Download A Physical Map of the Filamentous Bacteriophage Cf Genome

J. gen. Virol. (1984), 65, 1173-1181. Printedin Great Britain
1173
Key words: phage Cf/genome mapping/physicalmap
A Physical Map of the Filamentous Bacteriophage Cf Genome
By M E I - K W E I
Y A N G 1 AND T S O N G - T E H
K U O 1,2.
1Department of Botany, National Taiwan University, Taipei, Taiwan, Republic. of China and
2Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China
(Accepted 30 March 1984)
SUMMARY
Twenty-three restriction endonucleases were used to cleave the genome of
bacteriophage Cf. BamHI, EcoRI, HaelI, KpnI, PstI, Sail, SmaI and SstlI each cut Cf
D N A at one site, HindlII, PvuI, PvulI and XhoI cleaved at two sites, BgllI cleaved at
three sites, HinclI cleaved at four sites, HinfI, BstNI, AluI, HpalI and HaelII cleaved
at 14, 19, 20, 24 and 26 sites, respectively, whereas HpaI and XbaI have no cleavage site
on the Cf genome. HhaI and TaqI cleaved at more than 20 and more than 8 sites
respectively. Based on the sizes of the fragments produced by digestion with these
enzymes, Cf D N A was estimated to have 7.62 + 0.1 kilobase pairs. Using the EcoRI
cleavage site as a reference point, nine restriction enzymes, BamHI, BgllI, EcoRI,
HindlII, KpnI, PstI, PvulI, SstlI and XhoI were selected and a detailed physical m a p of
the Cf genome was constructed.
INTRODUCTION
Bacteriophage Cf is a filamentous phage isolated from Xanthomonas campestri pv. citri, which
is a pathogenic bacterium of citrus canker. It contains a single-stranded D N A molecule which,
upon infection, is converted into a double-stranded replicative form (RF) (Dai et al., 1980). In
general, phage C f is similar to other filamentous phages; however, it is longer (1008 n m length)
than some other filamentous phages such as M13. In addition, Cf D N A contains an unusual
nucieotide. These interesting properties led us to construct a physical m a p of the C f genome.
Cleavage maps for various restriction enzymes have been constructed for the genome of
phages M13 (van den Hondel & Schoenmakers, 1975; van den Hondel et al., 1976; Seeburg &
Schaller, 1975), fd (Seeburg & Schaller, 1975; T a k a n a m i et al., 1975) and fl (Horiuchi et al., 1975 ;
Seeburg & Schaller, 1975; Vovis et al., 1975). U p o n digestion with a single restriction endonuclease, characteristic differences were shown to exist between M13, fd, fl and Z J~2 D N A ,
providing a new and sensitive method of distinguishing these closely related coliphages (van den
Hondel & Schoenmakers, 1976). To characterize Cf further and to compare it with other
filamentous phages, a more-detailed physical m a p of its genome is necessary.
In this investigation, we screened the ability of 23 different restriction enzymes to cleave the
R F D N A of Cf and compared the restriction patterns with those of other filamentous phages.
Nine restriction enzymes (BamHI, BgllI, EcoRI, HindlII, KpnI, PstI, PvuII, SstlI and XhoI)
were then used to construct a physical m a p of the Cf genome.
METHODS
Bacterium and phage. X. campestri pv. eitri, obtained from Professor W. C. Wu, National Chung-Hsing
University, China, was used for cultivation of phage Cf and preparation of its RF. Phage Cf was isolated in this
laboratory. X. eampestripv. eitri was grown in potato-sucrose medium (PS medium), which contained, per litre:
fresh potato, 200 g; Ca(NO3)2, 0"5 g; Na:HPO4.12H20, 0.2 g; peptone, 5 g; sucrose, 15 g.
