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Physical map of the dispensable region of the genome of E. coli bacteriophage BF23
Gene, 8 (1980) 369--390 © Elsevier/North-Holland Biomedical Press
369
PHYSICAL MAP OF THE DISPENSABLE REGION OF THE GENOME OF E. coli BACTERIOPHAGE B F 2 3 * (Deletion mutants; gel electrophoresis; restriction endonucleases; single-strand interruptions; genetic crosses)
KIYOTAKA OKADA Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113 (Japan) (Received May 25th, 1979) (Revision received and accepted August 20th, 1979)
SUMMARY Using 13 deletion m ut a nt s of bacteriophage BF23, physical as well as genetic structures of that p o r t i o n o f the genome which is dispensable for phage growth were investigated. The dispensable region covers at least 15% of the genome of wild t y p e BF23, extending from a b o u t 0.2 to 0.35 map unit. Restriction endonuclease ( E coR I and HindIII) cleavage sites and the sites of single-strand interruptions in this dispensable region were localized. It was f o u n d th at the dispensable region contains an interruption site, which is missing in the m u t a n t B F23s t ( 0) used by Okada and Shimura (1980). Wild-type phage DNA is heterogeneous in the presence or absence of specific singlestrand interruptions in this or in a neighboring region of the genome.
INTRODUCTION Through the analyses of m a n y amber mutants, approx. 30 indispensable genes o f bacteriophage BF23 have been identified and m a p p e d (Mizobuchi et al., 1971; McCorquodale, 1975; Mizobuchi K., unpublished results). On the o t h er hand, a set of deletion mutants of BF23 has been isolated t hat do n o t synthesize specific phage-encoded tRNAs t ho ugh t h e y grow normally (Okada et al., 1978). The phage t R N A genes, which seem to be clustered on the phage genome (Ikemura et al., 1 9 7 8 ) , are n o t essential for normal phage growth. In an acco mp an y in g paper, we describe a n o t h e r viable m u t a n t which lacks a portion of the phage genome including a specific site of single-strand interruption. *This work was initiated at the Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606 (Japan).
370 This indicates that the phage genome has a region which is dispensable for normal phage propagation. The dispensable genes of the phage genome are generally more difficult to analyze genetically compared to the genes of indispensable functions. To analyze the dispensable region in the genome of BF23, I u n d e r t o o k a close examination of its physical structure, using several deletion mutants which can grow normally. All the deletions have been mapped in a rather restricted region on the phage genome. The sites cleaved by restriction endonucleases (EcoRI and HindIII) and a site of specific single-strand interruptions (Okada and Shimura, 1980), have been mapped within this region. MATERIALS AND METHODS
Media Nutrient broth (Difco) medium, adjusted to pH 7.2 with NaOH, was used for bacterial growth. Nutrient broth containing 1 mM CaC12 was used for phage growth. Low phosphate medium for preparation of 32p-labeled phage was made according to Sakano et al. (1974). TGAP buffer for phage suspension was prepared according to Mizobuchi et al. (1971). TE buffer containing 20 mM Tris • HC1, and 20 mM Na2EDTA, pH 8.2, was used for heat inactivation of phage particles. Bacterial and phage strains Escherichia coli strain K-12 C R 6 3 was used as host cell. Bacteriophage B F 2 3 strains used are listed in Table I. B F 2 3 s t ( 0 ) is a derivative o f B F 2 3 wild t y p e TABLE I BACTERIOPHAGE
BF23 STRAINS
Strain
Genotype a
wild type st(0) del-1 del-2 del-3 del-4 del-5 dcl-6 del-7 del-lO del-13 del-14 del-15 del-16
+ amber+ amber bfsul am177am218 bfsul am177am218 bfsul--aml 77am218 bfsu2 a m 9 1 a m 2 1 8 bfsu2 am91am218 bfsu3--am91am159 am91 am69 amlllaml41 am91 am91 am91
Buoyant density b (g/ml)
Heat inactivation r a t e c o n s t a n t e (k)
Reference
1.551 1.546 1.542 1.537 1.540 1.544 1.540 1.543 1.540 1.541 1.540 1.539 1.541 1.541
9.9 4.0 1.1-10 4.3"10 n.t. 4 . 6 " 10 n.t. 1.8- 10 5.9"10 n.t. 5.9"10 n.t. n.t. n.t.
M i z o b u c h i e t al. ( 1 9 7 1 ) Okada and Shimura (1980) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) this paper this paper this paper this paper this paper this paper
i : t ~ 2 ~
a A b b r e v i a t i o n s : a m , a m b e r n o n s e n s e m u t a t i o n ; bfsu, n o n s e n s e s u p p r e s s o r m u t a t i o n . b B u o y a n t d e n s i t i e s o f p h a g e p a r t i c l e s w e r e m e a s u r e d b y d e n s i t y g r a d i e n t e e n t r i f u g a t i o n i n CsCl u s i n g T 5 , T 4 , k +, a n d A b 2 b 5 p h a g e s as m a r k e r s . e t I e a t i n a c t i v a t i o n o f p h a g e s was p e r f o r m e d m T E b u f f e r a t 5 7 ~ C , R a t e c o n s t a n t ( k ) w a s c a l c u l a t e d as h = - l n (S/So)]T , w h e r e S / S o was f r a c t i o n of s u r v i v e d p h a g e s in t h e i n c u b a t i o n m i x t u r e w i t h d r a w n at t i m e T . T h e " k " v a l u e l i s t e d w a s c a l c u l a t e d u s i n g t h e v a l u e 7' ( m i n ) a t S]S~ = 0 . 3 7 (= e t ). n.t., not tested.
371 (BF23 ÷) and has been detailed in an accompanying paper (Okada and Shimura, 1980). Six suppressor negative (bfsu-) strains (del-1 to del-6) were isolated from strains carrying nonsense suppressor genes (bfsu +) by heattreatment in TE buffer (Okada et al., 1978). The six strains (del-7, del-lO, del-13 to del-16) were isolated from amber mutants as heat resistant mutants by Dr. K. Mizobuchi.
Preparation and purification of phage stocks BF23 phage stocks were prepared by the confluent lysis m e t h o d (Mizobuchi et al., 1971). Purification of phage stocks by density gradient centrifugation in CsC1 and preparation of 3~P-labeled phages were performed as described previously (Okada and Shimura, 1980).
Preparation of phage DNA and its digestion with restriction endonucleases Phage DNA was prepared from the purified phage suspension as described by Okada and Shimura (1980). Phage DNA was digested with EcoRI in 10 mM Tris- HC1 (pH 7.5), 100 mM NaC1, 7 mM MgC12, and 7 mM 2-mercaptoethanol. For digestion with HindIII, NaC1 was omitted from the reaction mixture for EcoRI digestion. Incubation was continued overnight at 37°C to complete digestion.
Gel electrophoresis Electrophoretic separation of DNA fragments cleaved with restriction endonucleases was done on 0.7% agarose or 5% polyacrylamide slab gels (0.2 × 30 × 30 cm). Agarose gel was prepared as described previously (Okada and Shimura, 1980). Polyacrylamide gel electrophoresis was carried out in 36 mM Tris, 32 mM KH2PO4,,1 mM EDTA, pH 7.8, at 4 V/cm for 24 h at 4°C.
Estimation of molecular weights of DNA fragments The molecular weights of the DNA fragments obtained by digestion with restriction endonucleases were determined from their electrophoretic mobilities in agarose and polyacrylamide gels using DNA fragments of known sizes as references. The fragments of ~ phage DNA cleaved with EcoRI (Thomas and Davis, 1975) and with HindIII (Robinson and Landy, 1977) were employed as the standards. The molecular weights of single-stranded DNA fragments were estimated as described by Okada and Shimura (1980).
Estimation of molar yields of DNA fragments Molar yields of single- or double-strand DNA fragments were estimated in the following ways. In the case of 32P-labeled DNA, the procedure described by Okada and Shimura (1980) was followed. In the case of non-labeled DNA, the molar yield was estimated from the fluorescence of the DNA band stained with ethidium bromide according to the m e t h o d of Pulleyblank et al. (1977). The stained gel was photographed using Fuji F film and traced with a Mitakakoki recording densitometer.
372 Genetic crosses between deletion mutants and isolation o f recombinant phages The procedure for genetic crosses was the same as that described by Okada et al. (1978). Cells were coinfected with two parental phages at a multiplicity of infection of 5 for each phage. After removing cell debris by centrifugation, the lysates were further centrifuged in a CsC1 density gradient, according to the procedure described by Okada et al. (1978). After centrifugation, the phage bands formed in the gradient were photographed and traced with a recording densitometer. Recombinant phages were isolated from the collected fractions of the gradient.
RESULTS Cleavage o f B F 2 3 D N A with restriction endonucleases Complete digestion of wild type DNA with E c o R I or HindIII produced 9 (Eco-A to I), or 12 (Hin-A to L) fragments, respectively. When the DNA was digested with the mixture of E c o R I and HindIII, 20 fragments (d-A to T) were generated. The electrophoretic separation of these fragments in a 0.7% agarose gel is shown in Fig. 1 (A, B, and C, lane 1). The small fragments (Eco-I, Hin-L, and d-Q to T) were detected in 5% polyacrylamide gels (data not shown). As noted in the figure, Hin-E and F fragments have the same electrophoretic mobility in a 0.7% agarose gel (Fig. 1B, lane 1). These two fragments, however, could be distinguished from each other by the following experiments. When the DNA band corresponding to the fragments was extracted from the gel and digested with E c oR I , 4 fragments corresponding to d-D, F, O, and S, were present in molar yield (Okada, K., unpublished results). The sum of the molecular weights of these 4 fragments (13.4 • 106 ) is two times that of the original band (6.7- 106 ). Furthermore, as will be seen later, E c o R I + HindIII digestion of del-1 or del-16 DNA produced d-D but not d-F, O, and S fragments. Since the molecular weight of d-D fragment is the same as that of the original HindIII-generated DNA band, these observations imply that the band contains two different species of DNA fragments of the same molecular weight: one fragment, designated Hin-E, is not cleaved with E c o R I , whereas the other, Hin-F, is cleaved to give rise to d-F, O, and S fragments. Molecular weights and molar yields of the fragments produced by E c o R I , HindIII, or E c o R I + HindIII digestion of BF23 DNA were estimated. The results obtained are summarized in Table II. The molecular weights of the fragments of each digestion sum to 73.5 - 106. This value is in good agreement with the molecular weight of BF23 DNA estimated by electron microscopy (Lang et al., 1976). The electrophoretic gel patterns of DNA fragments from the 13 deletion mutants are also shown in Fig. 1. It should be noted that certain specific frag-
Fig. 1. Electrophoretic separation of DNA fragments of deletion mutants produced by restriction endonucleases, EcoRI (A), HindIII (B), and EcoRI + HindIII (C). Thc fragments were separated in 0.7% agarose gels. (1) wild type; (2) del-4; (3) del-lO; (4) del-13; (5) del-6; (6) del-5; (7) del-1; (8) del-16; (9) del-7 ; (10) del-14; (11) del-15; (12) del-2; (13) del-3; (14) st(0).
