Studies on bacteriophage fd DNA

J. Mol. Biol. (1975) 95, 21-31

Studies on Bacteriophage fd DNA I. A Cleavage Map of the fd Genome M. TAEANAMI,

T. OKAMOTO, K. SUGIIEOTO AND H. SUGISAKI

Isstitute for Chemical Research Kyoto University, Uji, Kyoto, Japan

(Received 5 November 1974) In order to construct a physical map of the bacteriophage fd genome, the doubly closed replicative form (RFI) DNA of phage fd was cleaved into unique fragments by four different restriction endonucleases (Hap, Hga, HinH and Hind) prepared from Haemophilus strains H. aphirophilus, H. gallinarum, H. i@Zuemzae H-I and H. injluenzae Rd, respectively. As Hind cleaved RF1 DNA at a single site, this site was used as a reference point for mapping. H&H cleaved RF1 DNA at three sites, Hga at six sites and Hap at 13 sites, respectively. The 5’-termini of the fragments produced by either HinH or Hga were labelled with 32P in the polynucleotide kinase reaction. The labelled fragments were separated and further cleaved by other enzymes. The redigestion products of partially digested fragments were also analysed. On the basis of these data and estimates of the size of each fragment, a cleavage map of the phage fd genome was constructed.

1. Introduction The genome of bacteriophage fd is a single-stranded circular DNA about 6000 bases long (Hoffmann-Berling et aZ., 1963 ; Marvin & Hoffmann-Berling, 1963 ; Marvin 2% Schaller, 1966). Like other phages containing a single-stranded DNA, double-stranded replicative form (RF) DNA is formed upon infection of Escherichia cobi cells by this phage, and the RF DNA acts as template for messenger RNA synthesis. RF DNA is known to occur in two different forms; one is doubly closed rings (RFI) and the other is double-stranded rings in which only one strand is closed (RFII). We have previously analysed RNA transcribed in vitro on RF1 DNA by E. coli RNA polymerase and shown that several species of RNA with unique starting sequences and size are transcribed from the “minus” strand of the template (Takanami et al., 1970). The result suggests that RF1 DNA provides sets of specific sites for initiation and termination of RNA synthesis. In order to localize these sites on the phage fd genome and to study the DNA structure at these functional sites, we have attempted to cleave this DNA molecule into unique fragments by bacterial restriction endonucleases. In previous work, the action of Hind (Smith & Wilcox, 1970), Hae (Middleton et al., 1972) and three new Haemophilus enzymes isolated in our laboratory was examined, and each enzyme was shown to cleave RF1 DNA at digerent sites (Takanami & Kojo, 1973; Takanami, 1973). It was also shown that the number of cleavage sites increase additively by the combination of different enzymes. In the present study, the fragments produced by the cleavage with an enzyme were labelled with 32P at the 5’-termini and further cleaved by a second enzyme. The re-digestion products of 21

M. TAKANAMI

22

ET

AL.

partially digested fragments were also analysed. In this way, a cleavage map of the phage fd genome was constructed. Restriction endonucleases used in the present study have been prepared from H. aphirophilus, H. gallinarum, H. ir&enzae H-I and H. ir@uenzae Rd. These enzymes are abbreviated as Hap, Hga, HinH and Hind, respectively, according to the nomenclature proposed by Smith & Nathans (1973). It has been shown recently that H. aphirophilus and H. in$uenzae Rd, respectively, contain different enzymes (R. J. Roberts, J. R. Arrand, S. Zain & P. A. Myers, personal communication). appear to corresAccording to their classification, our Hap and Hind preparations pond to HapII and Hi&II, respectively. The fragments produced by the cleavage with an enzyme (e.g. Hap) were labelled as HapA, HapB etc., and those by a mixture of two enzymes (e.g. Hap and Hga) as Hap-HgaI, Hap-HgaII etc., in order of decreasing product size.