Preparationof the CfRF. Jd.campestripv. citriwas grown in 500 ml PS medium. The cells, at a density of 2 × 109
per ml, were infected with Cf phage at a multiplicity of 20 and treated with 170 ~tg/mlchloramphenicol at 10 min
post-infection. Chloramphenicol was added to increase the synthesis of RF DNA and to prevent chromosomal
DNA synthesis (Clewell, 1972). After 4h of incubation at 28 °C, the infected cells were harvested, chilled, washed
once with 250 ml buffer (10 mM-Tris-HCl pH 8.0, 0.1 mM-EDTA) and lysed with SDS and NaOH (Birnboim &
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M . - K . YANG AND T.-T. KUO
Doly, 1979). After lysis, the lysate was centrifuged at 44 000 g for 90 min, and 1/30 vol. of 3 M-sodium acetate and
0.6 vol. isopropanol were added to the supernatant. After centrifugation at 27200 g for 20 min, the pellet was
resuspended-in 5 ml Tris-glucose (25 mi-Tris-HCl pH 8.0, 10 mM-EDTA, 50 mi-glucose) and 15 ml 5 M-potassium acetate. The bacterial DNA and debris were spun down at 27200 g for 20 min. The supernatant, containing
the RF DNA, was precipitated with 2 vol. ethanol at - 2 0 °C for 2 to 4 h and recovered by centrifugation at 12000
g for 30 min. The pellet was dissolved in 8 ml TE buffer (10 mi-Tris-HC1 pH 8.0, 1 mM-EDTA). The DNA was
then purified by centrifugation to equilibrium in a CsCl-ethidium bromide density gradient (Radloffet al., 1967).
Each polyallomer centrifuge tube contained 8 ml of sample, 1.5 mg ethidium bromide and 7.5 g CsC1; the
remaining space in the centrifuge tube was filled with mineral oil. Centrifugation was performed at 130000g at 15
to 20 °C for 48 h. At the end of the run, the band containing closed supercoiled DNA was collected by inserting a
hypodermic needle into the side of the tube. Ethidium bromide was removed by five extractions with isopropanol
and the samples were dialysed against TE buffer. The DNA thus prepared was used directly for restriction
endonuclease digestion.
Digestion ofRF DNA with restriction endonucleases. Restriction endonucleases used in this study were purchased
from Bethesda Research Laboratories and used according to the manufacturer's specifications. The RF DNA of
Cf (1 to 5 ~tg) was completely digested in a 20 ~tl reaction mixture, with 1 to 5 ~tl of each enzyme, for 4 h at 37 °C.
Double-enzyme digestion of Cf DNA was carried out by restricting a primary DNA digest with a second enzyme
after adjustment of reaction mixtures. Reactions were stopped by heating at 70 °C for 10rain in 20 mM-EDTA,
15~ sucrose and 0-1~ bromophenol blue, followed by rapid cooling on ice.
Gel electrophoresis of restriction fragments. The RF DNA of Cf, digested with various restriction endonucleases,
was analysed by agarose gel electrophoresison horizontal slab gels (20 x 15 x 0.3 cm) or by PAGE on vertical slab
gels (18 × 16 × 0.15 cm). Electrophoresis was carried out in TBE buffer (89 mM-Tris-HCl, 89 mM-boric acid, 2.5
mM-disodium EDTA, pH 8.0), at 60 V (20 mA) for 4 to 6 h, and stained in 0.5 ~tg/ml ethidium bromide. DNA
fragments were visualized by illumination with a u.v. lamp and photographed using a red filter.
Purification of DNA restriction fragments for redigestion. After staining with 0.5 p.g/ml ethidium bromide, the
bands containing the DNA of interest were cut under long-wave u.v. light (330 nm) and weighed. The gel was
dissolved in 4 vol. 6 M-NaC104 at 45 °C for 30 rain. The dissolved agarose-DNA solution was transferred dropwise
to a GF/C filter paper (diam. 6 mm), washed with about 0.5 ml NaC10,-Tris solution, then with 0.5 ml
isopropanol and 0.5 ml ethanol, and finally air-dried. The filter paper was rolled up and placed into a 0.5 ml
Eppendorf tube with a small hole at the bottom. About 20 ~tl Tris-EDTA was added to the filter paper and after
incubation at room temperature for about ]0 min, the eluate was collected at the bottom of the tube by
centrifugation at 12000 g for 2 min. The elution process was repeated twice. The DNA in this low salt solution
could be used directly for further restriction enzyme digestion (Chen& Thomas, 1980).