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376 TABLE II CLEAVED FRAGMENTS OF WILD TYPE PHAGE DNA The molecular weights of fragments were estimated from electrophoretic mobilities in agarose an polyacrylamide gels using lambda phage DNA fragments obtained by digestion with EcoRI or HindIII as references. The values are averages of 12 independent experiments. The molar yields o the fragments were estimated using 3~P-labeled DNA fragments as described in MATERIALS ANI METHODS. The values are average of 5 independent experiments. Eco RI
Fragment
HindIII
Molecular weight
Molar yield
Fragment
(.10 -6) Eco-A Eco-B Eco-C Eco-D Eco-E Eco-F Eco-G Eco-H Eco-I
Total molecular weight
15.8 14.9 11.6 11.4 10.4 5.4 1.94 1.83 0.24
73.51
EcoRI + HindIII
Molecular weight
Molar yield
Fragment
(.10 6) 1.0 1.0 1.0 1.0 1.1 1.1 0.9 0.9 1.0
Hin-A Hin-B Hin-C Hin-D Hin-E Hin-F Hin-G Hin-H Hin-I Hin-J Hin-K Hin-L
Total molecular weight
14.7 11.3 10.5 7.9 6.7 6.7 4.1 3.9 2.57 2.41 2.29 0.48
73.55
Molecular weight
Molar yield
(-10 6) 0.9 1.1 0.9 1.1 1.0 1.0 1.0 1.0 1.1 1.1 1.1 1.1
d-A d-B d-C d-D d-E d-F d-G d-H d-I d-J d-K d-L d-M d-N d-O d-P d-Q d-R d-S d-T Total molecular weight
11.3 9.2 8.1 6.7 5.8 4.7 4.5 4.1 2.9 2.7 2.55 2.43 2.31 1.85 1.80 1.09 0.66 0.48 0.24 0.11
1.0 1.0 0.9 1.1 1.2 1.0 1.0 0.9 0.9 0.9 0.9 0.9 O.9 0.9 0.9 1.0 1.2 0.9 0.9 1.0
73.52
m e n t ( s ) p r e s e n t in w i l d t y p e is m i s s i n g in t h e r e s p e c t i v e m u t a n t s . I n s t e a d , a n e w f r a g m e n t is d e t e c t e d . F o r e x a m p l e , w h e n del-1 D N A w a s d i g e s t e d w i t h E c o R I , 3 f r a g m e n t s , E c o - F , G , a n d I, w e r e m i s s i n g a n d a n e w f r a g m e n t o f m o l e c u l a r w e i g h t o f 2 . 2 5 . 1 0 6 w a s o b s e r v e d ( F i g . 1 A , l a n e 7). S i m i l a r l y , w h e n digested with HindIII, the Hin-F fragment was absent but a new fragment of 1 . 4 5 - 106 w a s s e e n ( F i g . 1B, l a n e 7). U p o n d i g e s t i o n w i t h E c o R I + H i n d I I I , d - F , O, a n d S f r a g m e n t s w e r e a b s e n t b u t a f r a g m e n t o f 1 . 4 2 • 106 w a s p r e s e n t ( F i g . 1C, l a n e 7). T h u s , t h e t h r e e E c o R I f r a g m e n t s ( E c o - F , G, a n d I), o n e H i n d I I I f r a g m e n t ( H i n - F ) , a n d t h r e e E c o R I + H i n d I I I f r a g m e n t s ( d - F , O, a n d S) m u s t o v e r l a p , t o t a l l y o r p a r t i a l l y , e a c h o t h e r o n t h e B F 2 3 g e n o m e . T h e
del-2 del-3 st(0)
del-1 del-16 del-7 del-14 del-15
del-4 del-lO delbl3 del.6 clel-5
Strain
Eco-F,G,I Eco-D,F,G,I Eco-D,F Eco-F
Eco-F,G,H,I Eco-F,G,I Eco-F,G,I Eco-F,G,I Eco-F,G,I Hin-F Hin-A.F Hin-A,F Hin-A,F Hin-A,F Hin-A,F Hin~A,F
14.0 12.2 11.2 13.1 11.8 1.45 1.50 15.2 15.2 16.0 14.0 15.5 19.0
15.8 19.5 18,3 4.7 2.95 2.25 2.35 1.10 1.15 2.22 11.2 10.9 2.54
Hin-B,F Hin-B,F Hin-B,F Hin-B,F Hin-B,F Hin-F
Eco-A,G,H Eco-A,F,G,H,I Eco-A,F,G,H,I Eco-F,G,H,I
Molecular w e i g h t of newly appearing f r a g m e n t s (. 1 0 - 6 )
Disappearing fragments
Disappearing fragments
Molecular w e i g h t of newly appearing f r a g m e n t s (" 10 - ~ )
HindIII
EcoRI
d-B,N,O,T d-B,F,N,O,S,T d-B,F,N,O,S,T d-F,N,O,S,T d-F,N,O,S,T d-F,O,S d-F,O,S d-F,O,Q,S d-F,O,Q,S d-F,O,Q,S d-A,F,O,Q,S d-A,F,Q d-F,Q
Disappearing fragments
EcoRI + HindIII
9.2 12.5 11.3 4.1 2.35 1.42 1.45 1.00 0.98 2.10 11.0 11.0 2.50
Molecular w e i g h t of newly appearing f r a g m e n t s (, 10 - 6 ) 3.9 5.6 6.8 4.8 6.3 5.3 5.2 6,4 6.3 5.4 7.6 5.8 2.7
Molecular weight (" 1O - 6 )
5.3 7.6 9.3 6.5 8.6 7.2 7.1 8.7 8.6 7.3 10,3 7.9 3.7
% of w h o l e genome
Deleted region
D i s a p p e a r i n g f r a g m e n t s w e r e i d e n t i f i e d f r o m e l e c t r o p h o r e t i c P a t t e r n s in agarose a n d p o l y a c r y l a m i d e gels. T h e m o l e c u l a r w e i g h t s of t h e n e w l y a p p e a r e d f r a g m e n t s were e s t i m a t e d f r o m e l e c t r o p h o r e t i e m o b i l i t i e s in gels. The size of t h e d e l e t e d r e g i o n was c a l c u l a t e d as the s u m of t h e m o l e c u l a r w e i g h t s of d i s a p p e a r e d f r a g m e n t s m i n u s the m o l e c u l a r w e i g h t of the n e w l y a p p e a r e d f r a g m e n t . T h e sizes of the r e g i o n s listed are averages of the t h r e e v a l u e s c a l c u l a t e d i n d e p e n d e n t l y f r o m f r a g m e n t s d i g e s t e d w i t h E c o R L H i n d I I I , or E c o R I + H i n d f I I
A N A L Y S I S O F D E L E T I O N M U T A N T D N A s C L E A V E D BY R E S T R I C T I O N E N D O N U C L E A S E S
TABLE III
"-4
378 sizes of the deletion in the m u t a n t genome can be calculated by subtracting the molecular weight of the new fragment f r om the sum of the molecular weights o f the missing fragments. When this calculation is applied to the DNA fragments p r o d u ced by E c oR I , HindIII, or E c o R I + HindIII digestion of del-1 DNA, the sizes of the deletion of the m u t a n t are 5.33, 5.25, or 5.32 - 106, respectively. These values are in good agreement with each other. Similar analyses were p e r f o r m e d with the rest of the deletion mutants. The results are summarized in Table III. In all cases, the size of the deleted region in the m u t a n t DNA, which was calculated from the sizes of the fragments obtained by an y o n e of the three digestions (EcoRI, HindIII, and E c o R I + HindIII) was consistent with those obtained by the other digestions. The sizes of the deletions in the mutants increase in the order of st(0), del-4, del-6, del-16, del-1, del-15, del-lO, del-3 (del-5, del-14), del-7, del-13, and del-2. It is w ort h noting that this order is consistent with the order of decrease in the b u o y a n t densities of the mu tan ts as well as the increase in heat resistance (see Table I). Th e fact that only one non-canonical fragment was generated in each of the mutants indicates that each m u t a n t strain carries a single deletion m u t a t i o n on the phage genome. Therefore, the cleavage map of the dispensable region m ay be co n s tr u cted by grouping the missing fragments. In the case of E c o R I digestion, six of the nine fragments are affected by deletion mutations. The analyses of these six fragments indicate a fragment order of E co-A --H --G --I-F--D. Similarly, a fragment order of H i n- B - -F --A is indicated from the HindIII digests, and a fragment order of d-B--(N, T)--O--S--F--Q--A is shown from the E c o R I + HindIII digests. The arrangement of d-N and T fragments was determined by comparing the molecular weights of the E c o R I + HindIII fragments with those of the E c o R I fragments. T he molecular weight o f d-N is identical with that of Eco-H, and the sizes of d-S and Eco-I are the same. The molecular weights of d-T and d-O sum to that of Eco-G. It is concluded, therefore, th at the o r d e r is d-B--N--T--O--S--F--Q--A. Since E c o R I + HindIII fragments d-O, d-S, and d-F are pr oduced from the Hin-F fragment by E c o R I digestion, three fragments, d-B, d-N, and d-T, must be the c o m p o n e n t s of the Hin-B fragment. Likewise, both d-Q and d-A fragments are considered to constitute the Hin-A fragment. This interpretation was confirmed by E c o R I digests o f the purified HindIII fragments (Okada, K., unpublished results). The map locations of the fragments of B F23 DNA produced by E c o R I , HindIII, and E c o R I + HindIII digestions are shown in Fig. 6. Alkaline denaturation o f phage D N A We have extensively characterized the physical structure of BF23st(0) DNA (Okada and Shimura, 1980), and showed t h a t the phage DNA has several Fig. 2. Electrophoretic separation of alkali-denatured DNA fragments. Phage DNA was electrophoresed in 0.7% agarose gels containing 1 M urea after denaturation with 0.1 N NaOH. (1) wild type; (2) del-4; (3) del-6; (4) del-lO; (5) del-13; (6) st(0); (7) del-16; (8) del-1; (9) del-15; (10) del-3; (11) del-5; (12) del-14; (13) del-7; (14) del-2.
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380 single-stranded nicks ( 3 ' - h y d r o x y l , 5 ' - p h o s p h o r y l i n t e r r u p t i o n s ) at specific sites o f o n e o f the c o m p l e m e n t a r y strands. T h e i n t e r r u p t i o n s o f wild t y p e as well as m u t a n t DNAs were a n a l y z e d according to the p r o c e d u r e s e m p l o y e d w i t h B F 2 3 s t ( 0 ) DNA. Fig. 2 shows the e l e c t r o p h o r e t i c gel p a t t e r n s o f den a t u r e d D N A fragments. In wild t y p e B F 2 3 DNA, nine major f r a g m e n t s designated F r a g m e n t s I, II', III, IV', V, Xa, Xb, VI, and VII, and several m i n o r f r a g m e n t s were identified (Fig. 2, lane 1). F r a g m e n t s III, V, VI, and VII are the same as t h o s e originally f o u n d in B F 2 3 s t ( 0 ) D N A (Fig. 2, lane 6). F r a g m e n t s Xa and Xb have a l m o s t the same e l e c t r o p h o r e t i c mobilities in a 0.7% agarose gel. However, these t w o f r a g m e n t s c o u l d be distinguished f r o m each o t h e r in s o m e d e l e t i o n m u t a n t s . F o r e x a m p l e , in del-lO DNA, F r a g m e n t Xa is missing b u t F r a g m e n t Xb remains i n t a c t (Fig. 2, lane 4). T h e m o l e c u l a r weights and m o l a r yields o f the m a j o r f r a g m e n t s are listed in Table IV. T h e m o l e c u l a r weight o f F r a g m e n t I is a half o f the d u p l e x phage DNA. Thus, b y analogy w i t h the single-stranded f r a g m e n t s o f B F 2 3 s t ( 0 ) DNA, it is m o s t likely t h a t F r a g m e n t I is the i n t a c t strand, whereas the o t h e r fragments are the c o n s t i t u e n t s o f the complem e n t a r y strand. H o w e v e r , the facts t h a t the sum o f the m o l e c u l a r weights o f F r a g m e n t s II' t o VII (64.7 - 106 ) is a l m o s t t w o times t h a t o f F r a g m e n t I, and t h a t the m o l a r yields o f F r a g m e n t s II', IV', V, Xa, and Xb are significantly l o w e r t h a n t h o s e o f t h e o t h e r fragments ( F r a g m e n t s I, III, VI, and VII) suggest t h a t some o f these f r a g m e n t s r e p r e s e n t h e t e r o g e n e o u s D N A p o p u l a t i o n s , as is the case with B F 2 3 s t ( 0 ) DNA. TABLE IV ALKALINE-DENATURED SINGLE-STRANDED FRAGMENTS OF WILD TYPE PHAGE DNA The molecular weights of single-stranded fragments were estimated from electrophoretic mobilities in 0.7% agarose gels. The molar yields were estimated using 32P-labeled DNA fragments. The values are averages of five independent experiments. Fragment
Molecular weight (-10-')
Molar yield
I II' HI IV' V Xa Xb VI VII
39.0 14.0 13.6 10.3 8.5 5.4 5.4 4.4 3.1
1.0 0.5 1.0 0.1 0.6 1.3 a
0.8 b
0.5 5 1.2 l. 1
a Sum of molar yields of Fragments Xa and Xb calculated from wild type DNA. bIndividual molar yield of Fragments Xa and Xb calculated from del-lO DNA (see text).
