Ri
~-.93-~
\
F
I
Pst Eco
I
Rt
~.92-~-.94-~
Ava II-I- Eco RI
~-.77-~
~
Bgl I-I- Pst I
- - I
I ~ 1"2
4.6 I
1'44
I b63t ~--1.1--~--1.7-
FIG. 2. Restriction Maps of pKN36, pKN54, and pKN62. The central horizontal line on each map represents the chimeric plasmid DNA linearized by opening at the unique EcoRI site of the vector and oriented such t h a t the Mu DNA s e g m e n t s are in their normal left to right orientation (as in Fig. 1). The line represents pBR322 vector DNA; the filled box r e p r e s e n t s the cloned Mu DNA; and the open boxes represent other DNA cloned with the Mu DNA f r a g m e n t . In the case of pKN54 this other DNA is primarily ColEl DNA adjacent to approximately 0.1-0.3 kb of host DNA; whereas in pKN62 the other DNA is composed of two separate f r a g m e n t s of ColE1 DNA. The solid vertical lines on each m a p indicate the locations of BglI (above the line) and A v a I I (below the line) restriction sites, and the n u m b e r s located between and above or below these sites indicate the sizes (in kb) of restriction f r a g m e n t s produced by digestion with these enzymes. The extents of t h e f r a g m e n t s s p a n n i n g the EcoRI site are indicated by the thin horizontal lines extending between the restriction sites; the sizes of these f r a g m e n t s are given at only one end of the map. The dashed vertical lines indicate the locations of previously known sites of cleavage by other enzymes as indicated on each map. These m a p s (drawn to scale) were derived by analysis of f r a g m e n t s produced by single and double digestion of plasmid DNA with a variety of enzymes. Below each m a p are given the observed sizes and interpreted locations of (a) new restriction f r a g m e n t s arising by digestion of BglI- or AvaIIdigested DNA with a second enzyme or (b) the unique restriction f r a g m e n t s generated by digestion with enzymes other t h a n BglI or AvaII. The locations of BglI and A v a I I sites within the vector
AvaII AND BglI MAPS OF PHAGE MU
.23
3.0
.94
2.1
.97
569
2.2
,23
2.3
1.4
pKN50
I
Ii
t Eco RI E c o RI + Bgl I E c o RI + Ava II
Hpa I
I,
i .o9.2s.3+.28 Bgl II
Bam HI
Eco RI
i-.ss-I
1.sol
k.~st
9Po
[
Barn H I 4- Hpa I
4.4 - - [
I
B a m H I + Bgl II
4.o I--+.7
Barn HI + Bgl |
+
Barn HI + Ava II
4.6
I I.++I I.'+=t
Hpa 1+ Bgl I
"P?
H p a I + A v a II
--,,.8
t
Bg| II + Bgl I
~59 I-----
Bgl II + A v a II A v a II + Bgl I
4 I I I--90-t I~ 1.6 . 3 1 . 2 3 +26
I .9. I
4.4
20
I
I 97 I
~
.17
]\ ~
./,
. 0 9 . 0 5 ,21 +03.31 ,28
2.3
.23
1.3
4,1
.OS ,76
. 0 2 .22
1,7
pKN35
Eco RI
Hinc II
Hinc II
Barn HI
Hinc II
Hpa I
Eco RI
E c o RI + Bg! I E c o RI + A v a II
,05
1.641
If
H p a I -t- E c o RI
7.9
H p a I + Barn H I
4.3
I
H p a I + Bgl I H p a I + A v a It H i n c II H i n c I 1 + Bgl I
I
3.3 --
~,
~'421
:,.~
+2~--1"1~} - ' 1 " 0
H i n c II + A v a II
3.s
I I
2.2--~ 6.0--
1.621
2.7t
I J
~.~ 2.+. 2.s
I 1.3--~ L -~.~-1.62J t-1.3I ~.3-+ I-
Barn H I + Bgl I
I ~-,.~-~
Barn HI -t- A v a II A v a I 1 + Bgl I
3.3 / ~92 2 . 0 2
/
I
\""+~-
.28.31 .03 .21 .05 .09
[I.59[ ~.72 -}-
]\
+05
:d" 9
FIG. 3. Restriction maps of pKN50 and pKN35. See legend to Fig. 2.