2. Materials and Methods (a) Phagefd

RFI

DNA

The procedure used for preparation of RF1 DNA was essentially identical to that described previously (Sugiura et al., 1969). Phage fd was used to infect a growing culture of E. coli K38 (about log/ml), and replicated for 1 h. Chloramphenicol was added to 30 pg/ml. After 30 min, cells were harvested and gently lysed with lysozyme and sodium dodecyl sulphate. The resulting viscous solution was centrifuged for 30 min at 30,000 revs/min to remove host DNA. The supernatant was treated with 80% phenol. The aqueous layer was concentrated, treated with RNAase, and chromatographed on an Agarose column (BioRad A5m, 50 cm long). The RF DNA fraction was collected and centrifuged on a sucrose density-gradient to separate the RFI and RF11 DNAs. When DNA uniformly labelled with 32P was prepared, phage fd was replicated in cells growing in a Tris/glucose medium (Simon & Tessman, 1963) containing 32P, and the RF1 DNA fraction was prepared as described above.

TABLE 1 The sources of enzyme and number of cleavage sites on phage fcl RFI Enzyme

Hap Hw H&d Hi&I

No. of cleavage sites

Source H. H. H. H.

aphimphilus (ATCC-19415) gallina~um (XCTC-3438) influenzae Rd? in$uenzae H-1$

t Obtained from Dr J. K. Setlow. $ Isolated at Research Institute for Microbial

(b) Restriction

DNA

13 6 1 3

Diseases, Osaka University.

endonucleases

The source of Haemophilus strains is given in Table 1. These strains were grown in brain-heart infusion media containing NAD (2 pg/ml) and hemin (10 pg/ml), and enzymes were purified as described earlier (Takanami, 1973). The enzyme activity was assayed by transfection of RF1 DNA on lysozyme-spheroplasts. One unit of enzyme activity was expressed as the activity which destroys the infectivity of 0.01 Azso unit of RF1 DNA in 30 min at 37°C.

A CLEAVAGE

MAP

OF PHAGE

fd DNA

23

(c) Digestion of DNA For complete digestion, about 200 units of enzyme were added per 1 A,,, unit of DNA, and incubation was carried out for G h at 37°C in 0.01 M-Tris (pH 7*6), 7 m~-MgCl~, 7 m.M-mercaptoethanol. Reaction was terminated by shaking with 80% phenol. The aqueous layer was separated, treated with ethyl ether, briefly dialysed against 0.01 M-Tris (pH 7*6), 0.1 mM-EDTA, and used for subsequent experiments. (d) Label&g

of the 5’-termini

of

DNA

DNA fragments were treated with alkaline phosphatase and rephosphorylated with saP in the polynucleotide kinase reaction, according to the method of Richardson (1966). Alkaline phosphatase was the product of Worthington Biochem. Co. Polynucleotide kinase E. coli cells, as described by Richardson (1965). was prepared from phage T4-infected

(e) PolyacryEamide

gel electrophoresis

Samples were layered on 5% or 10% gel columns (0.6 cm x 12 cm) formed in running buffer (0.036 M-Tris, O-032 M-KH,PO,, 1 mm-EDTA, pH 7.8), and electrophoresed for 12 or 16 h at 2 mA/tube (Takanami & Kojo, 1973). For radioautography, gels were vertically split into halves, covered with thin plastic films, and exposed to X-ray films. When DNA bands were observed by staining, gels were stained for 1 h in 0.4% acridine orange and destained with 0.1 N-acetic acid. For the extraction of DNA fragments from gels, the band regions were sliced, homogenized and extracted with 0.01 IV-Tris (pH 7.9) by incubating for 5 to 6 h at 37°C.

