MGG

Molec. gen. Genet. 143, 279-290 (1976)

© by Springer-Verlag 1976

Physical Mapping of the HindIII, EcoRI, Sal and Sma Restriction Endonuclease Cleavage Fragments from Bacteriophage T5 DNA* Alexander yon Gabain, G a r y S. Hayward**, and H e r m a n n Bujard Molekulare Genetik, Universitfit Heidelberg; D-6900 Heidelberg 1, Im Neuenheimer Feld 230, Federal Republic of Germany

Summary. The D N A of bacteriophage T5 + (molecular weight 76 x 106 dalton) has been dissected by various specific endonucleases. The restriction enzymes H i n d I I I and E c o R I produce 16 and 7 fragments respectively, whereas Sal and Sma produce 4 fragments each. Complete cleavage maps were established for the enzymes E c o R I , S a l and S m a and an almost complete m a p for HindIII. Furthermore the location and size of the deletions St 20, St 14, b3, St 0 and bl were determined. The correlation of the genetic and functional m a p of the phage with the arrangement of fragments produced by the different enzymes has been established.

Introduction During its development bacteriophage T5 expresses 80 to 120 genes in a well defined sequence. Between infection and lysis of the host cell three classes of phage specific proteins (McCorquodale and Buchanan, 1968) and the corresponding classes of R N A (Moyer and Buchanan, 1969; Sirbasku and Buchanan, 1970) are synthesized in a fixed temporal sequence. The location of the transcriptional regions of the R N A classes I, II and III (" preearly ", " e a r l y " and " l a t e " respectively) along the phage D N A has been determined (Hendrickson and Bujard, 1973; H a y w a r d and Smith 1973) and correlations have been found between these regions and the genetic and functional m a p of the phage D N A . Our specific aims in the study of the regulation of T5 development are presently the determination of the distribution of regulating regions, especially * This paper is the 4rd publication in the series "Structure and Function of the Genome of Coliphage T5". ** Present Address: Dept. of Microbiology; Universityof Chicago, 939 East 57th St. Chicago, Ill. 60637, USA

promotors, throughout the genome as well as the elucidation of the mechanism of the switch from early to late transcription. A more precise knowledge of the physical arrangement of the genetic text within the T5 D N A would greatly facilitate the study of these two questions. We have therefore cleaved the T5 + c h r o m o s o m e with a number of restriction endonucleases including HindIII, E c o R l , Sal and Sma. These were found to produce respectively 16, 7, 4 and 4 chromosomal fragments. It was possible to m a p almost all the sites of attack of these enzymes. As a result we are now able to isolate and to study individually, chromosomal fragments comprising defined sections of only early or late genes as well as various combinations of all three gene classes.

Materials and Methods a) Chemicals

All chemicals were reagent grade from E. Merck Co (Darmstadt, Germany). Agarose for gel electrophoresis was purchased from Seakem MCI-Biochemicals (Rockland, Maine USA). Ethidium bromide was obtained from Serva (Heidelberg) and all radiochemicals from Amersham Buchler (Braunschweig, Germany). Yeast extract, beef extract, Tryptose and brain-heart-infusion were purchased from Difco Labs., (Detroit, Michigan, USA). c~-32p-DeoxyCytidintriphosphate (25 x 106 cpm/gMol) was a gift of H. Schaller (Universit/it Heidelberg). b) Growth of Phages and Bacteria

The origin and growth of phage T5 on E. coli F and B strains has been described previously (Bujard and Hendrickson, 1973; Hayward I974). The heat stable mutants T5st20, T5stl4, T5st0, T5bl and T5b3 were obtained from Y.T. Lanni (Institut Gustav Roussy, Paris). Haemophilus influenzae serotype d was obtained from H.G. Zachau and grown according to the procedure of Smith and Wilcox (1970). Streptomyces albus G, a gift of R.C. Roberts (Cold Spring Harbor Laboratory) was grown at 37° C to a density

A.v. Gabain et al. : Mapping of Restriction Sites in T5 DNA

280 of 1.0 at 550 nm in a medium containing per liter 0.3 g yeast extract, 0.3 g beef extract, 0.6 g tryptose, 0.3 g Glucose and 0.01 M Tris-HC1, pH 7.2.

c) Enzymes Restriction endonucleases II and III from Haemophilus influenzae d (HindII and HindIII) were isolated as described by Smith and Wilcox (1970). The endonuclease "Sal" from Streptomyces albus was purified following a procedure described elsewhere (Gabain, 1975), and EcoRI enzyme was isolated from E. coli RY-13 according to Yoshimori (1971). Endonuclease of Serratia marcescens ("Sma ") was a gift of C. Mulder (Cold Spring Harbor) and exonuclease III as well as DNA polymerase I were gifts of H. Schaller (Universitfit Heidelberg). All restriction enzymes appeared free of exonucleolytic activity as determined in the following way: 3 lag of 32P-labeled T5 DNA (specific activity 6 x 106 cpm per laMol) were mixed with an aliquot of the purified exonuclease which was sufficient to fully digest 3 lag of T5 DNA in 90 min. at 37° C. After incubation of this initial mixture for four hours at 37° C, the same amount of fresh enzyme was added and the incubation continued for four more hours. This process was repeated one more time. After this 12 hour treatment, less than 0.01% of the 3zp-labeled DNA was rendered acid soluble.

d) Analytical and Preparative Agarose Gel Electrophoresis of DNA Unlabeled and 32p-labeled T5 DNA was prepared from CsC1 purified phage as described previously (Bujard and Hendrickson, 1973; Hayward 1974). Analytical gel electrophoresis was carried out according to Hayward and Smith (1972a) and Hayward (1974) using agarose gel columns (diameter 1 cm) with agarose concentrations between 0.3 and 1.2%, as indicated in the text, and a DNA load of 1 to 3 lag. DNA bands were located in the gels either by autoradiography (Hayward, 1974) or by staining the gels for 30 rain in aqueous ethidium bromide solution (0.5 lag per ml) and subsequent illumination with short wave ultraviolet light. For records, ethidium bromide stained gels were photographed on Ilford Pan F film. For preparative gel electrophoresis of DNA, agarose columns of 2.5 cm in diameter were used. Up to 25 lag of DNA were applied per column. Electrophoresis in these gels was carried out at 2 - 4 ° C and at a constant Voltage of 30 V for 18 hours. DNA fragments separated in this way were reisolated from agarose by potassium iodide density gradients as described previously (Blin et al., 1975).

e) Digestion of T5 DNA by Restriction Endonucleases A typical reaction mixture (100 gl) contained 1.5 to 4 gg of T5 DNA in 0.01-M-MgC12 and 0.01-M-Tris-HC1 pH 7.6 (for EcoRI enzyme pH 7.6 and for Sma pH 9.0). The salt concentration used for each enzyme was as follows: 0.025 M-NaC1 for HindIII, 0.075 M-NaC1 for Sal, 0.01 M-KC1 for Sma and 0.05 M-KC1 for