Calculation of the size of restriction fragments. The size of restriction fragments in kilobase pairs (kb) was determined as described by Southern (1979). Lambda phage DNA (Bethesda Research Laboratories) fragments
generated by EcoRI or HindlII digestion, and phage ~bX174 RF DNA fragments produced by HaelII or HinfI
digestion were used as standards.
RESULTS
Restriction endonuclease cleavage of Cf RF
We tested the ability of 23 different enzymes to cleave the R F D N A of Cf. The corresponding
numbers of cleavage sites are given in Table 1. The restriction endonucleases BamHI, EcoRI,
HaelI, KpnI, PstI, Sail, SmaI and SstlI caused a single cut on Cf RF. HindlII, PvuI, PvulI and
XhoI cleaved Cf RF D N A into two fragments, whereas BgllI generated three distinct restriction
fragments. Both XbaI and HpaI had no cleavage sites on the Cf genome (Table 1, Fig. 1). For
each of the above digests, the sum of the sizes of the D N A fragments was approximately 7.62 ___
0.1 kb, indicating that the Cf D N A has a tool. wt. of about 2.5 × 106.
The restriction endonucleases AluI, BstNI, HaelII, HhaI, HinfI, HpalI and TaqI each
generated between 14 and 26 D N A fragments. The digests were analysed on a linear 5 to 1 5 ~
polyacrylamide gradient slab gel. As can be seen in Fig. 2, small D N A fragments were not
completely separated.
On the basis of these preliminary cleavage patterns, nine restriction enzymes, BamHI, EcoRI,
BgllI, HindlII, KpnI, PvulI, PstI, SstlI and XhoI, were chosen for map construction.
Mapping of the E c o R I and B a m H I cleavage sites
To obtain a restriction map, we determined the location of cleavage sites for those restriction
enzymes that had a few such sites on Cf RF. Both EcoRI and BamHI made one double-stranded
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Physical map of phage C f genome
+
+
+
I¸•¸ ~i'
Fig. 1. Agarose gel electrophoresis of restriction endonuclease digests of Cf RF. Cf RF was cleaved by
restriction enzymes and subjected to (a) 1~ or (b) 2~ agarose slab gel electrophoresis at 60 V for 4 h.
Phage lambda DNA digested with HindlII, and q~X174 RF DNA digested with HinfI and HaelII were
used as size markers.
break in Cf to produce a linear molecule that co-migrated with Cf R F I I I (Fig. 3). W e started our
m a p by measuring the size of the two fragments generated by an EcoRI/BamHI double
digestion.
The approximate fragment sizes were estimated from their mobility relative to HindIII
fragments of l a m b d a D N A . The size of a fragment was expressed as a percentage of the length of
phage Cf D N A . Phage Cf R F double digested with EcoRI and BamHl produced a 4.85 kb and a
2.70 kb fragment with sizes of 63.7 ~ and 36.3 ~ of the Cf RF, respectively. The EcoRI site was
chosen as the zero point and the BamHI cleavage site was located clockwise from it (Fig. 4).
