Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome

173

Gene, 30 (1984) 173-182 Elsevier GENE

1072

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the firstreplicating DNA with cloned fragments from the replication origin region of the chromosome (Recombinant DNA; initiation of replication; bacteriophage 1 Charon vector; 6-p-(hydroxy-phenylhydrazino)uracil; novobiocin; Ml3 cloning; spore germination; pBR322 plasmid)

Naotake Ogasawara, Sumi Mizumoto and Hiroshi Yoshikawa * Cancer Research Institute, Kanazawa University, 13-1, Takaramachi, Kanazawa 920 (Japan) Tel. (0762) 62-8151 (Received

March

(Accepted

April 23rd, 1984)

26th, 1984)

SUMMARY

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [3H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a 1 Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels. Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M 13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.

INTRODUCTION

An autonomously replicating fragment has been isolated from the replication origin (ori) of the Escherichia co/i chromosome (Yasuda and Hirota, 1977). Consequently the minimal 245 bp sequence

* To

whom

correspondence

and

reprint

requests

should

addressed. Abbreviations:

bp, base pairs;

drazino)-uracil;

kb, 1000 bp;

0378-I 119/84/$03.00

0

6HPU,

on', origin

1984 Elsevier

6-p-(hydroxy-phenylhyof DNA replication.

Science

Publishers

be

essential for replication (OK sequence) has been determined (Oka et al., 1980). In contrast, attempts to isolate the DNA sequence responsible for initiation of the chromosomal replication from the B. subtilis chromosome have not been successful. To study mechanism and regulation of initiation of replication, we first constructed a physical and genetic map of the ori region of the B. subtilis chromosome (Ogasawara et al., 1983a). We found that a ribosomal RNA operon, rrn0, is located within this region (Ogasawara et al., 1983a). In addition, the insertion of promoters of the n~0 into a composite

174

plasmid strongly inhibited replication of the plasmid in both E. coli and B. subtilis (Seiki et al., 1982). The failure to isolate autonomously replicating 0riC sequence from the B. subtilis chromosome may be because of the inhibitory effect of the rRNA promoters. For the further study, therefore, it is essential to determine the exact site of initiation of replication within the ori region. An approximate initiation site has been determined within a 5.7-kb BamHI fragment, B7, by determining the order of replication of the ori fragments during synchronous initiation (Ogasawara et al., 1979). Since a region of some 45-kb surrounding the putative ori is completely cloned (Ogasawara et al., 1983b), it is now possible to localize the initiation site more precisely by determining location of the first-replicating DNA strands on the chromosome using molecular hybridization techniques. The site of initiation of DNA replication was localized within a 3-kb region by this method. Moreover, analysis using separated single strands as hybridization probes showed that one major initiation site is located on each strand some 2-kb apart from each other. Since the newly identified initiation site is not in the first-replicating B7 fragment and is some 5-kb away from rrn0, the replication order of the ori fragments was reexamined with restriction enzymes which were not available in the previous experiment. The result is consistent with the data obtained by the hybridization experiment.

mosome, ChB SO1, ChB SO3 and ChB S 12, have been described previously (Ogasawara et al., 1983b). Chromosomal DNA fragments cloned in these phages were subcloned into pBR328 or pN021. Plasmid pN021 was derivative of pN02 (Seiki et al., 1981) which has lost one of the EcoRI sites at the left end of the Km’ gene insert. Sub-cloned fragments are listed in Fig. 1. Some fragments were transferred into M13mp8 or mp9 according to the procedure specified by the supplier. Fragments cloned in Ml3 phages are also shown in Fig. 1. (c) Chemicals

[ Methyl-3H]thymidine (TRK4 18, 40 Ci/mmol) was purchased from the Radiochemical Centre (Amersham, Bucks, England). T4 DNA ligase and restriction endonucleases were from Takara-shuzo Co. Ltd (Kyoto, Japan) or from Nippon Gene Co. Ltd (Toyama, Japan). Low-melting-point agarose was from Takara-shuzo Co. Ltd. Nitrocellulose membrane filter (BA85) was from Schleicher & Shuell GmbH (F.R.G.). 6-p-(Hydroxy-phenylhydrazino)-uracil(6HPU) was a gift from Dr. W. Langley, Imperial Chemical Industry Ltd. (d) Preparation of 3H-labeled first-replicating

DNA

B. subtilis CRK2000 (Zeu-8, trpC, thyAB) (Yoshikawa, 1967) was used throughout. E. coli C600 (thr, leu, thi, tonA, lac, supF) was used as a host for cloning B. subtilis chromosomal fragments. E. coli JM103 (supplied by New England BioLabs) was used as a host for Ml3 phage vectors.