EcoRI. The digestions were usually carried out at 37°C (30°C for

f) Double Digestion of T5 DNA by two Restriction Endonucleases Double digestion with HindlII and EcoRI as well as with HindIII and Sal were carried out by incubating T5 DNA with a mixture of the two enzymes under conditions described for HindIII. For a double digestion with EcoRI and Sal, the DNA was first treated with the EcoRI enzyme for 90 min. Then NaCI was added to a final concentration of 0.075 M followed by Sal enzyme and the incubation was continued for 90 rain. Double digestion by Sma and Sal enzyme was also sequential. The DNA was first incubated with Sma under Sma conditions. The pH of the reaction mixture was then lowered to 7.6 by adding 0.05 M-Tris-HC1, pH 7.2, the Sal enzyme added, and the incubation continued for 90 rain. All reactions were stopped as usual with EDTA.

g) Introduction of Radioactive Label into the Terminal Regions of T5 DNA Single strand breaks of T5 DNA were first sealed with DNA ligase as described previously (Bujard and Hendrickson, 1973). Subsequently this DNA was incubated with exonuclease III in order to prepare the substrate for the end labeling with c~-32plabeled nucleotides using DNA polymerase I. A typical reaction mixture (100 gL) contained 3 gg of DNA and 0.1 units of exonuclease III (Richardson et al., 1964) in 0.007 M MgC12, 0.001 M 2-mercaptoethanol and 0.07 M Tris-HC1, pH 8. Incubation was at 30° C. The rate of exonuclease III action under these conditions was determined in a pilot experiment in which 32p-labeled T5 DNA (specific activity 6 x 106 cts per min per gMol) was treated with exonuclease III for various lengths of time and the release of acid soluble radioactivity was monitored. The T5 DNA substrate was then prepared by digestion of unlabeled DNA with exonuclease III for 30 min. which was the time required to digest 2 to 3% of the T5 DNA. The exonuclease III digest mixture was pipetted into a tube containing a freeze dryed mixture of nucleoside triphosphates yielding a final concentration of 10 gM for dGTP, dATP and dTTP and of I gM for c~-a2P-dCTP (specific activity 2.5 x 107 cts per min per gMol). Then KC1 was added (final concentration of 0.02 M) followed by 0.02 units (Richardson and Kornberg, 1964) of DNA polymerase I. After incubating for 20 min. at 37° C, 2 to 4 x 104 cts per min. per gg of T5 DNA were incorporated. The DNA of the reaction mixture was then phenol extracted and dialyzed against 0.01 M Tris HC1, pH 7.6.

h) Electron Microscopy of DNA DNA was prepared for electron microscopy by the cytochrome C method as modified by Lang and Mitani (1970). DNA of Pseudomonas phage PM2 (Espejo and Canelo, 1968) was used as internal length standard. Photographs were taken with an Siemens Elmiskop I and evaluated as described previously (Bujard, 1970).

Results

a) The Strategy of Fragment Mapping

Srna) for 90 min. The endonucleases were added to the reaction mixture in two equal aliquots at the beginning of the incubation and after 45 min. The digestions were stopped by the addition of EDTA to a final concentration of 0.02 M. The total am'ounts of our enzyme preparations required for a complete digestion of 3 lag of T5 DNA within 90 min were 4 lal of HindIII, 0.4 lal of EcoRI, 6 gl of Sal and 6 gl of Sma.

T h e i n t e g r a t i o n o f t h e r e s u l t s o f five t y p e s o f e x p e r i mental approaches has established complete cleavage m a p s o f T5 D N A f o r t h e e n d o n u c l e a s e s E c o R I , SaI and Sma and a an almost complete map for endonuclease HindIII.

281

A.v. Gabain et al. : Mapping of Restriction Sites in T5 D N A

(i) The study was initiated by digesting to completion purified T5 ÷ DNA with each of the four enzymes. Conditions for agarose gel electrophoresis were then defined under which all DNA fragments of the digests could be completely resolved, their stoichiometry demonstrated and their molecular weights characterized. (ii) Next an analysis of digestions of DNA obtained from a set of T5 deletion mutants was made, which led to the identification of fragments within and near an internal deletable region of T5 ÷ chromosome. In the various digests of these mutant DNA's certain wild type fragments were altered, either being merely reduced in size (when the deletion did not eliminate a restriction site) or becoming fused to other fragments (when the deletion did eliminate a restriction site). A fusion of two fragments was assumed if in the digest of a deletion DNA two or more wild type bands disappeared and were replaced by a new band consisting of a DNA fragment whose molecular weight was less than the sum of molecular weights of the missing fragments. (iii) Introduction of radioactive label into the terminal regions of the DNA molecule before its cleavage with the restriction enzymes permitted the identification of the fragments originating from the ends of the T5 DNA molecule. (IV) Redigestion of isolated EcoRI fragments individually with the HindlII and Sal enzyme made possible the identification of the restriction sites of the latter two enzymes in each EcoRI fragment thereby revealing the regions of overlap in these three maps. (V) Double and triple digestions of T5 ÷ DNA with various enzyme combinations also yielded detailed information on the position of restriction sites of different enzymes relative to each other. The results of these digestions also served further to confirm all conclusions drawn from the first four experimental approaches.

b) Fragmentation of T5 DNA by Different Restriction Endonucleases T5 + DNA was digested with HindIII, EcoRI, Sal and Sma enzymes and the number and size of the resulting fragments were analyzed by electrophoresis on agarose gels. As shown in Fig. 1 the enzyme HindIII yields 16 fragments, EcoRI 7 and Sal and Sma 4 each. Although the DNA fragments were distributed over a wide range of molecular weights it was possible to resolve them by electrophoresis of the digests at different agarose concentrations ranging from 0.3 to 1.2% (compare for example the cases of the HindIII and EcoR1 digests in Fig. 1).

Table 1. Molecular weights of T5T DNA fragments produced by different restriction endonucleases Fragment

Restriction Enzyme HindlII

EcoRI

Sal

Sma

( x 106)

( x 106)

( × 106)

( × 106)

A

11.4

28

29.2*

35

34.1" (st 0 DNA)

30

B

10.1

18

19.7"

26

21.0" (st 0 DNA)

24

C

9.2

16

16.0"

9.7

16

D

8.6

9.0

8.7*

8.4

8.4

E

7.7

2.0

2.1"

~79.1

5278.4

F

7.1

1.6

1.6"

G

4.5

0.4

H

4.3

I

3.15

J

3.0

K

2.6

L

2.5

M

1.5

N

1.35

O

0.9

P

5275.0 77.3*

0.6 ~78.6

The molecular weights shown were estimated by electrophoresis using EcoRI digests of 2 D N A as internal marker (Allet et al., 1973; Thomas and Davis, 1975) or by direct electronmicroscopic length measurements of the fragments using PM2 D N A (6.3 x 106 dalton as calibrated with T7* D N A of molecular weight of 25.2 x 106 dalton) as internal length standard. The values obtained by electron microscopy are indicated by *. st 0 is a deletion mutant of T5 ÷ (see Fig. 8).