381 TABLE
V
MOLECULAR WEIGHTS OF THE NEWLY APPEARING SINGLE-STRANDED FRAGMENTS SIZES OF DELETIONS CALCULATED ACCORDING TO THE DNA STRUCTURE MODEL
AND
The molecular weights of new fragments were estimated from electrophoretic mobilities in 0.7% agarose gels. T h e v a l u e s a r c a v e r a g e s o f f i v e i n d e p e n d e n t e x p e r i m e n t s . T h e m o l e c u l a r w e i g h t o f d e l e t i o n w a s c a l c u l a t e d a c c o r d i n g t o t h e D N A s t r u c t u r e m o d e l s h o w n in F i g . 3. C a l c u l a t i o n s w e r e c a r r i e d o u t as follows: Upper new fragment of group 1 strains; the molecular weight of Fragment IV' minus that of u p p e r n e w f r a g m e n t . T h i s f r a g m e n t , o b s e r v e d b e t w e e n F r a g m e n t s V a n d X b i n F i g . 2, is c o n s i d e r e d t o be a shortened Fragment IV'. Lower new fragment of group 1 strains; the molecular weight of F r a g m e n t X a m i n u s t h a t o f l o w e r n e w f r a g m e n t . T h i s f r a g m e n t , o b s e r v e d u n d e r F r a g m e n t V I , is considered to be a shortened Fragment Xa. Upper new fragment of group 2 strains; the sum of molecular weights of Fragments Xa and II' minus that of upper new fragment. This fragment, observed b e t w e e n F r a g m e n t s I a n d I I I , is c o n s i d e r e d t o b e a f u s e d f r a g m e n t o f F r a g m e n t s X a a n d I I ' . L o w e r n e w fragment of group 2 strains; the sum of molecular weights of Fragments Xa and Xb minus that of lower n e w [ r a g m e n t . T h i s f r a g m e n t , o b s e r v e d b e t w e e n F r a g m e n t s I I I a n d V I , is c o n s i d e r e d t o b e a f u s e d fragment of Fragments Xa and Xb. Strain
M o l e c u l a r w e i g h t (" 1 0 Upper new fragment
6)
Size of deletion calculated
Lower new fragment
Size of deletion calculated
Size of deletion estimated from the cleaved fragments a
group 1
del-4 del-6 del-lO del-13
8.0 7.6 7.0 6.6
2.3 2.7 3.3 3.7
3.4 3.(} 2.5 2.0
2.0 2.4 2.9 3.4
1.95 2.4 2.8 3.4
group 2
st(0) del-16 dcl-1 del-15 del-3 del-5 del-14 del-7 del-2
18.4 16.8 16.5 16.8 16.8 16.1 16.5 16.3 15.5
1.0 2.6 2.9 2.6 2.6 3.3 2.9 3.1 3.9
9.9 8.3 8.4 8.2 8.2 7.8 7.6 7.'7 7.0
0.9 2.5 2.4 2.6 2.6 3.0 3.2 3.1 3.8
1.35 2.6 2.65 2.7 2.9 3.15 3.15 3.2 3.8
aMolecular weight of the deleted region estimated from the DNA fragments produced by digestion with r e s t r i c t i o n e n d o n u c l e a s e s . T h e v a l u e s o f t h i s c o l u m n a r e o n e h a l f o f t h e v a l u e s l i s t e d in T a b l e I I I , i n order to compare the sizes of single-stranded molecules.
In the electrophoretic gel patterns of single-stranded DNA fragments of the deletion mutants, it may be seen that several fragments are affected by the deletion mutations (Fig. 2). In view of the fragment species affected the mutant strains can be classified into two groups. In group 1 strains, Fragments IV' and Xa are simultaneously missing, while two new fragments, one between Fragments V and Xb, and the other below Fragment VI, are present. This group includes del-4, 6, 10, and 13 (Fig. 2, lanes 2 to 5). In group 2 strains, Fragments II', IV', Xa, and Xb are missing, whereas two new fragments are present; one between Fragments I and III, and the other between Fragments III and VI (Fig. 2, lanes 6 to 14). In four deletion mutants, del-16, 1, 15, and 3, the new fragment between Fragments III and VI overlaps with Fragment V (lanes 7 to 10). The molecular weights of the new fragments are variable in different mutants, as shown in Table V. The disappearance of Fragments II', IV', V, Xa, and Xb, as well as the appearance of new fragments in the deletion mutants, and also the fact that the
382 type 1
I
5" VII
Vl
IV
V
III
2
1 2
Xa
' deletiorl I, Xb
Xa
I1'
3 IV'
Fig. 3. Heterogeneous DNA structures of bacteriophage BF23 DNA. (A) Two types of st(0) DNA structures determined by Okada and Shimura (1980). In type 2 DNA, Fragment II replaces Fragments IV and V of type 1 DNA. A DNA portion that covers the single-strand interruption between Fragments Xa and Xb is deleted in st(0) DNA. (B) Three types of wild-type phage DNA. The single-strand interruptions between Fragments Xb and V and that between Fragments Xa and Xb are missing in type 2 and 3 DNA, respectively.
m o l a r yields o f t h e s e f r a g m e n t s are l o w e r t h a n the o t h e r single-stranded fragm e n t s o f wild t y p e p h a g e c o u l d be best e x p l a i n e d b y t h e f o l l o w i n g m o d e l w h i c h is based o n the findings o n t h e p h y s i c a l s t r u c t u r e o f B F 2 3 s t ( 0 ) D N A . We a s s u m e t h a t t h e r e are t h r e e d i f f e r e n t t y p e s o f D N A m o l e c u l e s in wild t y p e B F 2 3 differing in the nicking sites as s h o w n in Fig. 3B. In t y p e 1 D N A , Fragm e n t s Xa, Xb, a n d V are p r e s e n t in this order. In t y p e 2 D N A , F r a g m e n t s Xb a n d V o f t y p e 1 D N A are r e p l a c e d b y F r a g m e n t II'; a n d in t y p e 3 D N A , Fragm e n t s X a and X b of t y p e 1 D N A are s u b s t i t u t e d b y F r a g m e n t IV'. T h e m o l e c ular weights o f t h e f r a g m e n t s are c o n s i s t e n t w i t h these s t r u c t u r e s . T h e struct u r e o f st(0) D N A m a y be e x p l a i n e d if we a s s u m e t h a t the site o f the i n t e r r u p tion b e t w e e n F r a g m e n t s Xa and Xb o f wild t y p e D N A is d e l e t e d in the m u t a n t D N A (Fig. 3A). Since t h e d e l e t e d region o f st(0) D N A was e s t i m a t e d to be 1.35- 106 m o l e c u l a r w e i g h t as single-stranded D N A (Table I I I ) , t h e size o f t h e f u s e d f r a g m e n t o f F r a g m e n t s Xa a n d X b c r e a t e d b y the d e l e t i o n m u t a t i o n was c a l c u l a t e d to be 9.45 • 106, a n d t h a t o f t h e f u s e d f r a g m e n t o f F r a g m e n t s Xa and II' was 1 8 . 0 5 • 106. These values agree w i t h the m o l e c u l a r w e i g h t of Fragm e n t IV (9.9 • 106) a n d t h a t o f F r a g m e n t II (18.4 • 106) of s t ( 0 ) D N A , respectively. T h e d i s a p p e a r a n c e of the specific f r a g m e n t s and t h e a p p e a r a n c e o f n e w f r a g m e n t s in the o t h e r d e l e t i o n m u t a n t s c o u l d be also e x p l a i n e d on the basis of this m o d e l . If we a s s u m e t h a t t h e g r o u p 1 m u t a n t s have d e l e t i o n s w i t h i n F r a g m e n t Xa, F r a g m e n t s Xa a n d IV' s h o u l d d i s a p p e a r , b u t F r a g m e n t s II', III, V, Xb, VI, a n d V I I r e m a i n intact. I n s t e a d , we can e x p e c t the p r e s e n c e of t w o n e w f r a g m e n t s ; o n e derived f r o m F r a g m e n t IV' a n d the o t h e r f r o m F r a g m e n t Xa b y the deletions. In t h e case of g r o u p 2 m u t a n t s , we assume t h a t the delet i o n s c o v e r t h e n i c k i n g site b e t w e e n F r a g m e n t s Xa a n d Xb. C o n s e q u e n t l y , F r a g m e n t s II', IV', Xa, and Xb s h o u l d d i s a p p e a r , a n d t w o new f r a g m e n t s are g e n e r a t e d , one b y fusion o f F r a g m e n t s II' a n d Xa a n d t h e o t h e r b y fusion o f F r a g m e n t s Xa a n d Xb. T h e s e a s s u m p t i o n s w e r e s u p p o r t e d b y t h e sizes o f the d e l e t i o n s c a l c u l a t e d f r o m t h e sizes o f the new f r a g m e n t s . As s h o w n in T a b l e V,
383 the size of the deletion of an any given m u t a n t calculated from the size of one new fragment (upper new fragment) is comparable with that calculated from another new fragment (lower new fragment), indicating that the two new fragments are structurally related as expected from the model of BF23 DNA. Note that the sizes of the deletions thus calculated are in good agreement with the sizes of the deletions estimated from the analysis of the DNA fragments obtained by digestion of the m u t a n t DNAs with restriction endonucleases (Table V). Thus, it may be said that the dispensable region of the BF23 genome is divided into two parts by the site of the single-strand interruption between Fragments Xa and Xb (Fig. 6). The three types of DNA molecules of wild type and the group 1 mutants were apparently present in DNA preparations originally obtained from plaquepurified phages. The relative populations of the three heterogeneous DNAs may be roughly estimated from the molar yields of the single-stranded fragments to be about 40% (type 1), 50% (type 2), and 10% (type 3) in the wild type DNA population. In the group 2 mutants, two types of DNA are present in equal amounts as demonstrated with BF23st(0) (Okada and Shimura, 1980). It is likely that the efficiency of nick formation between Fragments Xb and V is the same in both wild type and st(0) DNAs.
Genetic analysis of overlapping of the deletions In order to construct the deletion maps of the mutants, it would be helpful if we knew whether any pairwise combinations of the mutants have deletions that overlap each other. If the deleted regions of two parental phages do not overlap each other, recombinant phages carrying both of the deletions of the parents as well as the recombinants carrying no deletion are expected to be produced. Such recombinants m a y be detected by centrifuging the progeny phages in CsC1 equilibrium density gradients. On the contrary, if the deleted regions overlap each other even partially, no such recombinants should be produced. First, a cross was carried o u t between st(0) and del-4 strains. From the results of cleavage mapping, the deletions of these two strains were shown not to overlap each other. When progeny phages of the cross were centrifuged in a CsC1 gradient, two small phage bands were observed at b u o y a n t densities of 1.551 and 1.539 in addition to the parental phage bands at 1.546 (st(0)) and 1.544 (del-4) (Fig. 4a). The newly appeared phage bands were not defective, since the ratio of the phages of b u o y a n t densities of 1.551 and 1.539 to the parental phages was increased when the phages of these b u o y a n t densities (indicated by bars 1 and 2 in Fig. 4a) were multiplied on host cells (Fig. 4b and c). Phages banded at a density of 1.551, which is the same as that of wild type phage (Table 1), were proved to be recombinant phages carrying no deletion, since the restriction endonuclease digests of their DNA showed an electrophoretic gel pattern identical to t h a t of wild type DNA. DNA of the phages banded at a density of 1.539, which was designated del-401, was digested with restriction endonucleases, EcoRI and HindIII. As shown in Fig. 5b, the EcoRI
384
I
d
a
iim 4
2
b
J C
/ 1.551 1.J544/ 1.546 1.539
155,1 1.546 1.541
Fig. 4. Profiles of phage bands formed in CsCl equilibrium density gradients. Two deletion strains were crossed and the resultant phage lysates centrifuged in CsC1 gradients. Phage bands were photographed and traced with a recording densitometer. Buoyant densities at the phage bands are indicated in the figure. Phage lysates applied are: (a) progeny phage lysate of a cross between st(0) and del-4 strains; (b) phages multiplied from the fractions indicated by bar 1 in Fig. 4a; (c) phages multiplied from the fractions indicated by bar 2 in Fig. 4a; (d) progeny phage lysate of a cross between st(0) and del-lO strains; (e) phages multiplied from the fractions indicated by bar 3 in Fig. 4d; (f) phages multiplied from the fractions indicated by bar 4 in Fig. 4d. d i g e s t s o f del-401 D N A p r o d u c e E c o - A * f r a g m e n t w h i c h is s p e c i f i c t o del-4 s t r a i n as w e l l as E c o - F * f r a g m e n t w h i c h is s p e c i f i c t o s t ( 0 ) s t r a i n , b u t l a c k E c o - A , F , G, a n d H f r a g m e n t s . B y e l e c t r o p h o r e s i s in a 5% p o l y a c r y l a m i d e gel, Eco-I f r a g m e n t w a s d e t e c t e d . W h e n d i g e s t e d w i t h H i n d I I I , del-401 D N A p r o d u c e s a v e r y l a r g e f r a g m e n t o f 2 6 • 106 d a l t o n s b u t l a c k s Hin-A, B, a n d F fragm e n t s ( F i g . 5e). T h e size o f t h e d e l e t e d r e g i o n o f del-401 D N A c a l c u l a t e d f r o m t h e s i z e o f t h e E c o R I f r a g m e n t s is 6 . 6 3 • 1 0 6 , w h i c h is in g o o d a g r e e m e n t w i t h t h a t c a l c u l a t e d f r o m t h e H i n d I I I f r a g m e n t s , 6 . 7 - 106. T h e s e v a l u e s a g r e e w e l l w i t h t h e s u m o f t h e d e l e t e d r e g i o n s o f b o t h p a r e n t a l s t r a i n s ( 6 . 6 • 106 ). Fig. 5. Electrophoretic separation of DNA fragments of a recombinant and its parent phages produced by restriction endonucleases. The DNA digests were separated in 0.7% agarose gels. (a) EcoRI-digested st(0) DNA; (b) EcoRI-digested DNA of del-401 strain (a recombinant strain of st(0) and del-4 mutants); (c) EcoRI-digested del-4 DNA; (d) HindIIL digested st(0) DNA; (e) HindIII-digested del-401 DNA; (f) HindIII-digested dcl-4 DNA.