Spores of B. subtilis CRK2000 were germinated in a synthetic medium GM 11 without thymine for 3 h as described previously (Seiki et al., 1979) and then 6HPU (100 PM) was added to the culture. After 5 min, [3H]thymidine (2.5 pCi/ml) was added to start DNA replication synchronously for various times. Cells were killed quickly by the addition of an ethanol-phenol mixture, and whole chromosomal DNA was purified as described (Seiki et al., 1979). Purified DNA was dissolved in T,,E, buffer (25 mM Tris . HCl, 1 mM EDTA, pH 8.0) and denatured in a boiling water bath for 5 min. 3H-labeled tirst-replieating DNA was separated from other chromosomal DNA by centrifugation in a 5-20x sucrose density gradient as described in the legend to Fig. 2.

(b) Phages and plasmids

(e) Hybridization

DNA from M 13mp8 and mp9 phages was from New England BioLabs. Derivatives of Charon 28 phage containing the ori region of the B. subtilis chro-

Each fragment cloned in Charon 28 phage or in pBR322 plasmid vectors was purified by electrophoresis in low-melting-point agarose gel after ap-

MATERIALS

AND METHODS

(a) Bacterial strains

experiments

175

,E PP I If I 0

E5 ‘-r

E20 ‘7

V PP

V

ES

E6 1

VE If

SVPB I( L

P I IO

5 P ,‘:

El9 7E221 EP II

E I IS

I

87 pSM2001 (pN021)

‘J

pSM2002 E L S I

E,

(pN021)

pSMlOOl (pBR328) :

V , E I

\(

pSM2003

(pN021)

pSM2004

( pN021) :

P L

‘:

pSM2005 E

pNOlOOl (pBR328 1 (pN02l)

J

f

E , P -

EB II

S S -

pNOlOl2 (pBR328) :

pN02004

(pBR328)

Ml3BSOl,02 (mp8, mpg) E Ml3BS03,04 (mp8,mp9) E , 7 Ml3BSOS,06 ( mp8, mpg) P ,

? Ml3BS07,08

(mp8,mp91

Fig.1. DNA fragments sub-cloned from Charon phages into pBR322 vectors and M13mp phages. Locations of fragments are shown in relation to the restriction map (top line) of the replication origin region(Ogasawaraet al., 1983b). Restrictionsitesare E, EcoRI;P, psrl; v, _&oRV;S, &/I; B, BumHI. Names of plasmids (or phages) containing each fragment together with vectors (in parenthesis) used for sub-cloning

are specified.

propriate

digestion with restriction enzymes. The fragment extracted and purified from the gel was put on a nitrocellulose membrane and fixed on it by baking at 80’ C for 2 h. Procedures for pretreatment of the fixed filter, hybridization with the 3H-labeled DNA and washing of filters after the hybridization were essentially same as described previously (Ogasawara et al., 1983a). Hybridization was performed at 42” C for more than 72 h until the 3H activity retained onto the filter reached plateau level. After washing, filters were dried and 3H activity on the filter was measured in a liquid scintillation counter using a toluene-PPO system.

ence of novobiocin to reduce elongation rate of replication as described previously (Ogasawara et al., 1979). The labeled DNA was purified and then digested with various restriction enzymes as described above. The resultant fragments were separated by electrophoresis in 0.7 y0 low-melting-point agarose as described. The labeled fragments in the gel were first detected by fluorography and then cut out from the dried gel. Each piece of gel was dissolved and 3H activity incorporated in DNA fragments,was determined as described previously (Ogasawara et al., 1983a).