The molecular weight of the DNA in each band (Table l) was estimated either by coelectrophoresis of the T5 DNA digests with an EcoRI digest of phage 2 DNA, the fragments of which have been well defined and served as molecular weight markers (Allet et al., 1973), or by direct electron microscopic measurements of isolated fragments using phage PM2 DNA as internal standard. By comparing the intensity of the individual bands with their electrophoretic mobility (Fig. 2) it appears that in all cases only one type of fragment is present per band. This conclusion is confirmed by the fact that the summation of the molecular weights of the fragments derived from each digest results in values in the range of 77 × 106 dalton _+5% (Table 1), which agrees well with our previously published value of 76.3 x 106 dalton for the intact molecule (Bujard and Hendrickson, 1973).

A.v. Gabain et al. : Mapping of Restriction Sites in T5 D N A

282

Hind

EcoRI

TIT

T5 + st20,14,

sto

sto

bl

A-E~

T5 +

SGI

T 5 + sto b l

T 5 + sto

Sma T5+sto

A-C__-

I / o

b

c

d

Fig. la-d, Agarose gel electrophoresis of T5 D N A after digestion with restriction endonucleases. Migration was from top to bottom.

(a) Digests of T5 D N A with Hind~lI endonuclease. The gel patterns grouped on the right were obtained using 0.7% agarose and show, from left to right, D N A digests of T5 + and of deletion m u t a n t s st 20, st 14, sr 0 and b I.-In order to demonstrate the difference in resolution obtained as a function of agarose concentration, the st 0 gel pattern obtained using 1.0% agarose is shown on the left. (b) Digests of T5 D N A with EcoRI endonuclease. The three gel patterns shown on the right were obtained using 0.7% agarose and show, from left to right, D N A digests of T5÷, T5 st 0 and T5 b 1. The gel pattern obtained using 1.2% agarose is shown to the left, again to enable a comparison of the resolution at different gel concentrations. (c) Digests of T5 D N A with Sal endonuclease. The patterns of T5 and T5 st 0 D N A were both obtained using 0.5% agarose. (d) Digests of T5 D N A with Sma endonuclease. The pattern of T5 ÷ and T5 st 0 D N A were both obtained using 0.5 agarose gels

1

a

A-E

I MN

c

O

P

i

4

A

A BCE

e

3

o

I

F

2

Z0 g) o_ 5"

1

~+

7: ~o

b

AB

Ae

Fig. 2a-d. Stoichiometry of fragments within the

CD o~

C 5 X

6

20 MigFaUon - - - - - 4 - -

40 60 80 Slice n u m b e r

100

different endonuclease digests. (a) and (b) show densitometer tracings of photographs taken of agarose gels in which a HindIII digest of T5 st 0 D N A (a) and a Sma digest of T5 ÷ D N A (b) were analysed by electrophoresis. (c) and (d) show the distribution of radioactivity in gel fractionations of 3/p-labeled T5 st 0 D N A digested with EcoRI enzyme (c) and with Sal enzyme (d). Gels were cut in 120 slices and the Chercnkov radiation of each slice was monitored. The proportionality of the staining intensity and of the radioactivity of the different bands with their relative electrophoretic mobility demonstrates that the fragments of each digest are present stoichiometrically

A.v. Gabain et al. : Mapping of Restriction Sites in T5 DNA

Hin d Ill T5 + st20 st14

b3

283

Sol

Eco RI sfO

bl

T5 + st20 s i l l

A

b3

st0

--

Smcl

T5 + st0

bl

B

A B C

- -

B

C --------

A B 6 D EF -

D

T5 + M 0

bl

-- - ~ - - - -

C D

6H

Fig. 3. Graphical display of the gel fractionation patterns of all digestions carried out with T5 ÷ and T5 deletion DNA's using restriction endonucleases HindIII, EcoRI, Sal and Sma. From this scheme it is readily seen that in the Sal and Sma digests only fragments B and A respectively are altered in the different deletion strains whereas, in the HindIII digest fragments A, O, F and H, and, in the EcoRI digest, fragments B, C, E and G are affected

Ij KL

c) Digestion o f D N A o f T5 Deletion Mutants by HindIII, EcoRI, Sal and Sma Enzyme Several h e a t stable m u t a n t s o f T5 have been described ( A d a m s , 1953; H e r t e l etal., 1962) a n d i t has been d e m o n s t r a t e d t h a t the i n c r e a s e d h e a t stability o f these m u t a n t s is due to the presence o f v a r i o u s deletions in the D N A m o l e c u l e ( R u b e n s t e i n , 1968). All deletions i n v e s t i g a t e d so far have been localized somewhere b e t w e e n m a p p o s i t i o n 0.19 a n d 0.36 o f the T5 ÷ c h r o m o s o m e (Fig. 4) ( H a y w a r d , 1974; Scheible a n d R h o a d e s , 1975; Delius, H. pers. c o m m . ; L o c k e r , M. a n d Weiss, S.B. pets. comm.). It w o u l d be expected therefore, t h a t d i g e s t i o n o f D N A f r o m deletion m u t a n t s s h o u l d yield i n f o r m a t i o n c o n c e r n i n g the a r r a n g e m e n t o f r e s t r i c t i o n f r a g m e n t s within a n d n e a r the d e l e t a b l e r e g i o n Figs. 1 a n d 3 a n d table 2 show h o w the v a r i o u s d e l e t i o n s alter the f r a g m e n t p a t t e r n s o b t a i n e d with the digests o f the different restriction enzymes. T h e d e l e t i o n s affect the f r a g m e n t s HindIII A , F, H a n d O, EcoRI B, C, E a n d G , Sal B a n d

Sma A. O f the five d e l e t i o n s s t u d i e d here (st 20, st 14, st 0, b 3 a n d b 1) we k n o w ( H a y w a r d , 1974; H a y w a r d , u n p u b l i s h e d results) t h a t two, st 0 a n d st 20, e l i m i n a t e the n a t u r a l single s t r a n d b r e a k in the T5 c h r o m o s o m e l o c a t e d at m a p p o s i t i o n 0.31 (Figs. 4 a n d 8). Since d e l e t i o n st 20 affects exclusively HindIII f r a g m e n t F (Figs. 1 a n d 3) it can be c o n c l u d e d t h a t this f r a g m e n t spans the single s t r a n d b r e a k at 0.31 f r a c t i o n a l length. S i m i l a r l y d e l e t i o n b 3 affects only HindIII f r a g m e n t F a n d reduces its m o l e c u l a r weight b y 4.1 × 106 d a l t o n . This deletion, however, is k n o w n to e x t e n d to the left o f the single s t r a n d b r e a k at

Table 2. DNA restriction fragments of T5+ altered by the presence of different deletions Restriction Fragenzyme ment

HindIII

EcoRI

Sal Sma

Deletion mutants of T5 st 20

st 14

A

-

-

F

-2.6

st0

b t

-

AF

-4.1

FH

FA

H O

-

-

-

HF -

absent

-

-

-

3.1

b3

(Ko)

A

/x.