Mapping of the P v u / / a n d Bgl//cleavage sites
Phage Cf R F was cleaved by PvulI into two pieces of about 5.22 kb and 2.40 kb (Fig. 3). In
order to locate the PvulI cleavage site, fragments A and B were purified from the agarose gel and
redigested separately with BamHI and EcoRI. The PvulI-A fragment, whose size is 6 8 . 5 ~ of Cf
R F , was cut by BamHI into 44.5 ~ and 24.3 ~ C f R F size fragments, whereas PvulI-B, 31.45 ~ of
C f RF, was not affected by BamHI. EcoRI cut only the PvulI-A fragment and resulted in the
formation of 4.72 kb and 0.49 kb fragments. Combining these two results, the location of the two
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M.-K. YANG AND T.-T. KUO
Table 1. Comparison o f the number o f cleavage sites o f C f with that o f other filamentous phages
Number of cleavage sites
A
Enzyme
AluI
BamHI
BgllI
BstNI
EcoRI
HaelI
HaelII
HhaI
HinclI
HindlII
Hinfl
Hpa I
HpalI
KpnI
PstI
PvuI
PvulI
Sali
Sinai
SstlI
Taql
XbaI
Xhol
Cf
20
M13*
18
fd*
16
fl*
18
1
3
1
0
2
0
1
0
19
2
-
-
1
1
0
4
0
4
0
4
26
> 20
10
20
10
23
9
21
4
2
1
0
0
0
0
14
24
24
24
0
1
-
0
24
-
-
-
1
1
2
2
1
1
l
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
>8
10
10
10
0
2
0
0
0
0
0
0
* M13 from Van Wezenbeck et al. (1980). fd from Beck et al. (1978) and fl from Hill & Petersen (1982).
PvulI cleavage sites in relation to E c o R I and B a m H I cleavage sites was established as shown in
Fig. 4.
Cleavage of Cf R F with B g l l I produced three fragments (Fig. 1). B g l l I - A and B g l l I - B
fragments were isolated from agarose gels and subsequently digested with E c o R I . B g l I I - A
yielded two fragments of approximately the same size: 2.72 kb and 2.43 kb. Further cleavage of
B g l l I - A fragment with P v u l I resulted in two fragments of 3.16 kb and 1.99 kb, whereas BglII-B
fragment was not cut by PvulI. The three B g l l I cleavage sites in relation to those of E c o R I and
PvulI are shown in Fig. 4.
Mapping o f the X h o I and H i n d l I I cleavage sites
Digestion o f C f R F with XhoI produced two fragments of 6-30 kb and 1.32 kb, which were
82.4 ~o and 17.6 ~ of the Cf genome respectively. W h e n Cf R F was double-digested with E c o R I
and XhoI, the X h o I - A fragment disappeared and two new fragments of 5-09 k b and 1-42 kb were
obtained. Two new fragments of 4.10 kb and 1.92 kb were produced from double digestion of
X h o I - A with B a m H I . Their sizes were 56.1 ~ and 26.3 ~ of the Cf genome. W i t h these results,
the cleavage site of XhoI on the circular Cf genome could be located (Fig. 4). In order to confirm
the cleavage site of XhoI, a similar experiment was carried out using X h o I / P v u l I double
digestion. The P v u l I - A fragment was not affected by cleavage with XhoI, but the PvulI-B
fragment was apparently shortened. Therefore, the two XhoI cleavage sites could be located
within the PvulI-B fragment.
C f R F cleaved with H i n d l I I generated two fragments of 5.75 kb and 1.63 kb, their sizes being
7 7 . 9 1 ~ and 2 2 . 0 9 ~ of Cf RF, respectively. In order to determine which H i n d l I I fragment
contained the cleavage site for B a m H I , H i n d l I I / B a m H I double digestion of Cf R F was analysed.
The H i n d l I I - A fragment wascleaved with B a m H I and yielded two fragments o f 4.12 kb and 1.63
kb. U p o n digestion with HindlII, P v u l I - A was cut whereas PvuII-B remained intact. Thus,
P v u I I - A covers the H i n d l I I site as shown in Fig. 4.
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Physical map of phage Cf genome
1177
r~
+
Fig. 2. Polyacrylamide gel electrophoresis of restriction endonuclease digests of Cf RF. Cf RF was
cleaved by the indicated restriction enzymes and subjected to a linear 5 to 15~ polyacrylamide gradient
slab gel. Phage ¢X174 RF II DNA cleaved by HaelII was used as a size marker.