(f) Labeling, detection and quantitative measurement of 3H activity of the first-replicating fragments of the chromosome

RESULTS (a) Isolation of the first-replicating

Replication origin region of the B. subtilischromosome was labeled with [3H]thymidine in the pres-

Using the spore germination system to achieve synchrony of initiation of replication (Ogasawara

DNAs

116

EcoRI fragments (not

shown).

pooled

in the ori region were not detected

Fractions

indicated

as the first-replicating

in Fig. 2 were

DNA

and used for

further analyses.

(b) Hybridization of the first-replicating DNA with cloned DNA fragments in the ori region To determine replicating cloned

the site of the synthesis

DNA, we hybridized

from the ori region.

large fragments

of the tirst-

it with the fragments

For a primary

cloned in the A Charon

used to cover the ori region of some 45kb Efficiency replicating

survey,

vector were (Fig. 3).

of hybridization of the 3H-labeled firstDNA with the mixture of cloned DNA

from the ori region was approx. 25-30 %, while it was 30-40x when the labeled DNA was hybridized with the whole B. subtilis chromosomal DNA. In contrast, when the nascent DNA was labeled in an exponentially growing cells and used for the same hybridization experiment, less than 1% of the label hybridized with the cloned ori DNA while 30-40%

l

Fraction

Fig. 2. Sucrose-gradient ments

3H-labeled

centrifugation

during

section

as described

were germinated 2.5 @/ml

added to initiate replication.

in MATERIALS

AND METHODS,

on 34 ml of a 5-20%

rotor. Fractions were assayed

frag-

in the absence of

AND and

1 min (open circles)

DNA was extracted

fuged at 24000 rev./min

DNA

of replication. METHODS,

40 Ci/mmol,

was

Cells were killed after the labeling

circles) at 37°C was layered

initiation

in MATERIALS

d. [3H]Thymidine,

for 0.5 min (triangles),

of nascent

synchronous

Spores ofB. subrih CRK2000 thymine

number

and 2 min (closed

and purified

as described

section d. A 2 ml sample sucrose

gradient

and centri-

for 16 h at 18°C in a Beckman

SW28

are taken from the top of the tube and aliquots for 3H counts

in DNA.

left to right. For further analyses, as the first-replicating

Sedimentation

fractions

was from

l-10 have been pooled

DNA.

et al., 1979; Seiki et al., 1979) the first-replicating DNA was labeled in the presence of 6HPU, an inhibitor of DNA polymerase III (Gass et al., 1973), as to reduce the rate of elongation by a factor of 50 (not shown). Fig. 2 shows the pattern of a sucrose gradient centrifugation of DNA strands synthesized in the presence of 6HPU. An analysis of DNA in peak fractions by electrophoresis in agarose gel from samples labeled for 1 and 2 min revealed a broad distribution of sizes up to 1000 bases (data not shown). Up to 2 min, replication of any complete

of the label DNA (see 3H-labeled fically with

hybridized with the whole chromosomal Fig. 4A). These results show that the first-replicating DNA hybridized specithe DNA in the ori region. Fig. 3A shows

that the largest amount of the first-replicating DNA hybridized to a DNA derived from Charon-BS03. Hybridization with smaller fragments produced by EcoRI and Sal1 digestion also shows that the same region covered by Charon-BS03 fragment trapped the largest amount of the first-replicating DNA (Fig. 3B). No other sites in the 45 kb region show specific aflinity with the first-replicating DNA. This result suggests that the replication of the chromosome is initiated within this region. Unexpectedly only a part of the first-replicating fragment B7 is included in this region. To define further the region of the initiation site, hybridization experiments were performed extensively within the 15-kb region. Smaller fragments shown in Fig. 4 were purified from the low-meltingagarose gel after digestion with various restriction enzymes shown in Fig. 4. The result indicates clearly that the site of initiation is located within a 3-kb S&I-Pst I fragment. Hybridization with sub-fragments derived from this fragment did not resolve the initiation site any further (Fig. 4B). A symmetrical distribution of the hybridized counts indicates a bidirectional replication.