B C

-2.6

E G

-

BC (~ 30) CE CE CE BE ())~5.3) (14.3) (11~2.0) (~ 30) EC EC EC absent absent absent absent absent

A

-2.6

-3.1

A

4.1

-5.4

-6.6

- 5.4

The table presents a summary of the alterations observed in the usual T5* pattern when DNA's of deletion strains were digested. The symbol /x is used to designate fragment fusion, e.g. A~ indicates that fragment A is fused to fragment F. The numbers in parentheses under each fusion is the molecular weight (x 106 dalton) of the fused fragment. Negative numbers indicate the diminiution in size ( × i06 dalton) of a fragment due to the presence of the deletion.

m a p p o s i t i o n 0.31 ( H a y w a r d , 1974). HindIII f r a g m e n t F t h e r e f o r e extends at least 4.1 x 106 d a l t o n to the left o f m a p p o s i t i o n 0.31. T h e r e are two deletions, b 1 a n d st 0, the presence o f which cause the fusion o f two HindIII f r a g m e n t s (Figs. 1, 3 a n d T a b l e 2). T h e b 1 d e l e t i o n fuses HindIII f r a g m e n t F w i t h f r a g m e n t A while f r a g m e n t 0 is lost

284

A.v. Gabain et aL : Mapping of Restriction Sites in T5 DNA I~ E

A

H i n d l][

dr

-I

o

F

H

i

IE~

E c o R]: D

Sal

b

I

I

Q1

D

Q2

(GIJL)

F I I

0.t3

M II

D

(p)

A

A A

I

0

C

B

(NK) I i

B

i

Sma

C I

C

1

C

I

0.4 Q5 f r a c t i o n a l length

o16

0.7

and the st 0 deletion fuses HindlII fragment F and H. Since the b 1 deletion neither deletes the single strand break at map position 0.31 (Hayward, unpublished results) nor affects HindIII fragment H it must extend to the left of map position 0.31 deleting HindIII fragment 0 completely and fusing HindIII F with A. The st 0 deletion on the other hand eliminates the single strand break at 0.31 fractional length and fuses HindIII fragment F to H. HindIII fragment H has therefore to be located adjacent to the right end of fragment F. From these arguments we conclude that HindIII 0 is located between HindIII A and F and that the order of HindlII fragments near the deletable region is A O F H, from left to right (Figs. 4 and 8). Studying the fragment patterns of the DNA of the different deletion mutants obtained with EcoR1 enzyme we found that deletion st 20 is apparently located completely within EcoRI fragment C, whereas the deletions st 14, b 3 and st 0 fuse fragment C with fragment E deleting fragment G. The large deletion b 1 which obviously extends further to the left than all the other deletions, leads to the fusion of EcoRI fragment C with fragment B and to the deletion of fragments E and G (Figs. 1 and 3, Table 2). It can be concluded from these results that the EcoRI fragments affected by the different deletions are arranged in the following way: B E G C (independent evidence for the arrangement E G will be presented in section (j)). The correlation of the molecular weight of fragments B, E, G and C and the distance of the deletable region from the left terminus of the T5 DNA molecule suggest that fragment B consitutes the left terminal part of the T5 DNA. The only change found in the fragment patterns of the Sal digests of the st 0 and b 1 deletion DNA's was a reduction in size of fragment B (Figs. 1 and 3, Table 2) indicating that this fragment covers the entire deletable region. If st 0 DNA is digested by Sma the situation is analogous: only fragment A is affected by the deletion thereby defining its position within the DNA molecule as also spanning the deletable region.

I

I

I

0.8

0.9

10

Fig. 4. Correlation of the deletable region in T5 with the cleavage maps of HindlII, EcoRI, Sal and Sma. As will be discussed later the deletable region (dr) in T5 ÷ spans from about 0.19 to 0.34 fractional length. The single strand break within the deletable region lies at map position 0.31 (Fig. 8). The map positions, expressed in fractional length for each cleavage site, was calculated after the sizes of the fragments of all four restriction digests had been normalized

d) Determination of Terminal Fragments in HindlII and Sal Digests Limited digestion of the termini of T5 DNA by exonuclease III and refilling of the arising terminal single stranded regions with e_32p labeled nucleotides by the action of DNA polymerase I were used to label specifically the terminal regions of the DNA. In order to avoid an attack of exonuclease III at the internal single strand breaks, the T5 DNA was first treated with ligase which quantitatively closes the interruptions in the r-strand (Jacquemin-Sablon and Richardson, 1970; Bujard and Hendrickson 1973). Under the conditions used (see Material and Methods) a stretch of DNA corresponding to about 1 to 2% of the DNA molecules should be labeled at each terminus by repair synthesis with DNA polymerase I. Fig. 5 shows that the incorporation of radioactive dCTP into the partially single stranded DNA reaches a plateau after 15 rain. indicating that the label was preferentially incorporated at the termini of the DNA molecule. Up to 6 x 10v cts per min could be introduced into 0.02 pMol of T5 DNA. Digestion of such terminally labeled DNA with restriction enzymes HindlII and Sal and subsequent

";3 c 2 2 2 v

l I

5

I I qo 15 Time (rain)

I 20

1 25

Fig. 5. Incorporation of c~-32p-labeled dCTP into the terminal regions of T5 DNA. Ligase treated T5 + DNA posessing single stranded termini each corresponding to about 1% of the molecule, was prepared as template for DNA polymerase I. The figure represents the kinetics of the labeling of the single stranded regions by DNA polymerase I using c~-32p-labeled dCTP. Details are described in the material and methods section

285

A.v. Gabain et al. : Mapping of Restriction Sites in T5 DNA

a n a l y s i s o f t h e f r a g m e n t e d m a t e r i a l b y a g a r o s e gel electrophoresis revealed that some radioactive label w a s i n c o r p o r a t e d i n t o all t h e f r a g m e n t s p r o b a b l y d u e to repair synthesis at randomly located nicks. In each digest, however, two fragments were labeled preferentially, a f a c t w h i c h w a s e v e n m o r e a p p a r e n t i f t h e r a d i o a c t i v i t y i n c o r p o r a t e d w a s n o r m a l i z e d t o t h e size of the fragment. As shown in Table 3 the preferent i a l l y l a b e l e d f r a g m e n t s a r e HindIII E a n d D a n d Sal f r a g m e n t s C a n d D . W e c o n c l u d e t h e r e f o r e t h a t t h e s e f r a g m e n t s lie a t t h e t e r m i n i o f t h e T 5 D N A molecule.