Mapping of PstL K p n I and SstH cleavage sites
Digestion of Cf R F with PstI, KpnI and SstII produced a linear molecule of 7.62 kb (Fig. 1). In
order to locate these three cleavage sites relative to each other, we used the same methods as
described above. The PstI, KpnI and SstlI digests were isolated from gels and redigested with
other enzymes. The redigestion products were then compared with the respective doubledigestion products.
Combined digestions of PstI and EcoRI produced two fragments of 5.10 kb and 2-49 kb,
which were 67.2~ and 32.8 % of Cf RF. PstI/BamHI double digestion of Cf RF also yielded two
fragments of 5.46 kb and 2.10 kb (Table 2 and Fig. 5). From these two results, the PstI cleavage
site was determined (Fig. 4). A similar experiment was carried out by digesting Cf R F with a
combination of KpnI/EcoRI, KpnI/BamHI, SstlI/EcoRI and SstlI/BamHI (Table 2). The location
of cleavage sites of KpnI and SstlI was then determined (Fig. 4).
DISCUSSION
A physical map of the circular genome of bacteriophage Cf was constructed through analysis
of single- and double-digestion data and by redigestion of purified fragments with a second
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M.'K.
+
YANG AND T.'T.
KUO
+
Z
~
Fig. 3. Agarose gel electrophoresis in 1 ~ agarose of fragments of C f R F produced by single and double
digestion. HindlII-digested lambda D N A was used as a size marker.
enzyme. In the present study, agarose gel electrophoresis was performed to estimate the
molecular weight of digest fragments. The mobility of double-stranded D N A on agarose gel
electrophoresis is dependent on its molecular weight (Daniels et al., 1980; Thomas & Davis,
1975). By measuring the exact band location, the size of each digest fragment was calculated.
Combining the results obtained from many digests, the molecular size of Cf RF D N A
was determined to be approximately 7.62 kilobase pairs, which corresponds to a mol. wt. of
2.5 x 106.
A comparison of the 23 restriction patterns of Cf with the corresponding patterns of other
filamentous phages M 13, fd and fl revealed characteristic differences. Among those we tested,
the restriction enzymes EcoRI, KpnI, PstI, Sail, SmaI and SstII cleaved Cf RF at one site. This
is in contrast to the filamentous coliphages M13 and fl, which have no cleavage sites for these
enzymes (van Wezenbeck et al., 1980; Beck et al., 1978; Hill & Petersen, 1982). Of the nine
restriction endonucleases chosen to construct a cleavage map of Cf, all but BamHI have no
cleavage site on M 13, fd or ft. A cleavage map of the M 13 genome was constructed with HaelII,
HaplI and HindlI, which cleaves M13 RF into 10, 13 and 1 fragment, respectively (van den
Hondel & Schoenmakers, 1975). The cleavage sites of AluII and HaelI were then determined by
using the previously constructed HaplI and HaelII maps of the M13 RF (van den Hondel et al.,
1976). These maps have been used as a guide for constructing the similar maps of the closely
related filamentous coliphages fd, fl and Z J/2 (van den Hondel & Schoenmakers, 1976). It was
found that restriction enzyme cleavage patterns provide a sensitive method for distinguishing
closely related filamentous phages. In order to determine the relationship between Cf and other
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Physical map of phage Cf genome
PvulI.
EcoRI
HindIII~~~
-
,o'W
~p'I~.7o
BgllI" ~
3o-./
60
40
Xhol/PstI"~i~-Sg~
~
50
y
I~
•
~.
~amHl
Bgm
Pvull/
Fig. 4. A physical map of bacteriophage Cf genome constructed by cleavage with the indicated
restriction endonucleases. The EcoRIcleavage site was chosen as the zero point and fractional genome
lengths plotted in a clockwise direction.
~
~
+
+
+
Fig. 5. Agarose gel electrophoresis in 1~ agarose (4 h at 60 V) of fragments of Cf RF produced by single
and double digestion. The purified XhoI-Afragment was digested by the indicated restriction enzymes.