385
~rO
~w
\II
II
\/
\1/
I\ <,57"
II\
14,,,,
O~
10 I ©
I /\ Ii ",~ r n o O w
"~OOuJ \\ \II
U_
I
0
.0
(0
I 0
/I II\ ,~rnO(::)w
.I
U_
I\
386 These results indicate t h a t the d e l - 4 0 1 strain carries b o t h of the deletions o f the st(0) and d e l - 4 strains, as e x p e c t e d . Similar maalyses were p e r f o r m e d w i t h some o t h e r c o m b i n a t i o n s of the m u t a n t s and the results are s u m m a r i z e d in Table VI. T h e c e n t r i f u g a t i o n profile of p r o g e n y phages o f a cross b e t w e e n the st(0) and d e l - l O strains in a CsC1 d e n s i t y g r a d i e n t is s h o w n in Fig. 4d. Phages b a n d i n g at a b u o y a n t d e n s i t y o f 1 . 5 5 1 were s h o w n to c a r r y n o deletion as described previously. It is concluded, t h e r e f o r e , t h a t t h e deleted regions o f the st(0) and d e l - l O strains do n o t overlap each other. However, r e c o m b i n a n t phages carrying b o t h o f the p a r e n t a l deletions were n o t d e t e c t e d even after f o u r successive cycles of m u l t i p l i c a t i o n o f the fractions of the d e n s i t y w h e r e the r e c o m b i n a n t phages were e x p e c t e d to b a n d (Fig. 4f). N o t o n l y in the cross b e t w e e n st(0) and del-lO strains, b u t also in crosses of the c o m b i n a t i o n s o f d e l - 3 X d e l - 4 , d e l - 3 X del-lO, a n d del-4 X del-15, r e c o m b i n a n t phages c a r r y i n g b o t h o f the deletions o f the parental phages were n o t detectable, a l t h o u g h r e c o m b i n a n t phages carr y i n g no deletion were isolated (Table VI). Possible e x p l a n a t i o n s o f this p h e n o m e n o n are discussed later.
TABLE VI GENETIC CROSSES BETWEEN DELETION MUTANTS Production of recombinant phages was examined by CsCl equilibrium density gradient centrifugation of the progeny phage lysates, as described in Materials and Methods Cross
Detection of recombinant phages a Recombinants having no deletion
Recombinants having both deletions
st(0) x st(0) x
del-4
+
+
del-lO
+
-
del-3
x
del-4
+
-
del-3
x
del-lO
+
-
del-4
x
deI-15
+
-
del-1
-
-
del-6
-
-
del-13
-
-
st(0) x st(0) x st(0) x del-1
x
del-4
-
-
del-2
x
del-4
-
-
del-3
x
del-6
-
-
del-3
x
del-13
-
-
del-4
x
del-7
-
-
del-4
× del-14
-
-
del-4
x
-
-
del-16
a +, detected;-, not detected.
Sum of the deleted regions of both parental strains Molecular weight (- 10 6)
% of genome
6.6 8.3 9.7 11.4 9.3 8.0 7.5 9.5 9.2 11.5 10.6 12.6 10.3 10.2 9.1
9.0 11.3 13.2 15.5 12.6 10.9 10.2 13.0 12.5 15.6 14.4 17.2 14.0 13.9 12.4
387
In the o th er crosses listed in Table VI, no recom bi nant s of either t y p e were detected. Accordingly, the deleted regions of the parental strains of those combinations overlap each other.
Physical map of the dispensable region of the chromosome of BF23 On the basis of the experimental results described in the preceding sections the physical structure of the dispensable region of BF23 DNA was det erm i ned and the results are summarized in Fig. 6. The six E c o R I fragments, three HindIII fragments, and eight E c o R I + HindIII fragments were m apped in this region. One o f th e single-strand interruptions of BF23 DNA, located bet w een Fragments Xa and Xb, was m a p p e d within the d-F fragment. This site is present in the genome of group I m u t a n t s (del-4, 6, 10, and 13), but deleted in the genome of group 2 m u t a n t s (other 9 mutants). The deleted regions of these m u t a n t s were also m a p p e d as illustrated in Fig. 6. Although the exact size of the dispensable region on the phage genome is n o t known, it may be roughly estimated to be at least 11.4 - 106 daltons (15.5% of whole genome). This value was calculated as the size from the left end of the deletion of del-lO to the right end of the deletion of del-3, since the deletions of these two strains were e x p e c t e d to cover the leftmost and rightmost regions of the deletions, respectively. Thus, the value must be an underestimate o f the size of the dispensable region. According to the complete cleavage map of BF23 whole genome, the dispensable region estimated single-strand fragment EcoR, EcoRI.Hindlll
Hmdlll
A
H
Xa I G
,
F
B
N
T
S
F
B
0
[ F
Xb
I 0
D h
A
del-4 de1.10 del.13 del- 6 de l. 5 del. 1.16 del. 7.14
del.15 de/. 2 del- 3 st(O)
Fig. 6. Physical m a p s of t h e dispensable region a n d d e l e t i o n m a p s o f t h e m u t a n t s o n t h e c h r o m o s o m e o f b a c t e r i o p h a g e B F 2 3 . T h e site o f single-strand i n t e r r u p t i o n a n d r e s u l t i n g s i n g l e - s t r a n d e d f r a g m e n t s ( F r a g m e n t s Xa a n d Xb) are s h o w n o n the top. T h e sites o f cleavages b y E c o R I , H i n d I I I , a n d E c o R I + H i n d I I I a n d resulting f r a g m e n t s are s h o w n b e l o w t h e s i n g l e - s t r a n d e d fragments. In t h e l o w e r p a r t of t h e figure, t h e d e l e t i o n s of various m u t a n t s are m a p p e d as i n d i c a t e d b y b l a c k bars. F o r t h e sake of c o n v e n i e n c e in discussion, t h e over-all region deleted in t h e m u t a n t s e m p l o y e d in t h e p r e s e n t studies is a r b i t r a r i l y subdivided into 10 p o r t i o n s (regions i to 10), c o n s i d e r i n g t h e degrees o f overlaps o f t h e deletions.
388 as above extends from 0.2 to 0.35 map unit within the early gene region (K. Okada, N. Koizumi, and K. Mizobuchi, unpublished results). DISCUSSION A fine physical map of the dispensable region of the bacteriophage BF23 chromosome has been constructed. In order to localize the dispensable region on the phage genome, the genome structures of m a n y deletion mutants that propagate normally on host cells have been compared with that of wild type. The analyses have revealed that the deletions of these m u t a n t s extend from 0.2 to 0.35 map unit covering at least 15.5% of the whole BF23 genome. Although this region is dispensable for phage growth under the laboratory conditions and its genetic c o n t e x t is therefore difficult to analyze, several genes have been identified in this region. From analysis of the phage-encoded transfer RNA species synthesized in cells infected with several deletion mutants, genes for 14 tRNA species whose amino acid specificities are not known have been shown to be located in the dispensable region (Ikemura et al., 1978). Combining those results with the physical map shown in Fig. 6, genes for t R N A species 13, 14, and 15 are mapped at region 2 of Fig. 6, and those for species 16 and 17 at region 3 of the figure. Four tRNA species (1, 8, 10, and 20) and one RNA species of about 7S are synthesized in cells infected with del-1, but not in cells infected with four deletion mutants (del-2, 4, 5, and 6). Five other tRNA species (3, 6, 7, 12, and 18) were synthesized in cells infected with del-4 and st(0), but not in cells infected with five mutants (del-1, 2, 3, 5, and 6). These results suggest that the left endpoints of the del-2 and del-1 deletions are n o t exactly the same, namely the endpoint of the del-2 deletion is at the left side of that of the del-1 deletion, and that genes for the four t R N A species and 7S RNA are located at the interspaced region between the two left endpoints. Similarly, it is suggested that the left endpoint of the del-3 deletion is at the left side of that of the st(0) deletion, and that genes for the five tRNA species are mapped at the interspaced region between the left endpoints of these deletions. Consequently, it is concluded that tRNA genes are clustered in two distinct regions, one extends from regions 2 to 3, and the other is at the left end of the deleted region of del-3. In addition to the tRNA genes, a gene coding for a protein of about 3 • 104 molecular weight appears to be mapped in the region 8 and/or 9, since this protein is synthesized in cells infected with del-lO and del-13, but n o t in cells infected with st(0), del-1, and del-2 (Nagasu, T. and Mizobuchi, M., unpublished results). The structure of the dispensable region of the BF23 chromosome appears to be comparable, in m a n y respects, to that of bacteriophage T5. Von Gabaln et al. (1976) reported that the deletable region of the T5 chromosome spans from 0.19 to 0.34 map unit, comprising 15% of the total chromosome of T5 ÷. More than 16 tRNA genes were mapped in the dispensable region of T5 genome in four discrete portions (Chen et al., 1976; Hunt et al., 1976; Desai et al., 1978). In the genetic crosses of deletion mutants, two types of recombinant
389 phages could be expected, if the deleted regions of the parental strains do n o t overlap each other; t y p e 1 having no deletion, and t y p e 2 having b o t h of the parental deletions. In a cross be t w een the st(0) and del-4 strains, b o t h types o f r e c o m b i n a n t phages emerged. However, in four crosses (st(0) X del-lO, del-3 X del-4, del-3 X del-lO, and del-4 X del-15), t y p e 2 r e c o m b i n a n t phages were n o t d etected , although t y p e I r e c o m b i n a n t phages were isolated. In these crosses, the sum of the deletions o f the t w o parental strains are in the range of 11.3% (st(0) x del-lO) to 15.5% (del-3 X del-lO) of the phage genome (Table VI). The absence of the t y p e 2 r e c o m b i n a n t phages in the four crosses is p r o b ab l y best explained on an assumption t h a t there is a limit in size of the BF23 phage genome which is to be packaged t o produce mature phage particles. The genomes of the t y p e 2 r e c o m b i n a n t phages which are smaller than t h a t o f wild t y p e phage by 11.3 to 15.5%, m a y be t oo small to be packaged. The m u t a n t of BF23 having the largest deletion is del-2, whose deletion spans approx. 10.3% of the genome (Table III). Therefore, it is possible to assume th at BF23 phage DNA longer than 89.7% of wild t y p e DNA could be packaged, b u t phage DNA shorter than 88.7% of wild t y p e DNA could not. This p h e n o m e n o n is pr obabl y relevant to t h a t observed with bacteriophage lambda. When digested with restriction endonuclease EcoRI, lambda phage DNA is cleaved into six fragments: Eco-A to F. Although re-assembled phage DNA lacking either Eco-B (9.8% of phage genome) or Eco-C (11.3%) fragment can f o r m plaques by transfection, phage DNA lacking b o t h Eco-B and C fragments (total 21.1%) can not (Thomas et al., 1974). ACKNOWLEDGEMENTS I t h a n k Dr. K. Mizobuchi for his s u p p o r t with valuable discussions. I also t h a n k Dr. H. Ozeki for valuable discussions. I am indebted to Dr. Y. Shimura for his critical reading of and m a n y valuable c o m m e n t s on the manuscript. I am grateful to Dr. M. Takanami for the gift of enzymes. This w o r k was supp o r t e d by a Scientific Research G r ant f r om the Ministry of E ducat i on of Japan. REFERENCES
Chen, M-J., Locker, J. and Weiss, S.B., The physical mapping of bacteriophage T5 transfer RNAs, J. Biol. Chem., 25] (1976)536--547. Desai, S.M., Hunt, C., Locker, J. and Weiss, S.B., Localization of the arginine tRNA gene to the D segment of T5 bacteriophage DNA, J. Biol. Chem., 253 (1978) 6544--6550. Hunt, C., Hwang, L-T. and Weiss, S.B., Mapping of two isoleucine tRNA isoacceptor genes in bacteriophage T5 DNA, J. Virol., 20 (1976) 63--69. Ikemura, T., Okada, K. and Ozeki, H., Clustering of transfer RNA genes in bacteriophage BF23, Virology, 90 (1978) 142--146. Lang, D., Shaw, A.R. and McCorquodale, D.J., Molecular weights of DNA from bacteriophage T5, T5st(0), BF23, and BF23st(4), J. Virol., 17 (1976) 296--297. McCorquodale, D.J., The T-odd bacteriophages, CRC Crit. Rev. Microbiol., 4 (1975) 101--154.
390 Mizobuchi, K., Anderson, G.C. and MeCorquodale, D.J., Abortive infection by bacteriophage BF23 due to the colicin Ib factor, I. Genetic studies of nonrestricted and amber mutants of bacteriophage BF23, Genetics, 68 (1971) 323--340. Okada, K., Ohira, M. and Ozeki, H., Nonsense suppressor mutants of bacteriophage BF23, Virology, 90 (1978) 133--141. Okada, K. and Shimura, Y., Arrangement of the single-stranded fragments in E. coli bacteriophage BF23 DNA, Gene, 8 (1980) 345--368. Pulleyblank, D.E., Shure, M. and Vinograd, J., The quantitation of fluorescence by photography, Nucleic Acids Res., 4 (1977) 1409--1418. Robinson, J.H. and Landy, A., HindII, HindIII, and HpaI restriction fragment maps of bacteriophage ~ DNA, Gene, 2 (1977) 1--31. Sakano, H., Yamada, S., Ikemura, T., Shimura, Y. and Ozeki, H., Temperature sensitive mutants of Escherichia coli for t R N A synthesis, Nucl. Acids Res., 1 (1974) 355--371. Thomas, M., Cameron, J.R. and Davis, R.W., Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA, Proc. Natl. Acad. Sci. USA, 71 (1974) 4579--4583. Thomas, M. and Davis, R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91 (1975) 315--328. Von Gabain, A., Hayward, G.S. and Bujard, H., Physical mapping of the HindIII, EcoRI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA, Moi. Gen. Genet., 143 (1976) 279--290. Communicated by K. Matsubara.