(A)

ch_BS()3

-e-m

(B)

$

----_r: ?---- ch-BSO I I

I

E

E

I I ____

4

I

I

I

I

I I I

I+--I

I I I

I I

E

E

ch-BS12

---

+ I I I

04 E

t t 0

E E5

E20 I

5

E6

S

I

S

.I

El9 E22 E27

E4

I

s

I

El3 El7 E26 El4 E22 E21 E27

E

s

I

E3

I

I

IO

rrn0

rrnA

87 Fig. 3. Extent of hybridization of the first-replicating DNA with cloned ori fragments: a preliminary survey. The first-replicating DNA 3H-labeled for 2 min, fractionated and pooled as in Fig. 2 was used to hybridize with fragments cloned in the Charon vector as described in MATERIALS AND METHODS, section e. (A) Whole phage DNA was used. Fragments are arranged according to the or®ion map (Ogasawara et al., 1983b) as shown near the bottom (see E fragments). Locations of two ribosomal RNA operons, rrn0 and rrnA (heavy segments), and B7 fragment are also indicated on the map. (B) EcoRI and EcoRI-Sal1 fragments were used. Total radioactivities used for the hybridization were 4000 cpm in A and 10000 cpm in B, respectively. E, EcoRI; S, Sal1 site.

(c) Determination of the site of synthesis of the firstreplicating DNA on each DNA strand in the ori region Using M 13 vector, it is now possible to clone each complementary DNA strand separately and hybridize to the first-replicating DNA. A complete set of fragments was cloned from the lo-kb region shown in Fig. 5 as described in MATERIALS AND METHODS, section b. Hybridization of the first-replicating DNA strands to these single-stranded M13mp fragments shows that approximately half of the label hybridized to each strand (Fig. 5) even with the sample of the shortest labeling time, indicating that initiation of replication occurs simultaneously from both strands.

The site of synthesis of the first DNA strands in one direction, 5’ + 3’ from left to right, is located in a SalI-EcoRI sub-fragment of E20 and that in the other direction is located in a EcoRI-PstI fragment (Fig. 5). Results of the hybridization experiments with complementary strands are schematically shown in Fig. 6. Direction of synthesis of the first-replicating DNA strands and the amount of hybridization are represented in a semi-quantitative fashion. One major region on each strand shows the highest affinity to the first-replicating DNA. These regions may correspond to the sites for the synthesis of the first leading strands which replicate simultaneously from both strands.

178

t ,i

(A)

-

(A)

I

I

I

3

gP

I

I P

I E

5

I I

1 E

s

I E

I

, ES

s

E

E20

E6

87

4-

6-

6-

v v

v

.

E

PII

to-

v

ES

E6

E25

t I 0

5

El9 EE22E I5

IO

rrtl0

a7

Fig. 4. Extent of hybridization cloned DNA fragments ori

A limited indicated

region

fragments.

E5 through

E, EcoRI;

including

locations

procedure

is as indicated

labeled for

of rrn0

DNA with examination.

E22 was cleaved

sites is shown

and

B7 fragment.

at the bottom, Experimental

in Fig. 3. (A) First-replicating

1min (hatched

into

P, PstI; S, SalI; V, EcoRV; PII,

PvuII. A map of EcoRI cleavage

DNA is

area) or 2 min (open area) fraction-

ated and pooled as in Fig. 2. As a control,

DNA pulse-labeled

from exponentially

growing

in MATERIALS

AND METHODS,

section d (solid black area).

used for hybridization DNA

was labeled

solid or broken lines) or 30 s (hatched used for hybridization

DNA with

Both complementary

strands

(Ml3BS

fragment

DNA

amount

stranded

labeled

for

fragments

60000 cpm.

of 3’ + 5’ direction shown

are aligned according

are aligned as in A. Arrows

of the first-replicating

P

sample.