3. Results (a) Analysis

of DNA

fragments produced by cleavages with two cl#eren;c enzymes

Number of cleavage sites on phage fd RF1 DNA by restriction endonucleases used in the present study are given in Table 1. The size of DNA fragments produced has been estimated from the distribution of 32P in each fragment produced from RF1 DNA uniformly labelled with 32P (Takanami, 1973). The size of fragments created by HinH and Hga has also been confirmed by electron microscope measurements (T. Oda 82 M. Takanami, unpublished data). The plot of length versus electrophoretic mobility of fragments gives the correlation in Figure 1. The approximate size of fragments produced by digestion with a combination of different enzymes was estimated from the relative mobility to either Hap fragments or Hga fragments based on this correlation. For the determination of the relative mobility, 32P-labelled fragments were usually co-electrophoresed with non-labelled fragments produced by either Hap or Hga, and the position of non-labelled fragments was detected by staining before taking the radioautograph. The size of fragments was expressed as a percentage of the length of phage fd DNA in a given fragment. As the contour length of RF11 DNA is 1.84 pm f 0.07 pm (Oda et al., 1971), 1% fd unit length corresponds to about 3.5 x lo4 daltons or 60 base-pairs. The radioautographs of fragments produced by digestion with mixtures of different enzymes are shown in Plate I, in comparison with those of each enzyme. As has been shown previously (Takanami, 1973), the Hind cleavage site is located on H&H-A, Hga-A, and Hap-F in the fragments produced by each enzyme (Plate I(b), (d) and (g)). By other combinations, the number of fragments created appear to increase additively (Plate I(e), (h) and (j)). To analyse the cleavage sites by different enzymes, uniformly labelled and nonlabelled RF1 DNA were respectively digested by either HinH or Hga. The resulting fragments were separated by gel electrophoresis, and each fragment was recovered

24

M.

-0

60

TAKANAMI

ET

AL.

Hh%A

4-

l

HOPI \.WJJ.K

2-

‘0

FIG. The obtained in each in each to these

I 3

I 6 Migmtion

I 9

I 12

(cm)

1. Log length wrmza electrophoretic mobility of DNA fragments in 5% polyaorylamide ge1. length of eaoh fragment was expressed as a percentage of the length of fd DNA. (0) Values by electron microscope measurements; (0) values obtained by the distribution of 3aP fragment produced from uniformly labelled RF1 DNA. As HapC, HgaD(E) and H&H-C digest have about the same size, the migration distance of each fragment is shown relative fragments.

from the gels. The fragments obtained from non-labelled RF1 DNA were treated with alkaline phosphatase and rephosphorylated with 3aP in the polynucleotide kinase reaction. Uniformly labelled and terminal labelled fragments thus prepared were digested with a second enzyme, and the digests were resolved by gel electrophoresis. The results of this analysis are summrtrised in Figure 2. (i) Cleavage of HinH fragmenteby Hga Treatment of H&H-A by Hga principally yields three bands (Fig. 2(e), El&H-A + Hgs). However, the largest band of which the mobility was almost identical with HgaB was slightly separated into two bands by further electrophoresis. We concluded that HinH-A was cleaved into four pieces by Hga. The terminal label was found at two regions: Hga-HinH-I and Hgcz-HinH-VI. The mobilities of two other fragments correspond to HgaB and HgaB’. The sum of these four fragments is about equal to the size of HinH-A. Digestion of HinH-B by Hga yielded three bands, in which the terminal label wasdetected at Hga-H&H-II and Hga-HinH-III (Fig. 2(e), HinH-B + Hga). The mobility of the band produced from the inside of H&H-B corresponds to HgaD (or E). Digestion of HinH-C by Hga produced only a single band which migrates much faster than the original HinH-C (Fig. 2(e), H&H + Hga). The termins.1 label w&s also shifted to this region, indicating that Hga cleaved HhH-C right in the middle. The conclusion derived from the above analyses is that HgaA,

F(4.5%)

!$12%)

C(l2%)

(L)(k3%) (M)(l.O%)

=$(‘,2%)

-I (2.6%)

=G’(7.2%) -H(6,5%)

-B(l4%) -C(l2%) -D(IO%) -E(8+3%)

-A(25%)

T-

‘VI (3.5%)

:$GO%)

.me+5x.)