369
PHYSICAL MAP OF THE DISPENSABLE REGION OF THE GENOME OF E. coli BACTERIOPHAGE B F 2 3 * (Deletion mutants; gel electrophoresis; restriction endonucleases; single-strand interruptions; genetic crosses)
KIYOTAKA OKADA Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113 (Japan) (Received May 25th, 1979) (Revision received and accepted August 20th, 1979)
SUMMARY Using 13 deletion m ut a nt s of bacteriophage BF23, physical as well as genetic structures of that p o r t i o n o f the genome which is dispensable for phage growth were investigated. The dispensable region covers at least 15% of the genome of wild t y p e BF23, extending from a b o u t 0.2 to 0.35 map unit. Restriction endonuclease ( E coR I and HindIII) cleavage sites and the sites of single-strand interruptions in this dispensable region were localized. It was f o u n d th at the dispensable region contains an interruption site, which is missing in the m u t a n t B F23s t ( 0) used by Okada and Shimura (1980). Wild-type phage DNA is heterogeneous in the presence or absence of specific singlestrand interruptions in this or in a neighboring region of the genome.
INTRODUCTION Through the analyses of m a n y amber mutants, approx. 30 indispensable genes o f bacteriophage BF23 have been identified and m a p p e d (Mizobuchi et al., 1971; McCorquodale, 1975; Mizobuchi K., unpublished results). On the o t h er hand, a set of deletion mutants of BF23 has been isolated t hat do n o t synthesize specific phage-encoded tRNAs t ho ugh t h e y grow normally (Okada et al., 1978). The phage t R N A genes, which seem to be clustered on the phage genome (Ikemura et al., 1 9 7 8 ) , are n o t essential for normal phage growth. In an acco mp an y in g paper, we describe a n o t h e r viable m u t a n t which lacks a portion of the phage genome including a specific site of single-strand interruption. *This work was initiated at the Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606 (Japan).
370 This indicates that the phage genome has a region which is dispensable for normal phage propagation. The dispensable genes of the phage genome are generally more difficult to analyze genetically compared to the genes of indispensable functions. To analyze the dispensable region in the genome of BF23, I u n d e r t o o k a close examination of its physical structure, using several deletion mutants which can grow normally. All the deletions have been mapped in a rather restricted region on the phage genome. The sites cleaved by restriction endonucleases (EcoRI and HindIII) and a site of specific single-strand interruptions (Okada and Shimura, 1980), have been mapped within this region. MATERIALS AND METHODS
Media Nutrient broth (Difco) medium, adjusted to pH 7.2 with NaOH, was used for bacterial growth. Nutrient broth containing 1 mM CaC12 was used for phage growth. Low phosphate medium for preparation of 32p-labeled phage was made according to Sakano et al. (1974). TGAP buffer for phage suspension was prepared according to Mizobuchi et al. (1971). TE buffer containing 20 mM Tris • HC1, and 20 mM Na2EDTA, pH 8.2, was used for heat inactivation of phage particles. Bacterial and phage strains Escherichia coli strain K-12 C R 6 3 was used as host cell. Bacteriophage B F 2 3 strains used are listed in Table I. B F 2 3 s t ( 0 ) is a derivative o f B F 2 3 wild t y p e TABLE I BACTERIOPHAGE
BF23 STRAINS
Strain
Genotype a
wild type st(0) del-1 del-2 del-3 del-4 del-5 dcl-6 del-7 del-lO del-13 del-14 del-15 del-16
+ amber+ amber bfsul am177am218 bfsul am177am218 bfsul--aml 77am218 bfsu2 a m 9 1 a m 2 1 8 bfsu2 am91am218 bfsu3--am91am159 am91 am69 amlllaml41 am91 am91 am91
Buoyant density b (g/ml)
Heat inactivation r a t e c o n s t a n t e (k)
Reference
1.551 1.546 1.542 1.537 1.540 1.544 1.540 1.543 1.540 1.541 1.540 1.539 1.541 1.541
9.9 4.0 1.1-10 4.3"10 n.t. 4 . 6 " 10 n.t. 1.8- 10 5.9"10 n.t. 5.9"10 n.t. n.t. n.t.
M i z o b u c h i e t al. ( 1 9 7 1 ) Okada and Shimura (1980) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) O k a d a e t al. ( 1 9 7 8 ) this paper this paper this paper this paper this paper this paper
i : t ~ 2 ~
a A b b r e v i a t i o n s : a m , a m b e r n o n s e n s e m u t a t i o n ; bfsu, n o n s e n s e s u p p r e s s o r m u t a t i o n . b B u o y a n t d e n s i t i e s o f p h a g e p a r t i c l e s w e r e m e a s u r e d b y d e n s i t y g r a d i e n t e e n t r i f u g a t i o n i n CsCl u s i n g T 5 , T 4 , k +, a n d A b 2 b 5 p h a g e s as m a r k e r s . e t I e a t i n a c t i v a t i o n o f p h a g e s was p e r f o r m e d m T E b u f f e r a t 5 7 ~ C , R a t e c o n s t a n t ( k ) w a s c a l c u l a t e d as h = - l n (S/So)]T , w h e r e S / S o was f r a c t i o n of s u r v i v e d p h a g e s in t h e i n c u b a t i o n m i x t u r e w i t h d r a w n at t i m e T . T h e " k " v a l u e l i s t e d w a s c a l c u l a t e d u s i n g t h e v a l u e 7' ( m i n ) a t S]S~ = 0 . 3 7 (= e t ). n.t., not tested.
371 (BF23 ÷) and has been detailed in an accompanying paper (Okada and Shimura, 1980). Six suppressor negative (bfsu-) strains (del-1 to del-6) were isolated from strains carrying nonsense suppressor genes (bfsu +) by heattreatment in TE buffer (Okada et al., 1978). The six strains (del-7, del-lO, del-13 to del-16) were isolated from amber mutants as heat resistant mutants by Dr. K. Mizobuchi.
Preparation and purification of phage stocks BF23 phage stocks were prepared by the confluent lysis m e t h o d (Mizobuchi et al., 1971). Purification of phage stocks by density gradient centrifugation in CsC1 and preparation of 3~P-labeled phages were performed as described previously (Okada and Shimura, 1980).
Preparation of phage DNA and its digestion with restriction endonucleases Phage DNA was prepared from the purified phage suspension as described by Okada and Shimura (1980). Phage DNA was digested with EcoRI in 10 mM Tris- HC1 (pH 7.5), 100 mM NaC1, 7 mM MgC12, and 7 mM 2-mercaptoethanol. For digestion with HindIII, NaC1 was omitted from the reaction mixture for EcoRI digestion. Incubation was continued overnight at 37°C to complete digestion.
Gel electrophoresis Electrophoretic separation of DNA fragments cleaved with restriction endonucleases was done on 0.7% agarose or 5% polyacrylamide slab gels (0.2 × 30 × 30 cm). Agarose gel was prepared as described previously (Okada and Shimura, 1980). Polyacrylamide gel electrophoresis was carried out in 36 mM Tris, 32 mM KH2PO4,,1 mM EDTA, pH 7.8, at 4 V/cm for 24 h at 4°C.
Estimation of molecular weights of DNA fragments The molecular weights of the DNA fragments obtained by digestion with restriction endonucleases were determined from their electrophoretic mobilities in agarose and polyacrylamide gels using DNA fragments of known sizes as references. The fragments of ~ phage DNA cleaved with EcoRI (Thomas and Davis, 1975) and with HindIII (Robinson and Landy, 1977) were employed as the standards. The molecular weights of single-stranded DNA fragments were estimated as described by Okada and Shimura (1980).
Estimation of molar yields of DNA fragments Molar yields of single- or double-strand DNA fragments were estimated in the following ways. In the case of 32P-labeled DNA, the procedure described by Okada and Shimura (1980) was followed. In the case of non-labeled DNA, the molar yield was estimated from the fluorescence of the DNA band stained with ethidium bromide according to the m e t h o d of Pulleyblank et al. (1977). The stained gel was photographed using Fuji F film and traced with a Mitakakoki recording densitometer.
372 Genetic crosses between deletion mutants and isolation o f recombinant phages The procedure for genetic crosses was the same as that described by Okada et al. (1978). Cells were coinfected with two parental phages at a multiplicity of infection of 5 for each phage. After removing cell debris by centrifugation, the lysates were further centrifuged in a CsC1 density gradient, according to the procedure described by Okada et al. (1978). After centrifugation, the phage bands formed in the gradient were photographed and traced with a recording densitometer. Recombinant phages were isolated from the collected fractions of the gradient.
RESULTS Cleavage o f B F 2 3 D N A with restriction endonucleases Complete digestion of wild type DNA with E c o R I or HindIII produced 9 (Eco-A to I), or 12 (Hin-A to L) fragments, respectively. When the DNA was digested with the mixture of E c o R I and HindIII, 20 fragments (d-A to T) were generated. The electrophoretic separation of these fragments in a 0.7% agarose gel is shown in Fig. 1 (A, B, and C, lane 1). The small fragments (Eco-I, Hin-L, and d-Q to T) were detected in 5% polyacrylamide gels (data not shown). As noted in the figure, Hin-E and F fragments have the same electrophoretic mobility in a 0.7% agarose gel (Fig. 1B, lane 1). These two fragments, however, could be distinguished from each other by the following experiments. When the DNA band corresponding to the fragments was extracted from the gel and digested with E c oR I , 4 fragments corresponding to d-D, F, O, and S, were present in molar yield (Okada, K., unpublished results). The sum of the molecular weights of these 4 fragments (13.4 • 106 ) is two times that of the original band (6.7- 106 ). Furthermore, as will be seen later, E c o R I + HindIII digestion of del-1 or del-16 DNA produced d-D but not d-F, O, and S fragments. Since the molecular weight of d-D fragment is the same as that of the original HindIII-generated DNA band, these observations imply that the band contains two different species of DNA fragments of the same molecular weight: one fragment, designated Hin-E, is not cleaved with E c o R I , whereas the other, Hin-F, is cleaved to give rise to d-F, O, and S fragments. Molecular weights and molar yields of the fragments produced by E c o R I , HindIII, or E c o R I + HindIII digestion of BF23 DNA were estimated. The results obtained are summarized in Table II. The molecular weights of the fragments of each digestion sum to 73.5 - 106. This value is in good agreement with the molecular weight of BF23 DNA estimated by electron microscopy (Lang et al., 1976). The electrophoretic gel patterns of DNA fragments from the 13 deletion mutants are also shown in Fig. 1. It should be noted that certain specific frag-
Fig. 1. Electrophoretic separation of DNA fragments of deletion mutants produced by restriction endonucleases, EcoRI (A), HindIII (B), and EcoRI + HindIII (C). Thc fragments were separated in 0.7% agarose gels. (1) wild type; (2) del-4; (3) del-lO; (4) del-13; (5) del-6; (6) del-5; (7) del-1; (8) del-16; (9) del-7 ; (10) del-14; (11) del-15; (12) del-2; (13) del-3; (14) st(0).