actly according to their size in a c r y l a m i d e gels, the p o s i t i o n i n g of the 0.05-kb fragm e n t should be taken as t e n t a t i v e w i t h the
possibility t h a t it could be located b e t w e e n the 1.7- and 0.76-kb f r a g m e n t s or even bet w e e n the 4.1- and 1.3-kb f r a g m e n t s .
DNA were derived from the previously determined nucieotide sequence (Sutcliffe, 1979) except that the site dividing the 0.34-kb fragment into 0.30- and 0.04-kb fragments was left off the map because the fragment usually was not cleaved, presumably because the site was modified by the dcm methylase of E. coli (Backman, 1980).
570
MARRS
AND
HOWE
.24 .10 .10 2.2 ~ l /
1.0
1.9
2.3
.23 .97
pCM02 :.20 l Eco
1.6
.64
: 2.2 Ii ,
Hind
R,
.27,71 I 1.7//// / \.97/I //%% .28.34.25 ,09 / \ H a P t Hind Eco Pl ~ Ill RI
H a
Ill
.os r .16 "1r
Eco R I + Bgll Eco RI + Ava II Hind III + agll
k.gg-~
t-.8o~
.081
F-~2.0 --~27 r
Hind III + Ava II .201
Hpa I Hp~l I + Hind III
I-.B9-']
I.'81------ '.;' ~
F ;'9-i
I 1.6 I J . 9 1 - - ~ 1.6 } 16 .16 ~ r--l.6---ir
--3.0
Hpa I + B g l I Hp8 I+.Ava II
6.9-3.6
J
F1.4~I-.B9-{ 1.56~? J } \l-'69 "l--'9 0 Jr'6 4_..__----///\ 3L'73 "J ]1F ' 9 8 4, \}-'71Jl~ - ~ l 1.7 / \ -----~/" I , \ ~b- '"-~- - - - - - - I ,11 ,20 .24 .10 .10 .27 .02 .2B ,31 .03,21 .05 .09
Av8 " * B g l l
2,3
.24 .10.10 1"2X t / /
.23
2,5
1,3
(.90,,08)
:
6,2
pKN82
I Eco RI I
Eco RI t- Bgl I Eco RI + Ava H
~///I ,22[ 1.7 .28'34.25,091 I' ~'s I Pst Sal Hind I
I
l
"~
3.8 ~.~
I
Pst
III
12
l ~.sL~.~j
I
I
I
RI
I
l--1.3-4 }-,.o-~
5.0
I-
k.~o ~27r
Hind III + Bgl I Hind III 1- Ave II SsI I + Bgl I Sal I + Ava II Pst I + 8gl I Pst I + Ava II
-'~
I~'.~
I .~'-I
.11.11 ld"
1.60F
2.5
1,3
(.90,.08)
:
6.2
I4.2 [-3.93 - -
t
5.2
2.3
.23
pKN56
ECO Pst RI
Eco RI+ Bgl I Sal I -t- Bgl I Sal I + Ava II PSI I + Bgl I Pst I + Ava II
I
Sal
Pst
I
I
Sal Eco I
RI
F-ls--~
F-~o-t
-~.~-1--~.3
.28 "1 r - ,15 lr--
I
3.0
[2.7]
t 5.0
I ECO
Sal
,13 -It I - - - ~'' [3.0] - - ~ ' ?