-?l(lO.5%)

.1(24%)

fga-HfnH

--

F,

B,

i2

3

il

(e) Hinff-8

E-

- -

HhH-A

HinH-C

(M)

(L)

E-

c: DI

M(I.O%)

lap-HinH

--

(M)

(L)

-I-

0

(9)

E.

HinH-B t ffop

- -

10)

MM-A + Hop Hap

t

HinH-C

HgaA

Hga6

HpoC

0-j (M)

Ii

F H

(X)(1.3%) (L) 0(1~3%)(M)

IX ( 1.8%)

YlJI(22%b

!JI(6,6%) F H rkwO%)

Wh(p-Hgc I-1(17%)

U-4 Hgaf

Hgof

Pm. 2. Analysis of the fragments oreated by cleavages wit,h different enzymes. Uniformly labelled and terminal labelled fragments were prepared from each digest, and redigested with the second enzyme, as described in Materials and Methods. The species of fragments and enzymes used are indicated at the top of each column. The digests were electrophoresed on 6% gels for 16 h at 2 mA/tube, and the radioautographs were taken. As the references, the positions of fragments produced by each enzyme are shown in (a), (b) and (0). Total fragments created with mixtures of 2 different enzymes are given in (d), (f) and (h). Capitals by the side of each column indicate the fragments produced by cleavages with a single enzyme ((a) H&H; (b) Hga; (c) Hap; (d) and (e) Hga; (f) and (g) Hup; (h) and (i) Hap), and roman numerals on the right side indicate the fragments created by cleavages with 2 different enzymes ((d) and (e) Hga-HinH; (f) and (g) Hap-HinH; (h) and (i) Hap-Hga). AS seen in (g) and (i), HinH-C and HgaC were not digested by the second enzyme. Therefore, the fragments numbered &a Hap-H&H-I in (f) and (g) and Hap-HgaI in (h) and (i) should be BinH-C and HgaC, respectively. The size of each fragment, as a peroentage of the length of fd DNA, is given by the side of fragments. The sum of the fragments in each column was about equal to tho size of the original DNA. The fragments which run through the 5% gel were separately analysed on 10% gel, and indicated in parentheses at the bottom of each column. il and i2 in (e) and (i) are intermediates of digestion.

)-

l-

,-

A(30%) B (25%) C(l7%)

B(30%)

A(58%)

-_

(4 /f/h4

26

M. TAKANAMI

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HgaC and HgaD (or E) each contain one HinH cleavage site. As the size of HgaD and HgaE was about equal, the one which is cleaved by HinH was named HgaD. Thus, the fragment produced from the inside of H&H-B should be HgaE. (ii) Cleavage of HinH

fragments

by Hap

Upon treatment of each HiaH fragment by Hap, HinH-A and H&H-B were cleaved into smaller pieces, producing pairs of terminal fragments (Fig. 2(g)). However, HinH-G was not digested by Hap. This implies that HinH-C should be produced from one of the Hap fragments by the cleavage with HinH. Such a Hap fragment should be composed of H&H-C and one each of the termina.1 fragments produced from H&H-A and HinH-B. The only Hap fragment which is large enough to yield those three fragments is HapA. This is consistent with the observation that the band corresponding to HapA is missing in the Hap-H&H digest (Fig. 2(f)). The other band which is missing in the Hap-H&H digest is HapB, indicating that the other H&H cleavage site is located on HapB. Note that since HinH-C was not cleaved by Hap, the fragment named as Hap-H&H-I in Figure 2(f) and (g) should be H&H-C. (iii) Cleavage of Hga fragments

by Hap

fragments were Each Hga fragment was digested by Hap, and the resulting analysed. All Hga fragments, except HgaC, were digested into smaller pieces by Hap (Fig. 2(i)). Analysis of the fragments produced from the internal region of each Hga fragment indicated that the Hga cleavage sites are located on HapA, HapB, HapD, HapC and HapI. HgaC was not digested by Hap. This implies that HgaC was produced from HapA, because HapA is the only fragment which is large enough to produce HgaC. As Hap-HgaV and Hap-Hga VI were about the same size, the one produced from HgaA was named Hap-Hga V and the other produced from HgaB as Hap-Hga VI. The size of HapJ and HapK was also identical: the one produced from HgaA was named HapJ and that produced from HgaB as HapK. Note that since HgaC did not contain the Hap cleavage site, the fragment named as Hap-HgaI in Figure 2(h) and (i) should be HgaC. (b) A physical