HindlIl-digestedlambda DNA was used as a size marker.
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M.-K. YANG AND T.-T. KUO
Table 2. Fragments generated by cleavage o f C f R F with two restriction endonucteases*
First enzyme
PstI
Second enzyme
EcoRI
BamHI
KpnI
EcoRI
BamHI
SstII
EcoRl
BamHI
Fragment and
size (kb)
A 5.10
B 2.49
A 5.46
B 2.10
A 5.55
B 2.10
A 4.83
B 2.74
A 5.48
B 2.12
A 4-96
B 2.54
Fraction length
of Cf DNA (~)
67.2
32.8
72.2
27-3
72-6
27.5
63.8
36-2
71-8
28-2
66.1
33.9
* Cf RF was cleaved by the action of the two restriction enzymes indicated, and the products were analysed by
agarose gel electrophoresis (Fig. 5).
filamentous phages, similar studies were carried out with Cf. From the results of such analysis,
we suggest that Cf is indeed not closely related to the filamentous coliphages M13, ft and fd.
Several techniques have been used to construct physical maps of phage genomes, including (i)
combined digestion of exonucleases and restriction enzymes, (ii) incomplete digestion, (iii)
double digestion with two restriction enzymes and (iv) redigestion of the isolated fragments with
a second restriction enzyme (Hamlett et al., 1977). In constructing a detailed physical map of the
bacteriophage Cf genome, we chose the only EcoRI cleavage site as a reference point. The
cleavage sites of other restriction enzymes were then determined by reciprocal double digestion
or redigestion of isolated fragments with the enzymes EcoRI and BamHI. We chose to give
fragment sizes as fractional lengths rather than molecular weights, since the exact number of
base pairs of the circular R F molecule is not accurately known. However, the genome of Cf is
larger than that of filamentous bacteriophages of Escherichia coli; the genome of fl and M 13 was
found to comprise 6407 base pairs (Hill & Petersen, 1982; van Wezenbeck et al., 1980) and that
of fd 6408 base pairs (Beck et al., 1978). This agrees with earlier contour length measurements of
Cf (Dai et al., 1980), which yielded larger values than those for M13, fl and fd (Salivar et all,
1964). However, in the absence of sequence information, it is not possible to assess the overall
accuracy of the genome size estimate in this study.
The availability of a restriction enzyme cleavage map is useful for the analysis of the structure
and function of the phage genome. Van den Hondel & Schoenmakers (1975) have constructed a
cleavage map of the M13 genome and then assigned the genetic markers to this physical map
(van den Hondel et al., 1975). By use of the coupled transcription and translation system the
protein products encoded by specific fragments could be analysed (Konings et al., 1975). In
order to reach an understanding of the genetic organization of the Cf genome and its biological
function, the correlation of the physical map to the genetic markers of the Cf genome is now
being studied by cloning specified restriction fragments in E. coli.
This work was supported by grants from the National Science Council, Republic of China and China
Foundation for Education.
REFERENCES
BECK, E., SOMMER,R., AUERSWALD,E. A., KURZ, CH., ZINK, B., OSTERBURG, G., SCHALLER,H., SUGIMOTO. K., SUGISAKI,
rI., OKAMOTO,T. & TAKANAMI,M. (1978). Nucleotide sequence of bacteriophage fd DNA. Nucleic Acids
Research 5, 4495-4503.
~ZRNBO~M,/~.c. &DOLY,J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Research 7, 1513-1523.
CrlEN,C. W. &THOMAS,C. a., JR (1980). Recovery of DNA segments from agarose gel. Analytical Biochemistry 101,
339-341.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 12 Jun 2017 05:23:28
Physical map o f phage C f genome
1181
¢LEWELL, D. B. (1972). Nature of Col E1 plasmid replication in the presence of chloramphenicol. Journal of
Bacteriology 110, 667-676.
DAI, H., CHANG, K. S. & KUO, T. T. (1980). Characterization of a new filamentous phage Cf from Xanthomonas cirri.