Table 3. Incorporation by DNA polymerase I of a 32P-labeled dCTP into the exonuclease III treated T5 st 0 DNA 32p-radioactivity per 10 x of DNA (cts/min)

Fragment

HindlII A B C D E FH I+J K+L

10 6

dalton

Sal

•

290 390 1,600 1,670

420 450 450 1,770 1,650 410 230 160

e) Fragments E c o R I A and Sal C Lie at the Right Terminus and E e o R I B and S a l D at the L e f t Terminus o f the T5 Chromosome

All values were normalized with respect to the size of the different fragments. The actual counts per minute corrected for background of a gel slice containing a terminal fragment were between 1,500 and 2,000. Background varied between 220 and 50 counts per minute throughout the gel progressively diminishing with distance from the origin.

Sal

1

2

3

4

5

6

7

Eco

Hin

1

The EcoRI fragments A through D of T5 + DNA were separated by electrophoresis on preparative

2

3

4.

5

Eco

A B C

E F

cl b Fig. 6a and b. Redigestion of isolated EcoRI fragments with Sal and HindIII endonucleases and analysis of the digests by agarose gel electrophoresis. Migration was from top to bottom. (a) Digestion of EcoRI fragments A, B and D with Sal endonuclease. For reference, the positions of EcoRI fragments obtained from T5 + DNA are indicated at the right margin. Agarose concentration was 0.5%. Gel 1: Sal digests of T5 + DNA (control); 2: Isolated EcoRI fragment A; 3: Digests of isolated EcoRI fragment A by Sal enzyme; 4: Digests of isolated EcoRI fragment B by Sal enzyme; 5: Double digests of T5 + DNA with EcoRI and Sal enzyme; 6: Digest of isolated EcoRI fragment D by Sal enzyme; 7: Isolated EcoRI fragment D. (b) Redigestion of EcoRI fragments A, B and D with HindIII endonuclease. Agarose concentration was 0.7%. Due to the broad range of molecular weights of the fragments present in gels 1 4 , it was not possible to obtain satisfactory resolution of all in a single electrophoretic run. Thus the pictures of gels l ~ t presented here are composites of two photographs of the gels of two independent electrophoretic separations of different durations. The portion of the gel photograph above the arrow at the margin depicts the resolution of the larger molecular weight fragments obtained by electrophoresis for 23 hours, while that below the arrow of the smaller molecular weight fragments for 18 hours. Gel 1 : HindIII digest of T5 + DNA (control); 2: Digest of isolated EcoRI fragment A by HindIII. HindIII fragments D, G, I, J, L and M are present in this digest in addition to one new fragment banding above G; 3: Digest of isolated EcoRI fragment D by HindIIl. Besides HindlII fragments N and K two new fragments of molecular weight 3 x 1 0 6 and 2.1 x 1 0 6 appear. The larger of the two new fragments comigrates with HindIII fragment I in double digests of T5 + DNA with EcoRI and HindIII, whereas the smaller one is found just above EcoRI fragment E (see gel 4). 4: Double digest of T5 + by HindIII and EcoRI enzyme. 5: Digest of isolated EcoRI fragment B by HindIII. The fraction of HindIII fragment 0 which is expected in this digest is not visible in this particular gel, but see Fig. 7a, gel 1 and 3

286 agarose gels and isolated from these gels by the potassium iodide gradient technique (Blin et al., 1975). Upon redigestion with Sal enzyme EcoRI A is cut only once yielding two fragments of which one is Sal C (Figs. 6 a). Two conclusions can be drawn from this experiment: (i) EcoRI A must be a terminal fragment since it contains Sal C, which has already been shown above to be a terminal fragment. (ii) EcoRI A must be the right terminal fragment since it is too large to be located to the left of the deletable region and thus by implications Sal C must lie at the right end of the T5 DNA. Redigestion ofEcoRI B with Sal enzyme again produced only two fragments of which one appears to be Sal D (Fig. 6a). By deduction it can be concluded that EcoRI B and Sal D must lie at the left end of the T5 molecule.

f ) Determination of Fragment HindIII D as the Right Terminus and HindIII E as the Left Terminus by Cleavage of EcoRI Fragments A, B and D by HindlI1 Endonuclease The digestion of fragment EcoRI A with HindIII yielded the fragments D, G, I, J, M and a new fragment derived from the left end of EcoRI A (Fig. 6b). Since fragment HindIII D was found to be terminal (section (d)) this experiment shows that it is the right terminal fragment of the HindIII cleavage map. Consequently HindIII fragment E must represent the left end of the cleavage map. This was confirmed by its appearance together with fragment HindIII A (and a part of fragment HindIII O, which will be discussed in section (i)) upon digestion of EcoRI fragment B with HindIII enzyme (Fig. 6b). EcoRI fragment D is cut by HindIII three times yielding the HindIII fragments N and K together with two new fragments (Fig. 6b).

g) The Sal Endonuclease Cleavage Map of T5+ DNA It has been shown in section (e) above that the fragments Sal C and D represent the right and the left end of the T5 DNA respectively. Since Sal fragment B is the only one affected by the deletions (Figs. 1 and 3) it must lie adjacent to Sal fragment D. By deduction Sal A lies between Sal fragment B and C establishing the order D B A C as shown in Figs. 4 and 8. From this sequence and from the molecular weights of the fragments it can be concluded that Sal endonuclease cleaves the T5 + DNA molecule at sites located 0.11, 0.45 and 0.87 fractional length along the molecule.

A.v. Gabain et al. : Mapping of RestrictionSites in T5 DNA

h) The Sma endonuclease Cleavage Map of T5 + DNA The Sma endonuclease cuts T5 + DNA in 4 pieces as shown in Fig. 1 and Table 1. The only fragment affected by the T5 deletions is Sma A (Figs. 1 and 3) which therefore has to be placed covering the deletable region. The double digest of T5 ÷ DNA with Sal and Sma enzyme (Fig. 7c) shows that fragments Sma C and Sma D are unaffected by the action of Sal, whereas fragments Sma A and Sma B are cleaved. Considering the molecular weights of the new fragments there is only one way to arrange the Sma cleavage sites: Since one Sal cleavage site seems to have no effect in this double digest and since Sal D and Sma D have the same molecular weight (they appear in one band of double stoichiometry if Sal and Sma digest are mixed 1:1 and coelectrophoresed, Fig. 7c) we conclude that Sma D represents the left end of the molecule. Fragment Sma A lies adjacent to fragment Sma D since it covers the deletable region. This implies that fragment Sma A must be cut by Sal at 0.45 fractional length. The appearance of two new fragments of molecular weight 25 x 106 (Sal B) and 4 x 106 dalton in the Sal/Sma double digest fullfills exactly this prediction. We have to place Sma fragment C adjacent to Sma A since it does not contain a cleavage site for Sal. Fragment Sma B has therefore to lie at the right end of the DNA molecule and as expected it is cleaved in the Sal/Sma double digest at the Sal site located at map position 0.87 into two fragments yielding Sal C and a new fragment of 14 x 106 dalton. The cleavage sites of the Sma enzyme along the T5 + DNA are therefore located at 0.11, 0.49 and 0.69 fractional length.

i) The HindlII Endonuclease Cleavage Map of T5 + DNA In section (f) it was deduced that the HindIII fragments E and D represent the left and right end of the T5 DNA molecule respectively, and in section (c) it was established that within and near the deletable region the arrangement of the HindIII fragment is A O F H from left to right. Since EcoRI fragment B contains the HindlII fragments E, A and part of 0 (Figs. 6b and 7a) the leftmost 40% of the HinclIII cleavage map is established as E A O F H (from left to right, Figs. 4 and 8). Digestion of purified EcoRI fragment A resulted in the appearance of the terminal HindIIl fragment D and fragments G I J L and M (Fig. 6b) within this rightmost 40% of the molecule. A double digest of T5 + DNA with HindIII and Sal endonuclease yields further information (Fig. 7a).