AvalI AND BglI MAPS OF PHAGE MU pCM02 The presence of an 0.24-kb Mu-specific
BglI f r a g m e n t in pCM02 in addition to the 0.23-kb vector band was indicated by (1) the presence of a broad b r i g h t band at 0.230.24 kb in BglI digested pCM02 D N A and only a n a r r o w 0.23-kb band in BglI-digested pKN56 D N A and (2) the presence of an 0.24-kb band f r o m pKN48 (containing Mu D N A present in pCM02 and pKN56) but not f r o m its vector pRSF2124 w h i c h has no 0.23-kb vector band. The relative order of the 0.20-, 0.64-, a n d 1.6-kb AvaII f r a g m e n t s located between the EcoRI and HindIII sites was d e t e r m i n e d f r o m results of two types of experiments. First, t h r e e of the six possible orders were e l i m i n a t e d by the sizes of f r a g m e n t s resulting f r o m double digestion of pCM02 with BglI and AvaII since those orders would have gene r a t e d f r a g m e n t s of 1.3 or 1.5 kb which were not observed. The final order was then d e t e r m i n e d by partial AvaII digestion of the EcoRI-HindIII f r a g m e n t which was 5' end-labeled at the EcoRI site (Smith and Birnstiel, 1976) and produced a p a r t i a l digestion p r o d u c t of 0.35 kb and a complete digestion p r o d u c t of 0.20 kb. A s i m i l a r a p p r o a c h was used to define the relative order of the 0.24- and 0.10-kb BglI f r a g m e n t s , pCM02 was cleaved with either HindIII or HpaI, 5' end-labeled, subjected to no s e c o n d a r y digestion (HindIII) or digestion with PstI (HpaI) and then p a r t i a l l y digested with BglI. Besides f r a g m e n t s 0.11 kb or smaller and f r a g m e n t s 0.89 kb or larger produced by p a r t i a l or complete digestion in o t h e r regions of the plasmid, the a u t o r a d i o g r a m s showed partial digestion products in the size r a n g e 0.11 to 0.89 which could be used to o r d e r the f r a g m e n t s in the region of interest.
571
The a u t o r a d i o g r a m of pCM02 end-labeled at HindIII and p a r t i a l l y digested by BglI showed a complete digestion product at about 0.27 kb and a p a r t i a l digestion product at 0.53 kb, d e m o n s t r a t i n g t h a t the 0.24kb f r a g m e n t was closest to the HindIII site. The a u t o r a d i o g r a m of pCM02 end-labeled at HpaI and p a r t i a l l y digested by BglI showed b a n d s at 0.16, 0.37, a n d 0.61 kb. The only p a t t e r n which would a c c o m m o d a t e these d a t a was the location of two 0.10-kb f r a g m e n t s close to the HpaI site followed by the 0.24-kb f r a g m e n t close to the HindIII site. This was confirmed by the observed HindIII to HpaI distance of 0.91 kb as c o m p a r e d to predicted values of 0.77 kb for a single 0.10-kb f r a g m e n t and 0.87 kb for two 0.10-kb f r a g m e n t s .
pKN82 Cleavage of pKN82 by AvaII produced a series of f r a g m e n t s whose lengths s u m m e d to a size 8.6 kb l a r g e r t h a n the length of pKN82. This resulted f r o m the cleavage of molecules c o n t a i n i n g the G s e g m e n t in opposite o r i e n t a t i o n s t o produce different sized f r a g m e n t s . The i d e n t i t y and location of these f r a g m e n t s was d e t e r m i n e d f r o m the f r a g m e n t sizes r e s u l t i n g f r o m cleavage with AvaII and EcoRI, PstI, or SalI as shown in Fig. 4. D e t e r m i n a t i o n of the G(+) or G ( - ) orientation specificity of the f r a g m e n t s was made as follows: First, it was reasoned t h a t since G s e g m e n t inversion does not alter the total length of Mu DNA, the larger of the AvaII f r a g m e n t s s p a n n i n g the EcoRI site m u s t be derived f r o m the same G orientation as the smaller of the AvaII fragments s p a n n i n g the SalI site and vice versa. Thus, the 3.8- and 4.8-kb f r a g m e n t s m u s t be derived f r o m one o r i e n t a t i o n and the
FIG. 4. Restriction maps of pCM02, pKN82, and pKN56. See legend to Fig. 2. On the maps of pKN82 and pKN56 the large parentheses and the dashed arrow indicate the outer boundaries and the center, respectively, of the invertible G segment and the fragment sizes within brackets indicate sizes derived from the opposite orientation of the G segment. The grouping of the 0.90- and 0.08kb BglI fragments in parentheses with a small arrow below indicates that the order of these fragments, and therefore the precise location of the BglI site between them, is unknown. Not shown on these maps is the presence of a small amount (probably less than 0.2 kb) of host DNA between the ~ end of Mu and the pBR322 (pKN56) or pBR325 (pKN82) vector DNA.