map constructed by the cleavages with Hga, Hind and HinH

As described in the previous section, the three HinH cleavage sites are located within HgaA, HgaC and HgaD, respectively. Thus, these three fragments should be constructed from pairs of the six terminal fragments produced from three Hid2 fragments by the cleavage with Hga. Among HgaA, HgaC and HgaD, the smallest one, HgaD, has the mobility identical to that of HinH-C, and HinH-C is cleaved by Hga into two pieces with about the same size : Hga-HinH-I V and Hga-HiniY- V. On the basis of this observation, we simply ruled out the combination of Hga-H&H-IV (or V) and the smallest terminal fragment, Hga-Hi?zH-VI, because such a combination would yield a fragment which migrates much faster than HgaD. This leads to the conclusion that the terminus of HinH-A which yields Hga-HinHVI should be linked to H&H-B and the other terminus of HinH-A to H&H-C. This is consistent with the result that the fragment constructed with Hga-HinH-I and Hga-H&H-IV (or V) has a size about equal to HgaA. As Hga-H&H-IV and Hga-HinHV have about the same size, the one which links to H&H-A was named Hga-H&H-IV. For the construction of HgaC and HgaD, there are only two possibIe combinations. By comparing the sizes of these two fragments with those of the four Hga-HinH

A CLEAVAGE

MAP

OF PHAGE

TABLE

21

fd DNA

2

The construction of Hap and Hga fragmelzts from sub-fragments produced by cleavages with other enzymes

(A) Hga fragments HsaA (30%) HgaC (17%) HwD (12%) (X) Hap fragments H~PA HapB HapD HapC HapI

(25%) (14%) (10%) (7.2%) (2.6%)

sum of constituentst (%I

Constituents

Fragments

containing

HinH

sites 30 16.5 12

Nga-H&-I (24%) + Hga-HinH-IV (6%) Hga-HinH-II (10.5%) + Hga-HinH-V (6%) Hga-HirzH-III (8.5%) + Hga-H&H-VI (3.6%) containing

Hga sites

HgaC (17%) Hap-HgaII Hap-HgaIV Hap-HgaVI Hap-HgaIX

+ Hap-HgaIII (6.8%) + Hap-HgaXI (114%) + Hap-HgaVIII (2.2%) (6%) + Hap-HgaV (3.8%) (3.8%) + Hap-HgaVII (3%) (1.8%) + Hap-HgaX (1.3%)

p The length of each fragment

is indicated

as a percentage

(1.3%)

25.1 13.7 9.8 6.8 3.1

of the length of fd DNA.

fragments remaining, we concluded that HgaC is formed by Hga-H&H-II and Hga-HinH-V and that HgaD is formed by Hga-H&H-III and Hga-HinH-VI (Table 2 (A)). The cleavage of H&H-A by Hga produces two Hga fragments, HgaB and HgaP, from the internal region of HinH-A. We noted that an incomplete digestion of HinH-A by Hga often yielded two intermediates, il and i2 (Fig. 2 (e), HinH-A + Hga). These two intermediates were isolated and re-digested by Hga. Digestion of il yielded HgaB and i2, and i2 was split into Hga.F and Hga-HinH-VI by further digestion. Thus, the four fragments were arranged in the following order : Hga-HinH-I

-

HgaB -

HgaF - Hga-HinH- VI.