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376 TABLE II CLEAVED FRAGMENTS OF WILD TYPE PHAGE DNA The molecular weights of fragments were estimated from electrophoretic mobilities in agarose an polyacrylamide gels using lambda phage DNA fragments obtained by digestion with EcoRI or HindIII as references. The values are averages of 12 independent experiments. The molar yields o the fragments were estimated using 3~P-labeled DNA fragments as described in MATERIALS ANI METHODS. The values are average of 5 independent experiments. Eco RI
Fragment
HindIII
Molecular weight
Molar yield
Fragment
(.10 -6) Eco-A Eco-B Eco-C Eco-D Eco-E Eco-F Eco-G Eco-H Eco-I
Total molecular weight
15.8 14.9 11.6 11.4 10.4 5.4 1.94 1.83 0.24
73.51
EcoRI + HindIII
Molecular weight
Molar yield
Fragment
(.10 6) 1.0 1.0 1.0 1.0 1.1 1.1 0.9 0.9 1.0
Hin-A Hin-B Hin-C Hin-D Hin-E Hin-F Hin-G Hin-H Hin-I Hin-J Hin-K Hin-L
Total molecular weight
14.7 11.3 10.5 7.9 6.7 6.7 4.1 3.9 2.57 2.41 2.29 0.48
73.55
Molecular weight
Molar yield
(-10 6) 0.9 1.1 0.9 1.1 1.0 1.0 1.0 1.0 1.1 1.1 1.1 1.1
d-A d-B d-C d-D d-E d-F d-G d-H d-I d-J d-K d-L d-M d-N d-O d-P d-Q d-R d-S d-T Total molecular weight
11.3 9.2 8.1 6.7 5.8 4.7 4.5 4.1 2.9 2.7 2.55 2.43 2.31 1.85 1.80 1.09 0.66 0.48 0.24 0.11
1.0 1.0 0.9 1.1 1.2 1.0 1.0 0.9 0.9 0.9 0.9 0.9 O.9 0.9 0.9 1.0 1.2 0.9 0.9 1.0
73.52
m e n t ( s ) p r e s e n t in w i l d t y p e is m i s s i n g in t h e r e s p e c t i v e m u t a n t s . I n s t e a d , a n e w f r a g m e n t is d e t e c t e d . F o r e x a m p l e , w h e n del-1 D N A w a s d i g e s t e d w i t h E c o R I , 3 f r a g m e n t s , E c o - F , G , a n d I, w e r e m i s s i n g a n d a n e w f r a g m e n t o f m o l e c u l a r w e i g h t o f 2 . 2 5 . 1 0 6 w a s o b s e r v e d ( F i g . 1 A , l a n e 7). S i m i l a r l y , w h e n digested with HindIII, the Hin-F fragment was absent but a new fragment of 1 . 4 5 - 106 w a s s e e n ( F i g . 1B, l a n e 7). U p o n d i g e s t i o n w i t h E c o R I + H i n d I I I , d - F , O, a n d S f r a g m e n t s w e r e a b s e n t b u t a f r a g m e n t o f 1 . 4 2 • 106 w a s p r e s e n t ( F i g . 1C, l a n e 7). T h u s , t h e t h r e e E c o R I f r a g m e n t s ( E c o - F , G, a n d I), o n e H i n d I I I f r a g m e n t ( H i n - F ) , a n d t h r e e E c o R I + H i n d I I I f r a g m e n t s ( d - F , O, a n d S) m u s t o v e r l a p , t o t a l l y o r p a r t i a l l y , e a c h o t h e r o n t h e B F 2 3 g e n o m e . T h e
del-2 del-3 st(0)
del-1 del-16 del-7 del-14 del-15
del-4 del-lO delbl3 del.6 clel-5
Strain
Eco-F,G,I Eco-D,F,G,I Eco-D,F Eco-F
Eco-F,G,H,I Eco-F,G,I Eco-F,G,I Eco-F,G,I Eco-F,G,I Hin-F Hin-A.F Hin-A,F Hin-A,F Hin-A,F Hin-A,F Hin~A,F
14.0 12.2 11.2 13.1 11.8 1.45 1.50 15.2 15.2 16.0 14.0 15.5 19.0
15.8 19.5 18,3 4.7 2.95 2.25 2.35 1.10 1.15 2.22 11.2 10.9 2.54
Hin-B,F Hin-B,F Hin-B,F Hin-B,F Hin-B,F Hin-F
Eco-A,G,H Eco-A,F,G,H,I Eco-A,F,G,H,I Eco-F,G,H,I
Molecular w e i g h t of newly appearing f r a g m e n t s (. 1 0 - 6 )
Disappearing fragments
Disappearing fragments
Molecular w e i g h t of newly appearing f r a g m e n t s (" 10 - ~ )
HindIII
EcoRI
d-B,N,O,T d-B,F,N,O,S,T d-B,F,N,O,S,T d-F,N,O,S,T d-F,N,O,S,T d-F,O,S d-F,O,S d-F,O,Q,S d-F,O,Q,S d-F,O,Q,S d-A,F,O,Q,S d-A,F,Q d-F,Q
Disappearing fragments
EcoRI + HindIII
9.2 12.5 11.3 4.1 2.35 1.42 1.45 1.00 0.98 2.10 11.0 11.0 2.50
Molecular w e i g h t of newly appearing f r a g m e n t s (, 10 - 6 ) 3.9 5.6 6.8 4.8 6.3 5.3 5.2 6,4 6.3 5.4 7.6 5.8 2.7
Molecular weight (" 1O - 6 )
5.3 7.6 9.3 6.5 8.6 7.2 7.1 8.7 8.6 7.3 10,3 7.9 3.7
% of w h o l e genome
Deleted region
D i s a p p e a r i n g f r a g m e n t s w e r e i d e n t i f i e d f r o m e l e c t r o p h o r e t i c P a t t e r n s in agarose a n d p o l y a c r y l a m i d e gels. T h e m o l e c u l a r w e i g h t s of t h e n e w l y a p p e a r e d f r a g m e n t s were e s t i m a t e d f r o m e l e c t r o p h o r e t i e m o b i l i t i e s in gels. The size of t h e d e l e t e d r e g i o n was c a l c u l a t e d as the s u m of t h e m o l e c u l a r w e i g h t s of d i s a p p e a r e d f r a g m e n t s m i n u s the m o l e c u l a r w e i g h t of the n e w l y a p p e a r e d f r a g m e n t . T h e sizes of the r e g i o n s listed are averages of the t h r e e v a l u e s c a l c u l a t e d i n d e p e n d e n t l y f r o m f r a g m e n t s d i g e s t e d w i t h E c o R L H i n d I I I , or E c o R I + H i n d f I I
A N A L Y S I S O F D E L E T I O N M U T A N T D N A s C L E A V E D BY R E S T R I C T I O N E N D O N U C L E A S E S
TABLE III
"-4
378 sizes of the deletion in the m u t a n t genome can be calculated by subtracting the molecular weight of the new fragment f r om the sum of the molecular weights o f the missing fragments. When this calculation is applied to the DNA fragments p r o d u ced by E c oR I , HindIII, or E c o R I + HindIII digestion of del-1 DNA, the sizes of the deletion of the m u t a n t are 5.33, 5.25, or 5.32 - 106, respectively. These values are in good agreement with each other. Similar analyses were p e r f o r m e d with the rest of the deletion mutants. The results are summarized in Table III. In all cases, the size of the deleted region in the m u t a n t DNA, which was calculated from the sizes of the fragments obtained by an y o n e of the three digestions (EcoRI, HindIII, and E c o R I + HindIII) was consistent with those obtained by the other digestions. The sizes of the deletions in the mutants increase in the order of st(0), del-4, del-6, del-16, del-1, del-15, del-lO, del-3 (del-5, del-14), del-7, del-13, and del-2. It is w ort h noting that this order is consistent with the order of decrease in the b u o y a n t densities of the mu tan ts as well as the increase in heat resistance (see Table I). Th e fact that only one non-canonical fragment was generated in each of the mutants indicates that each m u t a n t strain carries a single deletion m u t a t i o n on the phage genome. Therefore, the cleavage map of the dispensable region m ay be co n s tr u cted by grouping the missing fragments. In the case of E c o R I digestion, six of the nine fragments are affected by deletion mutations. The analyses of these six fragments indicate a fragment order of E co-A --H --G --I-F--D. Similarly, a fragment order of H i n- B - -F --A is indicated from the HindIII digests, and a fragment order of d-B--(N, T)--O--S--F--Q--A is shown from the E c o R I + HindIII digests. The arrangement of d-N and T fragments was determined by comparing the molecular weights of the E c o R I + HindIII fragments with those of the E c o R I fragments. T he molecular weight o f d-N is identical with that of Eco-H, and the sizes of d-S and Eco-I are the same. The molecular weights of d-T and d-O sum to that of Eco-G. It is concluded, therefore, th at the o r d e r is d-B--N--T--O--S--F--Q--A. Since E c o R I + HindIII fragments d-O, d-S, and d-F are pr oduced from the Hin-F fragment by E c o R I digestion, three fragments, d-B, d-N, and d-T, must be the c o m p o n e n t s of the Hin-B fragment. Likewise, both d-Q and d-A fragments are considered to constitute the Hin-A fragment. This interpretation was confirmed by E c o R I digests o f the purified HindIII fragments (Okada, K., unpublished results). The map locations of the fragments of B F23 DNA produced by E c o R I , HindIII, and E c o R I + HindIII digestions are shown in Fig. 6. Alkaline denaturation o f phage D N A We have extensively characterized the physical structure of BF23st(0) DNA (Okada and Shimura, 1980), and showed t h a t the phage DNA has several Fig. 2. Electrophoretic separation of alkali-denatured DNA fragments. Phage DNA was electrophoresed in 0.7% agarose gels containing 1 M urea after denaturation with 0.1 N NaOH. (1) wild type; (2) del-4; (3) del-6; (4) del-lO; (5) del-13; (6) st(0); (7) del-16; (8) del-1; (9) del-15; (10) del-3; (11) del-5; (12) del-14; (13) del-7; (14) del-2.
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380 single-stranded nicks ( 3 ' - h y d r o x y l , 5 ' - p h o s p h o r y l i n t e r r u p t i o n s ) at specific sites o f o n e o f the c o m p l e m e n t a r y strands. T h e i n t e r r u p t i o n s o f wild t y p e as well as m u t a n t DNAs were a n a l y z e d according to the p r o c e d u r e s e m p l o y e d w i t h B F 2 3 s t ( 0 ) DNA. Fig. 2 shows the e l e c t r o p h o r e t i c gel p a t t e r n s o f den a t u r e d D N A fragments. In wild t y p e B F 2 3 DNA, nine major f r a g m e n t s designated F r a g m e n t s I, II', III, IV', V, Xa, Xb, VI, and VII, and several m i n o r f r a g m e n t s were identified (Fig. 2, lane 1). F r a g m e n t s III, V, VI, and VII are the same as t h o s e originally f o u n d in B F 2 3 s t ( 0 ) D N A (Fig. 2, lane 6). F r a g m e n t s Xa and Xb have a l m o s t the same e l e c t r o p h o r e t i c mobilities in a 0.7% agarose gel. However, these t w o f r a g m e n t s c o u l d be distinguished f r o m each o t h e r in s o m e d e l e t i o n m u t a n t s . F o r e x a m p l e , in del-lO DNA, F r a g m e n t Xa is missing b u t F r a g m e n t Xb remains i n t a c t (Fig. 2, lane 4). T h e m o l e c u l a r weights and m o l a r yields o f the m a j o r f r a g m e n t s are listed in Table IV. T h e m o l e c u l a r weight o f F r a g m e n t I is a half o f the d u p l e x phage DNA. Thus, b y analogy w i t h the single-stranded f r a g m e n t s o f B F 2 3 s t ( 0 ) DNA, it is m o s t likely t h a t F r a g m e n t I is the i n t a c t strand, whereas the o t h e r fragments are the c o n s t i t u e n t s o f the complem e n t a r y strand. H o w e v e r , the facts t h a t the sum o f the m o l e c u l a r weights o f F r a g m e n t s II' t o VII (64.7 - 106 ) is a l m o s t t w o times t h a t o f F r a g m e n t I, and t h a t the m o l a r yields o f F r a g m e n t s II', IV', V, Xa, and Xb are significantly l o w e r t h a n t h o s e o f t h e o t h e r fragments ( F r a g m e n t s I, III, VI, and VII) suggest t h a t some o f these f r a g m e n t s r e p r e s e n t h e t e r o g e n e o u s D N A p o p u l a t i o n s , as is the case with B F 2 3 s t ( 0 ) DNA. TABLE IV ALKALINE-DENATURED SINGLE-STRANDED FRAGMENTS OF WILD TYPE PHAGE DNA The molecular weights of single-stranded fragments were estimated from electrophoretic mobilities in 0.7% agarose gels. The molar yields were estimated using 32P-labeled DNA fragments. The values are averages of five independent experiments. Fragment
Molecular weight (-10-')
Molar yield
I II' HI IV' V Xa Xb VI VII
39.0 14.0 13.6 10.3 8.5 5.4 5.4 4.4 3.1
1.0 0.5 1.0 0.1 0.6 1.3 a
0.8 b
0.5 5 1.2 l. 1
a Sum of molar yields of Fragments Xa and Xb calculated from wild type DNA. bIndividual molar yield of Fragments Xa and Xb calculated from del-lO DNA (see text).