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4.2- and 4.5-kb fragments from the other orientation. Identification of the f r a g m e n t pair present in G(+) DNA (the orientation of G allowing growth by infection of E. coli K12) was made by AvaII digestion of Mu cts62A445-3 DNA. This phage carries the 445-3 deletion which spans the G-/~ boundary and fixes the G segment in the G(+) orientation (Chow et al., 1977). Detection of the 4.8-kb fragment but not the 4.5-kb fragment defined the 4.8- and 3.8kb fragments as those arising from cleavage of G(+) DNA.
Construction and Confirmation of the BglI and A v a I I Maps of Mature Mu D N A The maps of BglI and AvaII restriction sites in Mu DNA (Fig. 5) were derived from the plasmid maps by placing the map of each individual cloned segment in its appropriate position in Mu DNA and determining the sizes of fragments spanning the cloning sites by summing the fragment sizes immediately flanking these sites. To test the size accuracy of these cloning junction fragments, the sizes of restriction fragments produced by AvaII or BglI cleavage of mature Mu DNA were compared to those predicted by the map. In addition, 32p-labeled probes prepared by nick translation of pKN001, pKN35, pKN50, pKN48, pCM02, pKN27, pKN82, pKN56, and Mu DNA (grown in Proteus mirabilis) were hybridized to AvaII and BglI restriction fragments of Mu DNA which had been grown in E. coli, cleaved, electrophoresed in agarose gels, and transferred to nitrocellulose paper by the method of Southern (1975). The latter experiment was designed to detect possible DNA rearrangements in the plasmids and potential comigrating fragments arising from different segments of the Mu genome. When the AvaII restriction pattern of Mu DNA grown in E. coli or P. mirabilis was compared to that predicted by the map, the majority of fragments were those predicted; however, there were two notable exceptions. Mu DNA grown in E. coli contained all the predicted fragments and two additional fragments of 2.9 and 1.1 kb. Mu DNA grown in P. mirabilis had only a small
573
amount of the expected 3.9-kb fragment and had two extra fragments of 2.9 and 1.1 kb. Both the 2.9- and 1.1-kb fragments hybridized nick-translated probe from pKN35, the plasmid containing the 3.9-kb fragment. These data could be most simply explained if the 3.9-kb fragment contained an additional AvaII site (GGTACC) which partially overlapped with an E. coli dcm methylation site (CC~eAGG) (as shown for pBR322 by Backman [1980]) such t h a t plasmid DNA was completely modified and replicating Mu DNA was only partially modified, thus allowing for production of all three fragments. Presumably in P. mirabilis this site was modified more slowly, if at all, thus producing primarily the 2.9and 1.1-kb fragments. The fragment patterns produced by hybridization of the remaining plasmids to AvaII-digested Mu DNA and those produced by hybridization of the plasmids to BglI-digested Mu DNA were completely consistent with the patterns predicted by the maps, thus confirming the accuracy of the maps. These detailed restriction maps should be helpful in continued dissection of Mu genome organization, allowing for more precise localization of proteins, transcripts, specific mutations, and regulatory sites. ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, and by National Science Foundation Grants PCM 75-02465 and PCM 79-04055 to M.M.H. and by a National Institutes of Health Predoctoral Traineeship to C.F.M. on Training Grant GM 07133. M.M.H. is the recipient of Research Career Development Award AI 00274 from the National Institute of Allergy and Infectious Diseases. The authors gratefully acknowledge the assistance of D. Daniels and F. Blattner in the end-labeling experiments. REFERENCES ALLET, B., BLATTNER, F., HOWE, M., MAGAZIN, M., MOORE, D., O'DAY, K., SCHULTZ, D., and SCHUMM, J. (1977). Genetic and physical map of bacteriophage Mu. In "DNA Insertion Elements, Plasmids, and Episomes" (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 745-748. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
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ALLET, B., and BUKHARI,A. I. (1975). Analysis of bacteriophage Mu and },-ME hybrid DNAs by specific endonucleases. J. Mol. BioL 92, 529-540. BACKMAN, K. (1980). A cautionary note on the use of certain restriction endonucleases with methylated substrates. Gene II, 169-171. BOLIVAR, F. (1978). Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique E c o R I sites for selection of EcoRI generated recombinant DNA molecules. Gene 4, 121-136. BOLIVAR, F., RODRIGUEZ,R., GREENE, P. J., BETLACH, M., HEYNEKER, H. L., BOYER, H. W., CROSA,J., and FALKOW, S. (1977). Construction and characterization of new cloning vehicles. Gene 2, 95-113. BUKHARI, A. I. (1976). Bacteriophage Mu as a transposition element. Ann. Rev. Genet. 10, 389-412. BUKHARI, A. I., and ALLET, B. (1975). Plaque-forming k-ME hybrids. Virology 63, 30-39. BUKHARI, A. I., FROSHAUER, S., and BOTCHAN, M. (1976). Ends of bacteriophage Mu DNA. N a t u r e (London) 264, 580-583. BUKHARI, A. I., and TAYLOR, A. L. (1975). Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA. Proc. Nat. Acad. Sci. USA 72, 4399-4403. CHOW, L. W., KAHMANN,R., and KAMP,D. (1977). Electron microscopic characterization of DNAs of nondefective deletion mutants of bacteriophage ME. J. Mol. Biol. 113, 591-609. COHEN, S. N., CHANG,A. C. Y., and Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by Rfactor DNA. Proc. Nat. Acad. Sci. USA 69, 21102114. DANIELL, E., ABELSON, J., KIM, J. S., and DAVIDSON, N. (1973). Heteroduplex structures of bacteriophage Mu DNA. Virology 51, 237-239. DANIELL, E., KOHNE, D. E., and ABELSON, J. (1975). Characterization of the inhomogeneous DNA in virions of bacteriophage Mu by DNA reannealing kinetics. J. Virol. 15, 739-743. DANIELS, D. L., DEWET, J. R., and BLATTNER, F. R. (1980). New map of bacteriophage ~ DNA. J. Virol. 33, 390-400. ENGLER, J. A., FORGIE, R. A., and HOWE, M. M. (1980). Restriction endonuclease B a m H I cleaves bacteriophage Mu DNA within cistrons E and F. Gene 10, 79-83. HATTMAN, S. (1979). Unusual modification of bacteriophage Mu DNA. J. Virol. 32, 468-475. HATTMAN, S. (1980). Specificity of the bacteriophage Mu mom+-controlled DNA modification. J. ViroL 34, 277-279. HELLING, R. B., GOODMAN, H. M., and BOYER, H. W. (1974). Analysis of endonuclease R Eco R1 fragments of DNA from lambdoid bacteriophages and