HgaA was cleaved by Hind into two pieces of about 8% and 22% fd unit lengths (Plate P(d)). On the other hand, the Hind cleavage site on HinH-A is located at about one-third of the distance from one end. Since the Hind cleavage site is unique, this site was designated the zero point. The cleavage map thus constructed is shown in Figure 3. Each cleavage site was numbered as Hgal, Hga2 etc. in the counterclockwise direction starting from the zero point, as RNA transcription proceeds in this direction (Okamoto et al., 1975, accompanying paper). The map distance from the zero point was also indicated in this map as the fraction of the length of phage fd DNA. (c) Localization

of Hap fragments

on the ctivage map

(i) HapA, HapB ano! HapE As described in section (a) above, one conclusion derived from the analysis of the Hap cleavage sites on HinH and Hga fragments was that both HgaC and HinH-C were produced from the inside of HapA. Therefore, HapA should be localized at the region which covers both HgaC and Hi%H-C. This also leads to the conclusion that

28

M. TAKANAMI

ET

AL.

FIG. 3. A physical map of the fd genome construebed by the cleavages with Hga, Hind and HinH. The Hind cleavage site was designated the zero point, and each cleavage site was numbered in the counterclockwise direction. The map distance from the zero point is shown as the function of the length of fd DNA.

HapB containing the other HinH cleavage site overlaps with HgaD which contains the H&H-I cleavage site. Upon digestion of HinH-B by Hap, only HapE was p~duced from the internal region of H&H-B, indicating that H&H-B contains two Hap cleavage sites (Fig. 2(g), H&H-B + Hap). Digestion of a mixture of HgaD and HgaE by Hap also produced HapE (Fig. 2(i), HgaD,E + Hap). As HgaD is overlapped with HapB, it is reasonable to conclude that HapE was produced from the inside of HgaE. On the basis of this information and estimates of the size of each fragment, HapA, HapB and HapE were mapped as shown in Figure 4. The possible constituents of HapA and HapB, and their sizes constructed are given in Table 2(B). (ii) HapD, HapG and Hap1 These fragments have been shown to contain the Hga cleavage sites. Among these fragments, the mapping of HapD is principally based on the analysis of DNA fragments whieh bind to RNA polymerase (Okamoto et al., 1975, accompanying paper). Upon mixing RNA polymerase with the Hap-Hga digest, two fragments, HapC and Hap-HgaV (or VI), were specifically bound to the polymerase with GTP present (see Plate III in the accompanying paper). The fragment corresponding to the smaller one was also isolated from either the digest of HapD by Hga or the digest of HgaA by Hap. As Hap-HgaV and Hap-HgaVI were about the same size, we previously determined that the one produced from HgaA is Hap-HgaV. Thus, we concluded that HapD overlaps with HgaA at the Hap-HgaV region. In addition, we observed that the cleavage of HapD by Hga yields two fragments with the sizes of Hap-HgaJV and Hap-HgaV. Hap-Hgal V has been shown to be produced from HgaB. The location of HapD is shown in Figure 4.

A CLEAVAGE

MAP

OF PHAGE

fd DNA

29

FIG. 4. Looalization of Hap frqpents on the cleavage map with Hya, Hind and HinH. The reman numerals on the map are Hap-Hga fragments (II to XI).

We already assigned seven of the eleven Hap-Hga fragments obtained from the Hga by digestion with Hap (see Table 2 (B)). The Hap-Hga fragments remaining are Hap-HgaVI, Hap-HgaVII, Hap-HgaIX and HapHgaX. HapG and HapI should be constructed with these four fragments. In addition, the regions which cover the Hga2 and Hga3 cleavage sites are left for these two Hap fragments (Fig. 4). With these informations and estimates of the size of each fragment, we constructed HapG and Hapl as shown in Table 2 (B). fragments