381 TABLE
V
MOLECULAR WEIGHTS OF THE NEWLY APPEARING SINGLE-STRANDED FRAGMENTS SIZES OF DELETIONS CALCULATED ACCORDING TO THE DNA STRUCTURE MODEL
AND
The molecular weights of new fragments were estimated from electrophoretic mobilities in 0.7% agarose gels. T h e v a l u e s a r c a v e r a g e s o f f i v e i n d e p e n d e n t e x p e r i m e n t s . T h e m o l e c u l a r w e i g h t o f d e l e t i o n w a s c a l c u l a t e d a c c o r d i n g t o t h e D N A s t r u c t u r e m o d e l s h o w n in F i g . 3. C a l c u l a t i o n s w e r e c a r r i e d o u t as follows: Upper new fragment of group 1 strains; the molecular weight of Fragment IV' minus that of u p p e r n e w f r a g m e n t . T h i s f r a g m e n t , o b s e r v e d b e t w e e n F r a g m e n t s V a n d X b i n F i g . 2, is c o n s i d e r e d t o be a shortened Fragment IV'. Lower new fragment of group 1 strains; the molecular weight of F r a g m e n t X a m i n u s t h a t o f l o w e r n e w f r a g m e n t . T h i s f r a g m e n t , o b s e r v e d u n d e r F r a g m e n t V I , is considered to be a shortened Fragment Xa. Upper new fragment of group 2 strains; the sum of molecular weights of Fragments Xa and II' minus that of upper new fragment. This fragment, observed b e t w e e n F r a g m e n t s I a n d I I I , is c o n s i d e r e d t o b e a f u s e d f r a g m e n t o f F r a g m e n t s X a a n d I I ' . L o w e r n e w fragment of group 2 strains; the sum of molecular weights of Fragments Xa and Xb minus that of lower n e w [ r a g m e n t . T h i s f r a g m e n t , o b s e r v e d b e t w e e n F r a g m e n t s I I I a n d V I , is c o n s i d e r e d t o b e a f u s e d fragment of Fragments Xa and Xb. Strain
M o l e c u l a r w e i g h t (" 1 0 Upper new fragment
6)
Size of deletion calculated
Lower new fragment
Size of deletion calculated
Size of deletion estimated from the cleaved fragments a
group 1
del-4 del-6 del-lO del-13
8.0 7.6 7.0 6.6
2.3 2.7 3.3 3.7
3.4 3.(} 2.5 2.0
2.0 2.4 2.9 3.4
1.95 2.4 2.8 3.4
group 2
st(0) del-16 dcl-1 del-15 del-3 del-5 del-14 del-7 del-2
18.4 16.8 16.5 16.8 16.8 16.1 16.5 16.3 15.5
1.0 2.6 2.9 2.6 2.6 3.3 2.9 3.1 3.9
9.9 8.3 8.4 8.2 8.2 7.8 7.6 7.'7 7.0
0.9 2.5 2.4 2.6 2.6 3.0 3.2 3.1 3.8
1.35 2.6 2.65 2.7 2.9 3.15 3.15 3.2 3.8
aMolecular weight of the deleted region estimated from the DNA fragments produced by digestion with r e s t r i c t i o n e n d o n u c l e a s e s . T h e v a l u e s o f t h i s c o l u m n a r e o n e h a l f o f t h e v a l u e s l i s t e d in T a b l e I I I , i n order to compare the sizes of single-stranded molecules.
In the electrophoretic gel patterns of single-stranded DNA fragments of the deletion mutants, it may be seen that several fragments are affected by the deletion mutations (Fig. 2). In view of the fragment species affected the mutant strains can be classified into two groups. In group 1 strains, Fragments IV' and Xa are simultaneously missing, while two new fragments, one between Fragments V and Xb, and the other below Fragment VI, are present. This group includes del-4, 6, 10, and 13 (Fig. 2, lanes 2 to 5). In group 2 strains, Fragments II', IV', Xa, and Xb are missing, whereas two new fragments are present; one between Fragments I and III, and the other between Fragments III and VI (Fig. 2, lanes 6 to 14). In four deletion mutants, del-16, 1, 15, and 3, the new fragment between Fragments III and VI overlaps with Fragment V (lanes 7 to 10). The molecular weights of the new fragments are variable in different mutants, as shown in Table V. The disappearance of Fragments II', IV', V, Xa, and Xb, as well as the appearance of new fragments in the deletion mutants, and also the fact that the
382 type 1
I
5" VII
Vl
IV
V
III
2
1 2
Xa
' deletiorl I, Xb
Xa
I1'
3 IV'
Fig. 3. Heterogeneous DNA structures of bacteriophage BF23 DNA. (A) Two types of st(0) DNA structures determined by Okada and Shimura (1980). In type 2 DNA, Fragment II replaces Fragments IV and V of type 1 DNA. A DNA portion that covers the single-strand interruption between Fragments Xa and Xb is deleted in st(0) DNA. (B) Three types of wild-type phage DNA. The single-strand interruptions between Fragments Xb and V and that between Fragments Xa and Xb are missing in type 2 and 3 DNA, respectively.
m o l a r yields o f t h e s e f r a g m e n t s are l o w e r t h a n the o t h e r single-stranded fragm e n t s o f wild t y p e p h a g e c o u l d be best e x p l a i n e d b y t h e f o l l o w i n g m o d e l w h i c h is based o n the findings o n t h e p h y s i c a l s t r u c t u r e o f B F 2 3 s t ( 0 ) D N A . We a s s u m e t h a t t h e r e are t h r e e d i f f e r e n t t y p e s o f D N A m o l e c u l e s in wild t y p e B F 2 3 differing in the nicking sites as s h o w n in Fig. 3B. In t y p e 1 D N A , Fragm e n t s Xa, Xb, a n d V are p r e s e n t in this order. In t y p e 2 D N A , F r a g m e n t s Xb a n d V o f t y p e 1 D N A are r e p l a c e d b y F r a g m e n t II'; a n d in t y p e 3 D N A , Fragm e n t s X a and X b of t y p e 1 D N A are s u b s t i t u t e d b y F r a g m e n t IV'. T h e m o l e c ular weights o f t h e f r a g m e n t s are c o n s i s t e n t w i t h these s t r u c t u r e s . T h e struct u r e o f st(0) D N A m a y be e x p l a i n e d if we a s s u m e t h a t the site o f the i n t e r r u p tion b e t w e e n F r a g m e n t s Xa and Xb o f wild t y p e D N A is d e l e t e d in the m u t a n t D N A (Fig. 3A). Since t h e d e l e t e d region o f st(0) D N A was e s t i m a t e d to be 1.35- 106 m o l e c u l a r w e i g h t as single-stranded D N A (Table I I I ) , t h e size o f t h e f u s e d f r a g m e n t o f F r a g m e n t s Xa a n d X b c r e a t e d b y the d e l e t i o n m u t a t i o n was c a l c u l a t e d to be 9.45 • 106, a n d t h a t o f t h e f u s e d f r a g m e n t o f F r a g m e n t s Xa and II' was 1 8 . 0 5 • 106. These values agree w i t h the m o l e c u l a r w e i g h t of Fragm e n t IV (9.9 • 106) a n d t h a t o f F r a g m e n t II (18.4 • 106) of s t ( 0 ) D N A , respectively. T h e d i s a p p e a r a n c e of the specific f r a g m e n t s and t h e a p p e a r a n c e o f n e w f r a g m e n t s in the o t h e r d e l e t i o n m u t a n t s c o u l d be also e x p l a i n e d on the basis of this m o d e l . If we a s s u m e t h a t t h e g r o u p 1 m u t a n t s have d e l e t i o n s w i t h i n F r a g m e n t Xa, F r a g m e n t s Xa a n d IV' s h o u l d d i s a p p e a r , b u t F r a g m e n t s II', III, V, Xb, VI, a n d V I I r e m a i n intact. I n s t e a d , we can e x p e c t the p r e s e n c e of t w o n e w f r a g m e n t s ; o n e derived f r o m F r a g m e n t IV' a n d the o t h e r f r o m F r a g m e n t Xa b y the deletions. In t h e case of g r o u p 2 m u t a n t s , we assume t h a t the delet i o n s c o v e r t h e n i c k i n g site b e t w e e n F r a g m e n t s Xa a n d Xb. C o n s e q u e n t l y , F r a g m e n t s II', IV', Xa, and Xb s h o u l d d i s a p p e a r , a n d t w o new f r a g m e n t s are g e n e r a t e d , one b y fusion o f F r a g m e n t s II' a n d Xa a n d t h e o t h e r b y fusion o f F r a g m e n t s Xa a n d Xb. T h e s e a s s u m p t i o n s w e r e s u p p o r t e d b y t h e sizes o f the d e l e t i o n s c a l c u l a t e d f r o m t h e sizes o f the new f r a g m e n t s . As s h o w n in T a b l e V,
383 the size of the deletion of an any given m u t a n t calculated from the size of one new fragment (upper new fragment) is comparable with that calculated from another new fragment (lower new fragment), indicating that the two new fragments are structurally related as expected from the model of BF23 DNA. Note that the sizes of the deletions thus calculated are in good agreement with the sizes of the deletions estimated from the analysis of the DNA fragments obtained by digestion of the m u t a n t DNAs with restriction endonucleases (Table V). Thus, it may be said that the dispensable region of the BF23 genome is divided into two parts by the site of the single-strand interruption between Fragments Xa and Xb (Fig. 6). The three types of DNA molecules of wild type and the group 1 mutants were apparently present in DNA preparations originally obtained from plaquepurified phages. The relative populations of the three heterogeneous DNAs may be roughly estimated from the molar yields of the single-stranded fragments to be about 40% (type 1), 50% (type 2), and 10% (type 3) in the wild type DNA population. In the group 2 mutants, two types of DNA are present in equal amounts as demonstrated with BF23st(0) (Okada and Shimura, 1980). It is likely that the efficiency of nick formation between Fragments Xb and V is the same in both wild type and st(0) DNAs.
Genetic analysis of overlapping of the deletions In order to construct the deletion maps of the mutants, it would be helpful if we knew whether any pairwise combinations of the mutants have deletions that overlap each other. If the deleted regions of two parental phages do not overlap each other, recombinant phages carrying both of the deletions of the parents as well as the recombinants carrying no deletion are expected to be produced. Such recombinants m a y be detected by centrifuging the progeny phages in CsC1 equilibrium density gradients. On the contrary, if the deleted regions overlap each other even partially, no such recombinants should be produced. First, a cross was carried o u t between st(0) and del-4 strains. From the results of cleavage mapping, the deletions of these two strains were shown not to overlap each other. When progeny phages of the cross were centrifuged in a CsC1 gradient, two small phage bands were observed at b u o y a n t densities of 1.551 and 1.539 in addition to the parental phage bands at 1.546 (st(0)) and 1.544 (del-4) (Fig. 4a). The newly appeared phage bands were not defective, since the ratio of the phages of b u o y a n t densities of 1.551 and 1.539 to the parental phages was increased when the phages of these b u o y a n t densities (indicated by bars 1 and 2 in Fig. 4a) were multiplied on host cells (Fig. 4b and c). Phages banded at a density of 1.551, which is the same as that of wild type phage (Table 1), were proved to be recombinant phages carrying no deletion, since the restriction endonuclease digests of their DNA showed an electrophoretic gel pattern identical to t h a t of wild type DNA. DNA of the phages banded at a density of 1.539, which was designated del-401, was digested with restriction endonucleases, EcoRI and HindIII. As shown in Fig. 5b, the EcoRI
384
I
d
a
iim 4
2
b
J C
/ 1.551 1.J544/ 1.546 1.539
155,1 1.546 1.541
Fig. 4. Profiles of phage bands formed in CsCl equilibrium density gradients. Two deletion strains were crossed and the resultant phage lysates centrifuged in CsC1 gradients. Phage bands were photographed and traced with a recording densitometer. Buoyant densities at the phage bands are indicated in the figure. Phage lysates applied are: (a) progeny phage lysate of a cross between st(0) and del-4 strains; (b) phages multiplied from the fractions indicated by bar 1 in Fig. 4a; (c) phages multiplied from the fractions indicated by bar 2 in Fig. 4a; (d) progeny phage lysate of a cross between st(0) and del-lO strains; (e) phages multiplied from the fractions indicated by bar 3 in Fig. 4d; (f) phages multiplied from the fractions indicated by bar 4 in Fig. 4d. d i g e s t s o f del-401 D N A p r o d u c e E c o - A * f r a g m e n t w h i c h is s p e c i f i c t o del-4 s t r a i n as w e l l as E c o - F * f r a g m e n t w h i c h is s p e c i f i c t o s t ( 0 ) s t r a i n , b u t l a c k E c o - A , F , G, a n d H f r a g m e n t s . B y e l e c t r o p h o r e s i s in a 5% p o l y a c r y l a m i d e gel, Eco-I f r a g m e n t w a s d e t e c t e d . W h e n d i g e s t e d w i t h H i n d I I I , del-401 D N A p r o d u c e s a v e r y l a r g e f r a g m e n t o f 2 6 • 106 d a l t o n s b u t l a c k s Hin-A, B, a n d F fragm e n t s ( F i g . 5e). T h e size o f t h e d e l e t e d r e g i o n o f del-401 D N A c a l c u l a t e d f r o m t h e s i z e o f t h e E c o R I f r a g m e n t s is 6 . 6 3 • 1 0 6 , w h i c h is in g o o d a g r e e m e n t w i t h t h a t c a l c u l a t e d f r o m t h e H i n d I I I f r a g m e n t s , 6 . 7 - 106. T h e s e v a l u e s a g r e e w e l l w i t h t h e s u m o f t h e d e l e t e d r e g i o n s o f b o t h p a r e n t a l s t r a i n s ( 6 . 6 • 106 ). Fig. 5. Electrophoretic separation of DNA fragments of a recombinant and its parent phages produced by restriction endonucleases. The DNA digests were separated in 0.7% agarose gels. (a) EcoRI-digested st(0) DNA; (b) EcoRI-digested DNA of del-401 strain (a recombinant strain of st(0) and del-4 mutants); (c) EcoRI-digested del-4 DNA; (d) HindIIL digested st(0) DNA; (e) HindIII-digested del-401 DNA; (f) HindIII-digested dcl-4 DNA.