A.v. Gabain et al. : Mapping of Restriction Sites in T5 DNA Hin

I

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Fig. 7a-e. Simultaneous digestion of T5 DNA with two or three restriction endonucleases and fractionation of the digests by agarose gel electrophoresis. Migration is from top to bottom. (a) T5 st 0 DNA digested with the following enzymes and analysed on 0.7% agarose: Gel 1: Hind III; 2: EcoRI; 3: HindIII+EcoRI; 4: HindIII+Sal; 5: HindlII+EcoRI+Sal. The three salient points to be stressed here are: (i) comparing gel 1 with gel 3 it can be seen that HindIII fragment 0 is reduced in size upon digestion with EcoRI (this can also be seen in gel 5). (ii) Comparing gel 1 with gel 4 and 5 it can be seen that HindIII fragment M is cleaved by Sal. (iii) Comparing gels 1 and 3 with gels 4 and 5 it can be seen that HindIII A is altered and moved to the position of HindIII B by the action of Sal. (b) Demonstration that EcoRI fragment E contains a HindIII cleavage site. Gels contain 0.5% agarose. Gel 1 : Digest of T5 + DNA with EcoRI; 2: Mixture consisting of T5 + DNA digested by EcoRI only and of T5 + DNA digested by both EcoRI and HindIII; 3: Double digest of T5 + DNA by EcoRI and HindIII. It can be seen that the band representing EcoRI fragment E in gel 1 and 2 is absent in gel 3. (c) Double digests of T 5 st 0 DNA with Sal and Srna enzyme. Gels contain 0.5% agarose Gel 1 : Sal digest; 2 : Mixture of Sal and Sma digests; 3 : Double digest by Sal and Srna. Gel 2 shows that Sa! D and Sma D comigrate. Gel 3 shows that Sal fragments B, C and D are preserved in the Sal/Sma double digest. Two new fragment with molecular weight 14 x 106 and 3 × 106 dalton appear (arrows)

HindIII f r a g m e n t A is r e d u c e d by a b o u t 1 x 10 6 d a l t o n as it w o u l d be expected f r o m the Sal cleavage site at 0.11 f r a c t i o n a l length. HindIII f r a g m e n t C on the o t h e r h a n d is cleaved into two f r a g m e n t s o f a b o u t 4.9 a n d 4.1 x 106 dalton. T h e o n l y o t h e r HindIII fragm e n t affected b y the Sal e n z y m e is f r a g m e n t M, which lies within the r i g h t m o s t 4 0 % o f the D N A (section (f)). T h u s it follows t h a t Hina'III f r a g m e n t C c o n t a i n s the Sal cleavage site at 0.45 f r a c t i o n a l length a n d HindIII f r a g m e n t M the Sal cleavage site at 0.87 fract i o n a l length. HindIII f r a g m e n t M is t h e r e f o r e situ a t e d a d j a c e n t to HindIII D w h e r e a s HindIII fragm e n t C is a d j a c e n t to HindIII f r a g m e n t H. W i t h this k n o w l e d g e it is p o s s i b l e to p l a c e f r a g m e n t s (N, K) a d j a c e n t to f r a g m e n t C f o l l o w e d by f r a g m e n t B, since the a l t e r n a t e a r r a n g e m e n t C B (N, K ) w o u l d place f r a g m e n t s N a n d K within the E c o R I f r a g m e n t A , where these f r a g m e n t s have n o t been f o u n d (Fig. 6b). A t the m o m e n t we have n o i n d i c a t i o n o f the m a p p o s i t i o n o f HindIII f r a g m e n t P n o r o f a m o r e d e t a i l e d

a r r a n g e m e n t o f f r a g m e n t s (N, K) a n d (G, I, J, L), c o m p r i s i n g a b o u t 2 0 % o f the T5 ÷ D N A molecule.

j ) The E c o R I Endonuclease Cleavage M a p o f T5 + DNA

D i g e s t i o n o f E c o R I f r a g m e n t B with Sal or HindlII e n d o n u c l e a s e yields in b o t h cases the t e r m i n a l fractions f r a g m e n t s Sal D or HindIII E respectively (Fig. 6) d e m o n s t r a t i n g the E c o R I B represents the left t e r m i n a l fragment. This f r a g m e n t is fused to E c o R I f r a g m e n t C by the b l deletion with the loss o f E c o R I f r a g m e n t G a n d E (Fig. 3). These latter two f r a g m e n t s m u s t be l o c a t e d between f r a g m e n t s B a n d C. W e p r o p o s e the sequence E G b a s e d on the f o l l o w i n g a r g u m e n t : T h e digestion o f E c o R I fragm e n t B with H i n d l I I e n z y m e reduces the size o f HindI I I f r a g m e n t 0 b y 0.25 x 106 dalton. T h e s a m e is f o u n d in d o u b l e digests o f c o m p l e t e T5 + D N A b y

288

A.v. Gabain et al. : Mapping of Restriction Sites in T5 DNA ? els 2

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Fig. 8. Correlation of the four restriction maps with the physical structure of T5 + DNA and with the genetic and functional map of the phage. The uppermost part shows some details of the segmented genetic map (Hendrickson and McCorquodale, 1971). The four linkage groups A, B, C and D have recently been reoriented by Doerman (cited by McCorquodale, 1975). The numbers in the genetic map denote genes in the corresponding groups (e.g. A i, A 2, C 2 etc). The genes called here "els" are prerequisite for a normal "early-late-switch" in transcription. A model of the physical structure of T5 ÷ DNA with it's "nick"-pattern (Bujard and Hendrickson, 1973; Hayward 1974) and tile regions of transcription for preearly, early and late RNA (Hendrickson and Bujard, 1973) is shown below the genetic map. The rightmost nick at fractional length 0.99 has been mapped by Delius (personal communication). The arrows above and below the double stranded DNA model delineate the known regions and the directions of transcription in T5. Included here are 3 strong binding sites of RNA polymerase in the preearly region which have been found recently (Blin, 1975). The lower part of the figure shows the four cleavage maps of the endonucleases HindIII, EcoRI, Sal and Sma and the map positions of the deletions bl, b3, st 14, st 0 and st 20. The dotted extensions of the lines indicating the size of the deletions give the range in which the map position of the b 1 and st 20 deletion can vary