E

S

P

s

indicate

E

\

/

\

i=

area,

area). Total radioactivities

were 42000 cpm for

1min sample and

10000 cpm for 30 s.

i

i

i

b

i

i

i

<

Fig. 6. Schematic

of the order of the replication of

first-replicating the synthesis 6HPU

We have reported previously that Barn HI-B7 fragment and its EcoRI subfragment, E19, are the firstreplicating fragments, respectively (Ogasawara et al., 1979). However, the present experiment with the first-replicating DNA revealed that E20 rather than

representation DNA. Structure

of the mode of synthesis of the replication

experiments

DNA

Single-stranded are

aligned

map shown in Fig. 5. Arrows

the DNA synthesis cate possible represented

E6

of the first-replicating

for hybridization

mapping

E20

is shown schematically.

restriction

(I(., ,

ES

(d) Reexamination the ori fragments

to

[3H]DNA.

c/

in Fig. 2). (A) Single-

in the middle of the figure. (B) Frag-

ments of 5’ + 3’ direction 5’ + 3’ directions

was

AND

with the first-

1min (pooled fraction

of radioactivity

the map position

were 13 400 cpm for for 1 min (open

in MATERIALS

section b, and used for hybridization

replicating

of

shown in this figure are cloned in M 13

clones in Fig. 1) as described

METHODS, Total

DNA.

cells, as described

1 min, 54 500 cpm for 2 min and 68 500 cpm for control (B) First-replicating

of the first-replicating

single-stranded

for

30 s was isolated Total radioactivities

Fig. 5. Extent of hybridization cloned

each restriction

of the first-replicating

in the’& region: a detailed from

(B)

k

i

of the

origin during

in the presence fragments according

to the

indicate the direction

during the pulse labeling. Open arrows

sites of initiation by broken

the initiation

of replication

of

indi-

with the 5’-termini

lines as the sign of the ambiguities sites.

of used

in

179

a bc

e f

d

g

h

i

k

j

I

- BSI -63

::

whm

- es2

ma

-*

- BS3 - BS4

rrdm

@ r,U@(I-B7

u(I)LI-BSs

Y)

90

#ilID-BS6

tmu

u

- E27-

f3SI

es4

BS6

S i

E3

Fig. 7. Fractionation (lOOpg/ml) BamHI

as described

of thymine

E6 fragments

El9

during

in MATERIALS

AND

METHODS,

section

k

E27 E22

synchronous

were pulse labeled by [3H]thymidine

initiation (10 @i/ml

E4 of replication.

Germinating

and 40 Ci/mmol)

f. The [3H]DNA

spores

in the presence

was digested

with BarnHI

of B. subtilis of novobiocin (lanes

+ Sal1 (lanes d-f), EcoRI (lanes g-i) or EcoRI + Sal1 (lanes j-l). For each set of three lanes, time of labeling periods

5 and 7.5 min from left to right, respectively. described

I3

; Ii ii

E20

of the first-replicating

in the absence

(831 8S2

s 8

Ii

i

E20’

CRK2000

BS7

S

i

(87) BS5

in MATERIALS

AND METHODS,

Fractionation

of the fragments

section f. At the bottom,

by electrophoresis

a restriction

and detection

map and fragment

numbers

by fluorography are shown.

a-c),

were 2, are

180

El9 is the first-replicating experiment

fragment.

apart from a slight M,. difference. as the first-replicating the

In the previous

(A)

l-l

El9 and E20 could not be distinguished

first-replicating

fragment

El9 was assigned because

B7 fragment.

fragment BO adjacent

to the left side of B7 is so large

(some 2%kb) that even if initiation near the right end, B7 fragment long before the replication We have now cloned detailed restriction