Journal of General Virology 46, 277-289.
DANIELS, D. L., DE WET, J. R. & BLArTER, r. R. (1980). New map of bacteriophage lambda D N A . Journal of Virology
33, 390~400.
HAMLETT, N. V., LANGE-GUFSTAFSON,B. & RHOADES,M. (1977). Physical map of the bacteriophage T5 genome based
on the cleavage products of the restriction endonucleases Sal I, Sma I, Bam HI and Hpa I. Journalof" Virology
24, 249-260.
rtILL, D. 3. & I'ETERSEN,G. B. (1982). Nucleotide sequence of bacteriophage ft DNA. Journal of Virology 44, 32-46.
I.-IORIUCH[, K., VOVIS, G. F., ENEA, V. & ZINDER, N~ D. (1975). Cleavage map of phage fl: location of the E. coli
B-specific modification site. Journal of Molecular Biology 95, 147-165.
KONINGS, R. N. H., HULSEBOS,T. & VAN DEN HONDEL,C. A. (1975). Identification and characterization of the in vitro
synthesized gene products of bacteriophage M13. Journal of Virology 15, 570-584.
RADLOFF, R., BAUER, W. & VINOGRAD,J. (1967). A dye-buoyant density method for the detection and isolation of
closed circular duplex D N A : the closed circular D N A in HeLa cells. Proceedings of the National Academy of
Sciences, U.S.A. 57, 1514-1521.
SALIVAR, W. O., TZAGOLOFF, a. & PRATT, D. (1964). Some physicochemical and biological properties of the rodshaped coliphage M13. Virology 24, 359-371.
SEEBURG, e. ft. & SCHALLER, H. (1975). Mapping and characterization of promoters in phages fd, fi and M13.
Journal of MolecularBiology 92, 261-279.
SOUTHERN, E. (1979). Gel electrophoresis of restriction fragments. Methods in Enzymology 68, 162-164.
TAKANAMI,M., OKAMOTO,T., SUGIMOTO,K. & SUGISAKI,H. (1975). Studies on bacteriophage fd D N A . I. A cleavage
map of the fd genome. Journal of Molecular Biology 95, 21-31.
THOMAS,M. & DAVIS,a. W. (1975). Studies of the cleavage of bacteriophage lambda D N A with EcoR I restriction
endonuclease. Journal of Molecular Biology 91, 315-328.
VAN DEN HONDEL, C. A. & SCHOENMAKERS,J. G. G. (1975). Studies on phage M13 DNA. I. A cleavage map of the
M13 genome. European Journal of Biochemistry 53, 547-558.
VANDEN HONDEL,C. A. & SCI-IOENMAKERS,J. G. G. (1976). Cleavage maps of the filamentous bacteriophage M13, fd,
fl and ZJ/2. Journal of Virology 18, 1024-1039.
VANDEN HONDEL,C. A., WEIJERS,A., KONINGS,R. N. H. & SCHOENMAKERS,J. G. G. (1975). Studies on phage M13. II.
The gene order of the M13 genome. European Journal of Biochemistry 53, 559-567.
VAN DEN HONDEL, C. A., PENNINGS, L. & SCHOENMAKERS, J. G. G. (1976). Restriction enzyme cleavage maps of
bacteriophage M 13 genome. European Journal of Biochemistry 68, 55-70.
VAN WEZENBECK, P. M. G. F., HULSEBOS, T. J. M. & SCHOENMAKERS, J. G. G. (1980). Nucleotide sequence of the
filamentous bacteriophage M13 D N A genome: comparison with phage fd. Gene 11, 129-148.
vovIs, G. F., HORXUCrtI,K. & ZINDER,N. D. (1975). Endonuclease R. EcoR II restriction of bacteriophage fl D N A in
vitro : ordering of gene V and VII, location of an R N A promoter for gene VIII. Journal of Virology 16, 674684.
(Received 14 October 1983)
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