EcoRI and HindIII enzyme (Fig. 7a). This indicates that fragment 0 contains an EcoRI cleavage site which divides it into a left fragment of 0.65 x 106 dalton and a right fragment of 0.25 x 106 dalton. Therefore the EcoRI fragment adjacent to B must contain a HindIII cleavage site which would reduce the fragment by 0.25 x 106 dalton when a double digest of T5 D N A with both enzymes is carried out. Fig. 7b shows such a double digest which demonstrates that the size of fragment EcoRI E is reduced by HindIII enzyme by about 0.25 x 10 6 dalton. We therefore conclude that EcoRI fragment E is adjacent to EcoRI fragment B and that the arrangement is B E G C. The position of EcoRI fragments D and F as they are shown in Fig. 4 and 8 were derived from the analysis of HindIII action on purified EcoRI fragment D. Since the position of HindIII fragment N and K is known from the HindIII cleavage map it is possible to predict which fragments would arise by digesting EcoRI D with HindIII if the position of EcoRI fragment F is to the left or the right of fragment D : If it were located to the left of EcoRI D digestion with HindIII enzyme would yield a fragment of about

5 × 10 6 dalton in addition to the HindIII fragments N and K. If fragment F were located to the right of fragment D, HindIII digestion would produce two new fragments one, of molecular weight of about 2 x 106, the other, of about 3 x 106 dalton. As seen in Fig. 6b the latter is the case establishing the arrangement shown in Fig. 4 and 8. The cleavage sites of EcoRI enzyme along the T5 + D N A are therefore at 0.25, 0.275, 0.285, 0.49, 0.61 and 0.63 fractional length. While this manuscript was in preparation a EcoRI cleavage map of T5 D N A was described by Rhoades (1975) which agrees with our data.

k) Size and Position o f the Deletions st 20, st 14, b 3 and b 1

The size of the different deletions can be deduced from the reduced molecular weights of certain restriction fragments which span the deletions or in those cases where two fragments were fused by a deletion, by the reduced sum of the molecular weights of the

A.v. Gabain et al, : M a p p i n g of Restriction Sites in T5 D N A

fragments affected. Table 2 shows the effects of the deletions on fragments in various digests. According to these data the amount of DNA deleted by the different mutations is 2.6x106 for st 20, 3.1x106 for st 14, 4.1x 106 for b 3, 5,4x 106 for st 0 and 6.6x 106 for b 1. Since the st 0 deletion eliminates the single strand break at 0.31 fractional length and in addition fuses the EcoRI fragments C and E as well as the HindIII fragments F and H its position is well defined (Fig. 8). The st 20 deletion also eliminates the single strand break at 0.31 fractional length but it does not affect EcoRI fragment E and G; it therefore spans somewhere between 0.29 and 0.34 fractional length. In all the other deletion DNA's the single strand break at fractional length 0.31 is present, indicating that the deletions st 14, b 3 and b 1 are located to the left of this "nick". Since neither the b 3 nor the st 14 deletion fuse EcoRI fragment C with B nor affect HindIII fragment 0 their position has to be between 0.27 and 0.31 of the fractional length of the T5 ÷ DNA. Finally, the b 1 deletion fuses HindlII fragment F with A and EcoRI fragment C with B eliminating HindIII fragment 0 as well as EcoRI fragments G and E. It extends therefore somewhere between 0.19 and 0.31 fractional length (Fig. 8).

Discussion

The aim of the work presented here was to dissect the chromosome of phage T5 into defined fragments which when studied individually might facilitate investigations into the regulatory mechanisms controlling the development of this phage. The T5 DNA was therefore digested with the restriction endonucleases HindIII, EcoRI, Sal and Sma and the cleavage maps of the enzymes were established using the strategies described in Results. An essential point in this work is the correlation of the cleavage maps with the physical structure of the T5 DNA and with the genetic and functional maps of the phage. The orientation of the cleavage map relative to the physical structure of the DNA was greatly facilitated by two unique properties of the T5 chromosome, namely, the presence of five specifically located single strand breaks along the linear chromosome, the positions of which have been mapped previously (Bujard, 1969; Hayward and Smith, 1972b; Bujard, Hendrickson, 1973) and the presence of an internal deletable region which spans the single strand break at map position 0.31 (Hayward and Smith, 1972b; Hayward, 1974). Using the DNA of five different deletion mutants

289

(namely b 1, b 3, st 14, st 20, st 0) specific alterations in the fragment patterns of the HindlII, EcoRI, Sal and Sma digests were observed. This enabled us to orient unequivocally the four cleavage maps relative to the physical structure of the T5 ÷ DNA (Fig. 8). Furthermore, once the cleavage maps were established it was possible to determine the limits of the deletable region defined by the five deletion mutants studied and to map the position of each deletion within this region. As seen in Fig. 8 the deletable region spans the chromosome from map position 0.19, the left end of the b 1 deletion, to map position 0.34, the right end of the st 0 deletion, comprising 15% of the total chromosome of T5 ÷ A correlation of the physical structure of T5 with the functional map of the phage has already been accomplished by the hybridisation of preearly, early and late phage-specific RNA, isolated from infected cells, to defined single stranded portions of T5 DNA (Hendrickson and Bujard, 1973; Hayward and Smith, 1973). It was found that the genes determining the three transcriptional classes are clustered, the clusters being arranged in a linear array according to their sequence of synthesis during the development of T5. Thus, as shown in Fig. 8 the genes controlling preearly functions are located between map position 0 and 0.08, and since the T5 DNA is terminally redundant (Rhoades and Rhoades, 1972), also between 0.92 and 1.0. The genes controlling early functions are located between map position 0.08 and 0.64 while those of late functions lie between map position 0.64 and 0.92. It should be mentioned here that the switch point between the early and late regions is not known precisely, its position shown in Fig. 8 at the single strand break at map position 0.64 being merely speculative. The combined knowledge of the sequential arrangement of the three transcriptional regions along the T5 DNA and of the position of certain preearly, early and late functions on the genetic map determined by recombinational analyses with mutants of the phage (Hendrickson and McCorquodale, 1971), enable us now to correlate the genetic map of T5 with the physical structure of T5 DNA and, consequently, with the four cleavage maps described herein. As can be seen in Fig. 8 there are a number of restriction fragments which carry exclusively early or late genes and others, e.g. HindlII E and D and Sal fragment C and D, which contain all preearly functions with only a relatively small portion of DNA from the early or late regions. HindIII fragment F should contain the genes for most of the T5 specific t-RNA's (Chen etal., 1975; Locker, M. and Weiss, S.B. pers, comun.). Fragment HindIII A is of special interest since it most likely contains gene C2, an essen-