it is within

Unfortunately, occurs in the BO

would be replicated

of entire BO is completed. all these fragments

and a

enzyme map has been completed

(see Fig. 1). Therefore

it is possible

E 19 and E20 by Sal1 digestion

to distinguish

which makes one cut

only in E20. Also several Sal1 sites are determined in BO and can be used for determining the order of replication within the BO fragment. The ori region was labeled with [ ‘Hlthymidine in the presence of an inhibitor for DNA gyrase, novobiocin, using synchronously germinating spores as reported previously (Ogasawara et al., 1979) (see also MATERIALS AND METHODS, section d). The labeled DNA was digested and fractionated as shown in Fig. 7. Quantitative analysis is shown in Fig. 8. Digestion with BumHI alone shows that B7 is the first-replicating fragment detectable and no BO fragment has been replicated. On the other hand, when fragments are produced by BarnHI + MI, new fragments labeled as strongly as B7 (which has no Sal1 site) appeared. Quantitative analysis (Fig. 8) shows that specific activities of BS6 and BS7 were higher than that of BS5 (= B7). In the EcoRI digestion experiment, the fragment originally assigned as El9 had the highest specific activity. However, this fragment was digestible by SalI, whereas E20 was not digestible. Therefore the first-replicating fragment detected by fluorography was identical to the cloned fragment E20. Since fragment El9 has been widely known as a subfragment of B7 containing promoters of rrn0, we leave the nomenclature unaltered in spite of the reversed sizes of El9 and E20. We conclude that E20 is the lirst-replieating fragment detectable and is slightly larger than E19. The present results demonstrate clearly that the site of initiation of chromosomal replication, as determined by the order of replication of restriction fragments, coincides perfectly with the determination by the synthesis of the first-replicating DNA strands.

6-

6-

IO

Fig. 8. Quantitative ments.

analysis

Fragments

digested

of the replication by EcoRI

of the ori frag-

(lane h in Fig. 7) and

EcoRI + Sal1 (lane k in Fig. 7) (see A) and those BamHI

and radioactivity plotted,

digested

in DNA was determined.

was obtained

Specific activity,

fragment

(in kb). Radioactivities

as follows. From lane h, El9 and E20 (actually

in E19, E20 and E20’ were

posed of E20 and E20’) bands were cut out separately activities portion

were

measured

(El9 = 755,

E20 + E20’ = 1882

When digested with EcoRI and Sal1 (lane k) the larger of E20 digested

by Sal1 became

inseparable

of E20’ digested

by Sal1 appeared

separated

band between

El9 and E22. Therefore,

for El9 + E20 and E20’ were measured

525 cpm/kb

respectively.

Total activities

were 2637 and 2607 cpm/kb Activities

map of this region.

from E19, as a

from lane k as 2082 and

for El9 + E20 t E20’

from lane h and k, respectively.

for each band were either measured

lated from these values. Fragments restriction

com-

and their

while the larger portion activities

as

by dividing the cpm by the length of each

estimated

cpm/kb).

by

+ Sal1 (lane e in Fig. 7) (see B) are cut out from the gel

are arranged

directly or calcuaccording

to the

n , EcoRI; 0, SalI; 0, BumHI.

DISCUSSION

Two specific single-stranded DNA fragments cloned from the ori region of the B. subtilis chromo-

some hybridize specifically to the first-replicating DNA strands. The first-replicating DNA was synthesized, using a trace amount of [ 3H]thymidine, after germinating thymine-requiring spores in the absence of thymine. Since the mutant has defects in two genes (Neuhard et al., 1978), thyA and thyB, no de novo synthesis of dTTP occurs during the germination. However, the initial DNA synthesis may take place by using a trace amount of cold thymine derivatives present in the spore before the addition of [ 3H]thymidine. The present results of hybridization of newly synthesized DNA with the specific DNA fragments indicate that the labeled short DNA strands synthesized in the presence of 6HPU were the first DNA strands synthesized synchronously from the chromosomal ori site. When double-stranded DNA was used for hybridization with the first-replicating DNA, a region as large as 3.7 kb showed homogeneously high affinity to the newly synthesized fragments (Fig. 4). However, when single DNA strands were used for hybridization, this region could be divided equally into two adjacent fragments (Fig. 5). One major region in each strand hybridized a maximal amount of the first-replicating DNA strands (Fig. 6). These replicating strands may be the first leading strands synthesized from the replication origin. If this is the case, one site of the on’ is located in each strand some 2-kb apart, and replication from these sites begins simultaneously after the addition of thymidine to the germinating spores starved for thymine. Replication proceeds inwards to pass each other. We found that there was an error in the assignment of the first-replicating EcoRI fragment in our previous experiment. El9 was assigned wrongly as the first-replicating fragment because of incomplete information on restriction sites and the error in size determination. It is now clear that E20 is the tirstreplicating fragment. The result is consistent with the initiation site determined by hybridization with the first-replicating DNA. The replication origin newly determined by the present experiment is located some 5-kb upstream from the promoters for a ribosomal RNA operon, rrn0. Therefore, it should be possible to isolate the oriC sequence as an autonomously replicating sequence separated from an inhibitory sar sequence of rrn0 promoters (Seiki et al., 1982). Our attempts