(iii) HapC amd HapK These two fragments were produced from HgaB by digestion with Hap (Fig. 2(i), HgaB + Hap). Their order within HgaB was determined by the analysis of partial digestion products. Gel electrophoresis of the partial Hap digests of HgaB always gave an additional band at il indicated in Figure 2(i), HgaB + Hap, whereas all other intermediates migrate slower than HapC. This il band was isolated and redigested with Hap. HapK and Hap-HgaIV were produced from il, indicating that HapK is linked to Hap-HgaIV. Thus, Hap? and HapK were localized as shown in Figure 4. (iv) HapF, HapH, HapJ, HapE and HapM These fragments are derived from HgaA (Fig. 2(i), HgaA + Hap). In addition, it has been shown that HapP contains the Hind cleavage site (Plate I(g)). To analyse the location of these fragments within HgaA, HgaA was cleaved into two fragments by Hind, and the resulting fragments were further digested by Hap. HapH, HapJ, HapL and HapM were produced from the larger fragment, indicating that these four Hap fragments are located between HapA and HapP (Fig. 4). However, the order of these four fragments is not determined yet.

30

M. TAKANAMI

ET

AL.

4. Discussion The discovery of restriction endonucleases has enabled us to cleave a DNA molecule into unique fragments. In addition, the DNA fragments can be ordered by analysis of overlapping fragments obtained either by partial digestion with an enzyme or by digestion with a second enzyme (Danna et al., 1973). In the present study, we applied the terminal labelling method for analysis of the cleavage sites among different enzymes. The physical map shown in Figure 3 is based on the cleavage sites with three different enzymes, Hga, Hind and H&H. Further confirmation for this map comes from evidence obtained by analysis of the Hap cleavage sites and by identification of the fragments which bind to RNA polymerase. For example, the evidence that HapA was the origin of both HgaC and H&H-C indicates that these two fragments overlap on the map. As shown in the accompanying paper, the fragments which formed stable complexes with RNA polymerase in the presence of GTP were HinH-A in the H&H digest, HgaA and HgaB in the Hga digest, HapC and HapD in the Hap digest, and HapC and Hap-HgaV in the Hap-Hga digest. The result is well explained on the basis of the cleavage map shown in Figures 3 and 4. Although we used only four species of enzymes for the construction of the cleavage map, many enzymes with different cleavage-site specificities have been isolated from a variety of bacterial strains (for review see Smith & Nathans, 1973). Therefore, it would be possible to cleave each fragment into smaller pieces by other enzymes in relation to this map: as we have done with Hap. It should be mentioned however that the efhciency of digestion with this type of enzyme appears to be greatly intluenced by the size of substrates. In general, the smaller the fragment, the longer the incubation period required for digestion. For the construction of an accurate physical map, it is of importance to determine the size of DNA fragments accurately. In the present study, the size of larger fragments has been confirmed by electron microscope measurements. However, the estimate of the size of smaller fragments is based on the mobility on gel electrophoresis. This would lead to some errors in estimations. The size of the smaller fragments should be determined by other direct methods. The creation of the cleavage map has allowed the assignment of markers of biological significance, as has been done with other viral DNAs (Danna & Nathans, 1972; Chen et al., 1973; Griffin et al., 1974). We have examined the affinity toward RNA polymerase as well as the template activity of each DNA fragment, and localized the RNA initiation sites on the cleavage map (see accompanying paper). It would be possible also to assign the genetic markers to the physical map, as the genetic map of the male-specific filamentous phages has been constructed (Lyons & Zinder, 1972). In this connection, we would like to mention the recent work by H. P. Seburg and H. Schaller (personal communication) who assigned the genetic markers to the physical map of phage fd constructed by the cleavage with HpaII from H. parainjluenxae. Both Hap and HpaII have been shown to cleave DNA at the same sequence (Sugisaki & Takanami, 1973; Garfin & Goodman, 1974; K. Murray, personal communication). Seburg and Schaller have used an entirely different procedure for mapping of the HpaII fragments, and obtained the cleavage map essentially identical to that obtained with Hap.

A CLEAVAGE We thank The results Organisation

MAP

OF PHAGE

fcl DNA

31

Drs H. P. &burg and H. Schaller for the exchange of unpublished information. reported in this paper were presented at the European Molecular Biology Workshop for restriction enzymes (Gent, Belgium) in May 1974. REFERENCES

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