290

tial gene for the early/late switch in the transcriptional program of T5 (Chinnadurai and McCorquodale,

1974a). Thus, a variety of in vitro and in vivo investigations of specific phage functions such as the early late switch now appear feasible using isolated T5 DNA fragments. In addition, using the information of the four restriction maps reported here, it might also now be possible to identify and isolate fragments of even greater specialisation using the restriction enzymes HpaI and II and HindII, which, in preliminary experiments, we have found to cleave the T5 chromosome more extensively, yielding from 23 (HpaI) to approximately 80 (HpaII and HindII) bands in agarose gels. Acknowledgments. We thank R. Roberts and C. Mulder for procedures helpful in the isolation of restriction enzymes and for a culture of Streptomyces albus and C. Mulder for a generous gift of the Sma enzyme. We are very indebted to H. Schaller for gifts of exonuclease III and DNA polymerase I and to R. Bauerle and H. Schaller for criticism and help in the preparation of the manuscript. This work was supported by grant no. Ba 384/6 from the Deutsche Forschungsgemeinscbaft. One of us (G.S.H.) was a holder of an EMBO postdoctoral fellowship.

References Adams, M.H.: The calcium requirements of the coliphage T5. J. Immunol. 62, 505-516 (1949) Allet, B., Jepessen, P.G.N., Katagiri, K.J., Delius, H.: Mapping the DNA fragments produced by cleavage of 2 DNA with Endonuclease RI. Nature (Lond.) 241, 120-123 (1973) Blin, N.: Die Promotoren auf dem Genom des Bakteriophagen T5 +. Dissertation, Universit/it Heidelberg (1975) Blin, N., v. Gabain, A., Bujard, H. : Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Letters 53, 84-86 (1975) Bujard, H.: Location of single-strand interruptions in the DNA of bacteriophage T5 ÷. Proc. nat. Acad. Sci (Wash.) 62, 1167 1174 (1969) Bujard, H. : Electron microscopy of single stranded DNA. J. molec. Biol. 49, 125-137 (1970) Bujard, H., Hendrickson, H.E.: Structure and function of the genom of coliphage T5.1. The physical structure of the chromosome ofT5 +. Europ. J. Biochem. 33, 517 528 (1973) Chen, M., Shiau, R.P., Hwang, L., Vaughan, J., Weiss, S.B. : Methionine and formylmethionine specific t-RNAs coded by bacteriophage T5. Proc. nat. Acad. Sci. (Wash.) 72, 558-562 (1975) Chinnadurai, G., McCorquodale, D.J. : Requirement of a phageinduced 5'-exonuclease for the expression of late genes of bacteriophage T5. Proc. nat. Acad. Sci. (Wash.) 70, 3502-3505 (1973) Chinnadurai, G., McCorquodale, D.J.: Regulation of expression of late genes of bacteriophage T5. J. Virol. 13, 85-93 (1974a) Chinnadurai, G., McCorquodale, D.J.: Dual role of gene D5 in the development of bacteriophage T5. Nature (Lond.) 247, 554 556 (1974b) Espeyo, R.T., Canelo, E.S.: Properties of bacteriophage PM2: A lipid-containing bacterial virus. Virology 34, 738-747 (1968) Gabain, A.v., Diplomarbeit, Univ. Heidelberg (1975)

A.v. Gabain et al. : Mapping of Restriction Sites in T5 DNA Hayward, G.S., Smith, M.G. : The chromosome of bacteriophage T5. I. Analysis of the single stranded DNA fragments by agarose gel electrophoresis. J. molec. Biol. 63, 383-395 (1972a) Hayward, G.S., Smith, M.G. : The chromosome of bacteriophage T5. II. Arrangement of the single stranded DNA fragments in the T5 + and T5 st(0) chromosomes. J. molec. Biol. 63, 397 407 (1972b) Hayward, S.D., Smith, M.G. : The chromosome of bacteriophage T5. III. Patterns of transcription from single stranded DNA fragments. J. molec. Biol. 80, 345-359 (1973) Hendrickson, H.E., Bujard, H.: Structure and function of the genome of coliphage T5. 2. Regions of transcription of the chromosome. Europ. J. Biochem. 33, 529 534 (1973) Hendrickson, H.E., McCorquodale, D.J. : Genetic and physiological studies of bacteriophage T5. I. An expanded genetic map ofT5. J. Virol. 7, 612 618 (1971) Hertel, R., Marchi, L. Muller, K.: Density mutants of phage T5. Virology 18, 576-581 (1962) Jacquemin-Sablon, A., Richardson, C.C. : Analysis of the interruptions in bacteriophage T5 DNA. J. molec. Biol. 47, 477-493 (1970) Lang, D., Mitani, M. : Simplified quantitative electron microscopy of biopolymers. Biopolymers 9, 373-379 (1970) McCorquodale, D.J. : The T-odd bacteriophages. CRC Press Critical Reviews in Microbiology in press. Caskin, A.I. and Lechevalier, H. eds. McCorquodale, D.J., Buchanan, J.M. : Patterns of protein synthesis in T5-infected Escherichia coli. J. biol. Chem. 243, 2550~559 (1968) Moyer, R.W., Buchanan, J.M. : Patterns of RNA synthesis in T5 infected cells. I. As studied by the technique of DNA-RNA hybridisation-competition. Proc. nat. Acad. Sci. (Wash.) 64, 1249-1256 (1969) Rhoades, M, : Cleavage of T5 DNA by the Esckerichia coli RI restriction endonuclease. Virology 64, 170-179 (1975) Rhoades, M., Rhoades, E.A.: Terminal repetition in the DNA of bacteriophage T5. J. molec. Biol. 69, 187200 (1972) Richardson, C.C., Lehmann, J.R., Kornberg, A. : A deoxyribonucleic Acid phosphatase-exonuclease from Escherichia coli. II. Characterisation of the exonuclease activity. J. biol. Chem. 239, 251-258 (1964) Richardson, C.C., Schildkraut, C.L., Aposhian, H., Kornberg, A. : Enzymatic synthesis of Deoxyribonucleic Acid. XIV. Further purification and properties of deoxyribonucleic acid polymerase of Esckerichia coll. J. biol. Chem. 239, 222-232 (1964) Rubenstein, I. : Heat-stable mutants of T5 phage. I. The physical properties of the phage and their DNA molecule. Virology 36, 356 376 (1968) Scheible, P.P., Rhoades, M. : Heteroduplex mapping of heat-resistant deletion mutants of bacteriophage T5. J. Virol. 15, 12761280 (1975) Smith, H.O., Wilcox, K.W. : A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. molec. Biol. 51, 379 391 (1970) Thomas, M., Davis, R.W.: Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. molec. Biol. 91, 315-328 (1975) Yoshimori, R.N. : P h . D . Thesis; Univ. of San Francisco Medical Center (1971)

Communicated by W. Arber Received September 22, 1975