other viruses by agarose-gel electrophoresis. J. Virol. 14, 1235-1244. HOWE, M. M. (1973). Prophage deletion mapping of bacteriophage ME-1. Virology 54, 93-101. HOWE, M. M., and BADE, E. G. (1975). Molecular biology of bacteriophage ME. Science 190, 624-632. HUMPHREYS, G. 0., WILLSHAW, G. A., and ANDERSON, E. S. (1975). A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 383, 457-463. HUTCHISON III, C. A., and SINSHEIMER, R. L. (1966). The process of infection with bacteriophage ~bX174. Mutations in a ~bX lysis gene. J. Mol. Biol. 18, 429447. KAHMANN, R., KAMP, D., and ZIPSER, D. (1977). Mapping of restriction sites in Mu DNA. In "DNA Insertion Elements, Plasmids, and Episomes" (A. I. Bukhari, J. Shapiro, and S. Adhya, eds.), pp. 335339. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. KAMP, D. (1981). Invertible deoxyribonucleic acid: The G segment of bacteriophage ME. I n "Microbiology1981" (D. Schlessinger, ed.), pp. 73-76. American Society for Microbiology, Washington, D. C. KHATOON, H., and BUKHARI, A. I. (1978). Bacteriophage ME-induced modification of DNA is dependent upon a host function. J. Bacteriol. 136, 423428. MACNEIL, D., HOWE, M. M., and BRILL, W. J. (1980). Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella p n e u m o n i a e n i f genes. J. Bacteriol. 141, 12641271. MAGAZIN, M., HOWE, M., and ALLET, B. (1977). Partial correlation of the genetic and physical maps of bacteriophage ME. Virology 77, 677-688. MANIATIS, T., JEFFREY, A., and KLEID, D. G. (1975). Nucleotide sequence of the rightward operator of phage h. Proc. Nat. Acad. Sci. USA 72, 1184-1188. MAXAM,A. M., and GILBERT, W. (1977). A new method for sequencing DNA. Proc. Nat. Acad. Sci. USA 74, 560-564. MILLER, J. H. (1972). I n "Experiments in Molecular Genetics," Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., p. 431. O'DAY, K., SCHULTZ,D., ERICSEN, W., RAWLUK,L., and HOWE, M. (1979). Correction and refinement of the genetic map of bacteriophage ME. Virology 93, 320328. PAGANO, J. S., and HUTCHISON III, C. A. (1971). Small, circular viral DNA: Preparation and analysis of SV40 and r DNA. Methods in Virology 5, 79123. SANGER, F., AIR, G. M., BARRELL, B. G., BROWN, N. L., COULSON, A. R., FIDDES, J. C., HUTCHISON, III, C. A., SLOCOMBE,P. M., and SMITH,M. (1977).
AvaII AND BglI MAPS OF PHAGE MU Nucleotide sequence of bacteriophage r DNA. Nature (London) 265, 687-695. SCHUMANN,W. (1979). Cloning and biological characterization of the immunity region of Escherichia coli phage Mu. Gene 5, 275-290. SCHUMANN, W., and BADE, E. G. (1979). In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions. Mol. Gen. Genet. 169, 97-105. SCHUMANN,W., BADE, a. G., DELIUS, S., and HUBERT, P. (1978). Cloning of a restriction fragment of phage Mu DNA coding for early functions. Mol. Gen. Genet. 160, 115-118. SCHUMANN,W., BADE, E. G., FORGIE, R. A., and HOWE, M. M. (1980). Cloning of DNA fragments of the right end of phage Mu and location of the HindIII, SaII, PstI, and BamHI restriction sites on the genetic map of Mu. Virology 104, 418-425. SCHUMANN,W., WESTPHAL, C., BADE, E. g., and HOLZER, L. (1979). Origin and binding specificity of proteins coded for by Mu prophages. Mol. Gen. Genet. 173, 189-196. SCHUMM, J. W., MOORE, D. D., BLATTNER, F. R., and HOWE, M. M. (1980). Correlation of the genetic and physical maps in the central region of the bacteriophage Mu genome. Virology 105, 185-195.
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