385
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386 These results indicate t h a t the d e l - 4 0 1 strain carries b o t h of the deletions o f the st(0) and d e l - 4 strains, as e x p e c t e d . Similar maalyses were p e r f o r m e d w i t h some o t h e r c o m b i n a t i o n s of the m u t a n t s and the results are s u m m a r i z e d in Table VI. T h e c e n t r i f u g a t i o n profile of p r o g e n y phages o f a cross b e t w e e n the st(0) and d e l - l O strains in a CsC1 d e n s i t y g r a d i e n t is s h o w n in Fig. 4d. Phages b a n d i n g at a b u o y a n t d e n s i t y o f 1 . 5 5 1 were s h o w n to c a r r y n o deletion as described previously. It is concluded, t h e r e f o r e , t h a t t h e deleted regions o f the st(0) and d e l - l O strains do n o t overlap each other. However, r e c o m b i n a n t phages carrying b o t h o f the p a r e n t a l deletions were n o t d e t e c t e d even after f o u r successive cycles of m u l t i p l i c a t i o n o f the fractions of the d e n s i t y w h e r e the r e c o m b i n a n t phages were e x p e c t e d to b a n d (Fig. 4f). N o t o n l y in the cross b e t w e e n st(0) and del-lO strains, b u t also in crosses of the c o m b i n a t i o n s o f d e l - 3 X d e l - 4 , d e l - 3 X del-lO, a n d del-4 X del-15, r e c o m b i n a n t phages c a r r y i n g b o t h o f the deletions o f the parental phages were n o t detectable, a l t h o u g h r e c o m b i n a n t phages carr y i n g no deletion were isolated (Table VI). Possible e x p l a n a t i o n s o f this p h e n o m e n o n are discussed later.
TABLE VI GENETIC CROSSES BETWEEN DELETION MUTANTS Production of recombinant phages was examined by CsCl equilibrium density gradient centrifugation of the progeny phage lysates, as described in Materials and Methods Cross
Detection of recombinant phages a Recombinants having no deletion
Recombinants having both deletions
st(0) x st(0) x
del-4
+
+
del-lO
+
-
del-3
x
del-4
+
-
del-3
x
del-lO
+
-
del-4
x
deI-15
+
-
del-1
-
-
del-6
-
-
del-13
-
-
st(0) x st(0) x st(0) x del-1
x
del-4
-
-
del-2
x
del-4
-
-
del-3
x
del-6
-
-
del-3
x
del-13
-
-
del-4
x
del-7
-
-
del-4
× del-14
-
-
del-4
x
-
-
del-16
a +, detected;-, not detected.
Sum of the deleted regions of both parental strains Molecular weight (- 10 6)
% of genome
6.6 8.3 9.7 11.4 9.3 8.0 7.5 9.5 9.2 11.5 10.6 12.6 10.3 10.2 9.1
9.0 11.3 13.2 15.5 12.6 10.9 10.2 13.0 12.5 15.6 14.4 17.2 14.0 13.9 12.4
387
In the o th er crosses listed in Table VI, no recom bi nant s of either t y p e were detected. Accordingly, the deleted regions of the parental strains of those combinations overlap each other.
Physical map of the dispensable region of the chromosome of BF23 On the basis of the experimental results described in the preceding sections the physical structure of the dispensable region of BF23 DNA was det erm i ned and the results are summarized in Fig. 6. The six E c o R I fragments, three HindIII fragments, and eight E c o R I + HindIII fragments were m apped in this region. One o f th e single-strand interruptions of BF23 DNA, located bet w een Fragments Xa and Xb, was m a p p e d within the d-F fragment. This site is present in the genome of group I m u t a n t s (del-4, 6, 10, and 13), but deleted in the genome of group 2 m u t a n t s (other 9 mutants). The deleted regions of these m u t a n t s were also m a p p e d as illustrated in Fig. 6. Although the exact size of the dispensable region on the phage genome is n o t known, it may be roughly estimated to be at least 11.4 - 106 daltons (15.5% of whole genome). This value was calculated as the size from the left end of the deletion of del-lO to the right end of the deletion of del-3, since the deletions of these two strains were e x p e c t e d to cover the leftmost and rightmost regions of the deletions, respectively. Thus, the value must be an underestimate o f the size of the dispensable region. According to the complete cleavage map of BF23 whole genome, the dispensable region estimated single-strand fragment EcoR, EcoRI.Hindlll
Hmdlll
A
H
Xa I G
,
F
B
N
T
S
F
B
0
[ F
Xb
I 0
D h
A
del-4 de1.10 del.13 del- 6 de l. 5 del. 1.16 del. 7.14
del.15 de/. 2 del- 3 st(O)
Fig. 6. Physical m a p s of t h e dispensable region a n d d e l e t i o n m a p s o f t h e m u t a n t s o n t h e c h r o m o s o m e o f b a c t e r i o p h a g e B F 2 3 . T h e site o f single-strand i n t e r r u p t i o n a n d r e s u l t i n g s i n g l e - s t r a n d e d f r a g m e n t s ( F r a g m e n t s Xa a n d Xb) are s h o w n o n the top. T h e sites o f cleavages b y E c o R I , H i n d I I I , a n d E c o R I + H i n d I I I a n d resulting f r a g m e n t s are s h o w n b e l o w t h e s i n g l e - s t r a n d e d fragments. In t h e l o w e r p a r t of t h e figure, t h e d e l e t i o n s of various m u t a n t s are m a p p e d as i n d i c a t e d b y b l a c k bars. F o r t h e sake of c o n v e n i e n c e in discussion, t h e over-all region deleted in t h e m u t a n t s e m p l o y e d in t h e p r e s e n t studies is a r b i t r a r i l y subdivided into 10 p o r t i o n s (regions i to 10), c o n s i d e r i n g t h e degrees o f overlaps o f t h e deletions.
388 as above extends from 0.2 to 0.35 map unit within the early gene region (K. Okada, N. Koizumi, and K. Mizobuchi, unpublished results). DISCUSSION A fine physical map of the dispensable region of the bacteriophage BF23 chromosome has been constructed. In order to localize the dispensable region on the phage genome, the genome structures of m a n y deletion mutants that propagate normally on host cells have been compared with that of wild type. The analyses have revealed that the deletions of these m u t a n t s extend from 0.2 to 0.35 map unit covering at least 15.5% of the whole BF23 genome. Although this region is dispensable for phage growth under the laboratory conditions and its genetic c o n t e x t is therefore difficult to analyze, several genes have been identified in this region. From analysis of the phage-encoded transfer RNA species synthesized in cells infected with several deletion mutants, genes for 14 tRNA species whose amino acid specificities are not known have been shown to be located in the dispensable region (Ikemura et al., 1978). Combining those results with the physical map shown in Fig. 6, genes for t R N A species 13, 14, and 15 are mapped at region 2 of Fig. 6, and those for species 16 and 17 at region 3 of the figure. Four tRNA species (1, 8, 10, and 20) and one RNA species of about 7S are synthesized in cells infected with del-1, but not in cells infected with four deletion mutants (del-2, 4, 5, and 6). Five other tRNA species (3, 6, 7, 12, and 18) were synthesized in cells infected with del-4 and st(0), but not in cells infected with five mutants (del-1, 2, 3, 5, and 6). These results suggest that the left endpoints of the del-2 and del-1 deletions are n o t exactly the same, namely the endpoint of the del-2 deletion is at the left side of that of the del-1 deletion, and that genes for the four t R N A species and 7S RNA are located at the interspaced region between the two left endpoints. Similarly, it is suggested that the left endpoint of the del-3 deletion is at the left side of that of the st(0) deletion, and that genes for the five tRNA species are mapped at the interspaced region between the left endpoints of these deletions. Consequently, it is concluded that tRNA genes are clustered in two distinct regions, one extends from regions 2 to 3, and the other is at the left end of the deleted region of del-3. In addition to the tRNA genes, a gene coding for a protein of about 3 • 104 molecular weight appears to be mapped in the region 8 and/or 9, since this protein is synthesized in cells infected with del-lO and del-13, but n o t in cells infected with st(0), del-1, and del-2 (Nagasu, T. and Mizobuchi, M., unpublished results). The structure of the dispensable region of the BF23 chromosome appears to be comparable, in m a n y respects, to that of bacteriophage T5. Von Gabaln et al. (1976) reported that the deletable region of the T5 chromosome spans from 0.19 to 0.34 map unit, comprising 15% of the total chromosome of T5 ÷. More than 16 tRNA genes were mapped in the dispensable region of T5 genome in four discrete portions (Chen et al., 1976; Hunt et al., 1976; Desai et al., 1978). In the genetic crosses of deletion mutants, two types of recombinant
389 phages could be expected, if the deleted regions of the parental strains do n o t overlap each other; t y p e 1 having no deletion, and t y p e 2 having b o t h of the parental deletions. In a cross be t w een the st(0) and del-4 strains, b o t h types o f r e c o m b i n a n t phages emerged. However, in four crosses (st(0) X del-lO, del-3 X del-4, del-3 X del-lO, and del-4 X del-15), t y p e 2 r e c o m b i n a n t phages were n o t d etected , although t y p e I r e c o m b i n a n t phages were isolated. In these crosses, the sum of the deletions o f the t w o parental strains are in the range of 11.3% (st(0) x del-lO) to 15.5% (del-3 X del-lO) of the phage genome (Table VI). The absence of the t y p e 2 r e c o m b i n a n t phages in the four crosses is p r o b ab l y best explained on an assumption t h a t there is a limit in size of the BF23 phage genome which is to be packaged t o produce mature phage particles. The genomes of the t y p e 2 r e c o m b i n a n t phages which are smaller than t h a t o f wild t y p e phage by 11.3 to 15.5%, m a y be t oo small to be packaged. The m u t a n t of BF23 having the largest deletion is del-2, whose deletion spans approx. 10.3% of the genome (Table III). Therefore, it is possible to assume th at BF23 phage DNA longer than 89.7% of wild t y p e DNA could be packaged, b u t phage DNA shorter than 88.7% of wild t y p e DNA could not. This p h e n o m e n o n is pr obabl y relevant to t h a t observed with bacteriophage lambda. When digested with restriction endonuclease EcoRI, lambda phage DNA is cleaved into six fragments: Eco-A to F. Although re-assembled phage DNA lacking either Eco-B (9.8% of phage genome) or Eco-C (11.3%) fragment can f o r m plaques by transfection, phage DNA lacking b o t h Eco-B and C fragments (total 21.1%) can not (Thomas et al., 1974). ACKNOWLEDGEMENTS I t h a n k Dr. K. Mizobuchi for his s u p p o r t with valuable discussions. I also t h a n k Dr. H. Ozeki for valuable discussions. I am indebted to Dr. Y. Shimura for his critical reading of and m a n y valuable c o m m e n t s on the manuscript. I am grateful to Dr. M. Takanami for the gift of enzymes. This w o r k was supp o r t e d by a Scientific Research G r ant f r om the Ministry of E ducat i on of Japan. REFERENCES
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390 Mizobuchi, K., Anderson, G.C. and MeCorquodale, D.J., Abortive infection by bacteriophage BF23 due to the colicin Ib factor, I. Genetic studies of nonrestricted and amber mutants of bacteriophage BF23, Genetics, 68 (1971) 323--340. Okada, K., Ohira, M. and Ozeki, H., Nonsense suppressor mutants of bacteriophage BF23, Virology, 90 (1978) 133--141. Okada, K. and Shimura, Y., Arrangement of the single-stranded fragments in E. coli bacteriophage BF23 DNA, Gene, 8 (1980) 345--368. Pulleyblank, D.E., Shure, M. and Vinograd, J., The quantitation of fluorescence by photography, Nucleic Acids Res., 4 (1977) 1409--1418. Robinson, J.H. and Landy, A., HindII, HindIII, and HpaI restriction fragment maps of bacteriophage ~ DNA, Gene, 2 (1977) 1--31. Sakano, H., Yamada, S., Ikemura, T., Shimura, Y. and Ozeki, H., Temperature sensitive mutants of Escherichia coli for t R N A synthesis, Nucl. Acids Res., 1 (1974) 355--371. Thomas, M., Cameron, J.R. and Davis, R.W., Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA, Proc. Natl. Acad. Sci. USA, 71 (1974) 4579--4583. Thomas, M. and Davis, R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91 (1975) 315--328. Von Gabain, A., Hayward, G.S. and Bujard, H., Physical mapping of the HindIII, EcoRI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA, Moi. Gen. Genet., 143 (1976) 279--290. Communicated by K. Matsubara.