to isolate an autonomously replicating sequence from the ori region of the chromosome have been so far unsuccessful. A possible reason of the failure may be the presence of another sar sequence close to the ori. Alternatively, at least two independent functions may be required for autonomous replication of oriC plasmids in B. subtilis. One is a conventional ori function to initiate the first primer RNA synthesis through interaction with initiation proteins, and the other is a mechanism by which the replicon is stably maintained in the cell. Binding of the chromosome as well as plasmids to the cell membrane has been known to be controlled by dnaB gene which is essential for initiation of chromosomal replication in B. subtilis (Winston and Sueoka, 1980). Experiments to examine these possibilities are now underway.

ACKNOWLEDGEMENTS

We thank Dr. T.H. Jukes for critical reading of the manuscript. We thank Drs. S. Murakami and K. Yamaguchi and other colleagues in our laboratory for discussion and Mrs. K. Terada and Mrs. K. Koshimura for help. This work was supported by a Grant-in-aid for Special Project Research, for Cooperative Research, and for Scientific Research from the Ministry of Education, Science and Culture, Japan.

REFERENCES Gass, B.K., Low, R.L. and Cozzarelli, midines. Ogasawara, biocin Nature Ogasawara, region operons. Ogasawara,

N.R.: Inhibition

ofa DNA

from Bacillus subfilis by hydroxyphenylazopyri-

polymerase

Proc. Natl. Acad.

Sci. USA 70 (1973) 103-107.

N., Seiki, M. and Yoshikawa, on initiation

of DNA

H.: Effect of novoin Bacillus subtilis.

replication

281 (1979) 702-704. N., Seiki, M. and Yoshikawa,

H.: Replication

of Bacillus subtilis chromosome J. Bacterial.

154 (1983a)

N., Moriya,

S. and Yoshikawa,

organization

of rRNA operons

contains

origin

two rRNA

50-57. H.: Structure

and

in the region of the replication

origin ofthe Bacillus subtilischromosome.

Nucl. Acids Res.

11

(1983b) 6301-6318. Oka, A., Sugimoto,

K., Takanami,

M. and Hirota,

origin of the Escherichia coli K-12 chromosome: structure

of the minimum

DNA segment

Y.: Replication the size and

carrying

the infor-

182

mation

for autonomous

replication.

Mol. Gen. Genet.

178

Neuhard,

J., Price, A.R., Schack,

thymidylate

synthetases

L. and Thomassen,

in Bacillus sub&.

E.: Two

Proc. Natl. Acad.

Sci. USA 75 (1978) 1194-l 198. Seiki, M., Ogasawara, some. Nature

H.: Structure

of the

origin of the Bacillus subfilis chromo-

281 (1979) 699-701.

Seiki, M., Ogasawara,

sequence

for DNA replication

in the replication

of the region of the replication

mids containing

I. Isolation

the origin

H.: Structure

origin of the Bacillus

and characterization

region.

and

Mol. Gen.

ofplas-

Genet.

183

S. and

Sueoka,

N.: DNA-membrane

for initiation

of chromosomal

in Bacillus subtilis. Proc.

cation

N. and Yoshikawa,

H.: Identification

of

Natl.

association

and plasmid Acad.

is

repli-

Sci. USA

77

(1980) 2834-2838. S. and Hirota,

cation

Y.: Cloning

and mapping

of the repli-

origin of Escherichia coli. Proc. Natl. Acad.

Sci. USA

74 (1977) 5458-5462. Yoshikawa,

H.: The initiation

subtilis. Proc. Natl. Acad.

of DNA

Communicated

replication

in Bacillus

Sci. USA 58 (1967) 312-319.

(1981) 220-226. Seiki, M., Ogasawara,

Proc. Natl.

Sci. USA 79 (1982) 4285-4289.

Winston,

Yasuda,

N. and Yoshikawa,

subtilischromosome,

Acad.

necessary

N. and Yoshikawa,

region of the replication

function

a suppressor

origin region of the Bacillus sub&s chromosome.

(1980) 9-20.

by K. Matsubara