Gene,60(1987)129-135

Elsevier 129

GEN 02186

Short Communications

Construction of an ordered overlapping library of bacteriophage Pl DNA in phage vector 1D69*

(Pl genomic library; L-P1 hybrid phage; recombinant DNA; genetic map; restriction fragment; chlorampheni-

co1 resistance; transposon)

Gerard T. O’Regan”, Nat L. Sternbergb and Gerald Cohena

a Microbiology Department, Tel Aviv University, Ramat Aviv, 69978 (Israel) Tel. (03)420-833 and b E.I. DuPont de Nemours

and Co., Central Research and Development, Experimental Station, 3281151, Wilmington, DE 19898 (U.S.A.) Tel. (302)695- 1081

Received 8 June 1987

Accepted 30 August 1987

SUMMARY

A library of bacteriophage Pl DNA was constructed in the phage vector 1D69. The DNA of some 150

randomly chosen il-Pl hybrid phages containing Pl DNA fragments 5-10 kb in size was analyzed by restriction

endonuclease digestion using enzymes EcoRI, BgZII, and BumHI that cleave Pl DNA at known positions on

the physical map of Pl. Approximately one third of the phages contained Pl DNA inserts having two or more

restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with

respect to the physical map. Genetic tests allowed detection of L-P1 hybrid phages possessing inserts with

functional Pl ban and CmR genes. A subset of 18 phages was analyzed in more detail; their Pl DNA inserts

comprise an ordered collection of overlapping Pl DNA fragments that cover almost 98% of the Pl genome.

INTRODUCTION

Bacteriophage Pl is a large temperate virus of

Escherichia coli with the unusual property of existing

Correspondence to: Dr. G. Cohen, Microbiology Department, Tel

Aviv University, Ramat Aviv, 69978 (Israel) Tel. (03)420-833.

* On request, the authors will supply detailed experimental evi-

dence for the conclusions reached in this short presentation.

Abbreviations: bp, base pair(s); Cm, chloramphenicol; kb,

1000 bp; mu, map unit(s) (0.9 kb; see Table I and Fig. 1); R,

resistance; Tn, transposon.

as an autonomously replicating plasmid in the

lysogenic state. The Pl viral genome is close to

100 kb in size, possesses a long terminal redundancy

of about 10 kb and is cyclically permuted in phage

particles. The circular prophage species is approx.

90 kb in size (Ikeda and Tomizawa, 1968). Many of

the phage functions involved in vegetative growth,

maintenance and control of the prophage state,

incompatibility, immunity and a variety of other sys-

tems have been identified and the genes encoding

them have been localized on the Pl chromosome.

Much of this information is contained in the physical

0378-l 119/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

130

PI

Pl EcoRI- 1 1 12 116 1 4 I 10 12 . / \

30 /’ ‘\ ,-‘.“*l** a \\ . . . ‘,5 . I I

PlCm cl 100 EcoRl- 11 12 116 [ 8” 1 14*1 8’ 1 10 I 2

6 141

I I

Fig. 1. Physical alignment of Pl DNA inserts with respect to the Pl physical map in 18 I-P1 hybrid phage DNAs that cover the Pl

genome. The locations of the inserts (open boxes) have been superimposed on the recent restriction and genetic map of Yarmolinsky

(1987), to which the reader is referred for gene definitions and relevant references. Genes are indicated outside the circles by bold-face

and genetic map shown in Fig. 1 (Y~o~nsky, 1987) and is discussed in detail in a recent compre- hensive review (Yarmolinsky and Sternberg, 1987).

A major gap in our current understanding of Pl biology is the lack of knowledge of P 1 structural and functional components required for the lytic replica- tion cycle. In contrast, the general features of plas- mid replication are well established (Chattoraj et al., 1985). In an attempt to determine which PI DNA sequences are necessary for lytic replication, we con- structed a library of Pl DNA fragments in a I vector with the aim of devising a functional test to screen these sequences for ability to promote replication. In this report we describe the construction of an order- ed, overlapping, collection of Pl DNA sequences in A-P1 hybrid phages suitable for this purpose. Else- where we describe the use of this library for isolating the P 1 lytic replicon. The library should prove useful for isolating other PI genes.

~X~~RIMENTA~ AND DISCUSSION

(a) Construction of a L-P1 library

The vector AD69 was chosen for construction of the Pl library (Mizusawa and Ward, 1982). It possesses a single BumHI site in the 1 int gene and the immunity of phage 21. The DNA of phage P 1 Cmc 1.100 was partially digested with restriction endonuclease Sau3AI and fractionated by gel elec- trophoresis. Fragments of 5-10 kb were purified, ligated to the BamHI-cut vector and phage were recovered by in vitro packaging. Approximately 87 % of the phage were 1-P 1 hybrid phages as judged by a test for int function, the red-plaque test (Enquist and Weisberg, 1976), which detects &-mediated

131

restitution of an inte~upted gut gene. A collection of 150 white recombinant plaques were randomly pick- ed to generate a large enough collection of PI se- quences for a library. A similar collection was made with A-PI hybrids having inserts of 3-5 kb, but was not further employed in this study.

(b) Analysis of Pl nucleotide sequences in J-PI

hybrids

The origin of P 1 nucleotide sequences in I-P1 hybrids was determined from restriction endonu- clease digestion analysis of R-PI DNA and genetic tests. For the former the enzymes used were EcoRI, BgZII, and BamHI for which detailed restriction maps exist (Fig. 1). Cleavage patterns of some 150 hybrid phage DNAs were examined by electro- phoresis in 0.8% agarose gels and compared with the corresponding restriction pattern of PlCmc I. 100 DNA to identify specific Pl fragments. Positive identi~cation of a Pl fragment in a A-PI hybrid requires the presence of two or more restriction sites within the Pl insert. Inspection of single enzyme cleavage patterns of the collection of d-P1 hybrid DNAs showed that some 54 of the hybrids contained at least one identifiable Pl fragment. The Pl DNA in the remaining hybrids either had no restriction sites, or only one, for each of the above enzymes. A typical restriction pattern analysis is shown in Fig. 2.

Fig. 1 depicts the size and location of each of the PI DNA inserts of 18 R-PI hybrid phages with re- spect to the Pl physical map. The size of the Pl insert was calculated from the EcoRI restriction di- gestion pattern of the hybrid phage DNAs, i.e., from the size of the new EcoRI fragments generated by the Pl insert into /ZD69-EcoRI fragment-2. This infor- mation, together with the identification of specific P 1 restriction fragments in the insert, was used to pre-

--..

numbers or acronyms, or both. Bracketed symbols denote genes that have been identified only in the related phage P7 but are assumed

to correspond to similarly positioned homologues in Pl. The cl protein-binding sites or operator sequences (Op) are numbered according

to their integral position on the map. Bold-face letters and blackened boxes indicate insertions and substitutions relative to Pl; C

designates the invertible segment of Pt. The IS2 element (part of B) is shown as an open bar and an arrow depicts the site and direction

of packaging of Pl DNA. The map shows the known definite locations of the end points of each PI insertion in the 18 d-P1 hybrids.

Numerical values for co-ordinates of definite end points, the insert sizes and the extent of end contribution are tabuiated in Table I.

The PI genome is, by convention, taken as 90 kb and is sub-divided into 100 map units (mu). In the lower part of the figure, the PI

region surrounding the positions of the TnP tandem dimer in PlCmc1.100 DNA (De Bruijn and Bukhari, 1978) used in this study is

shown with the Pl DNA inserts of d-P1 hybrid phages that cover this region. The tandem dimer of Tn9 in Pl EcoRI-4 generates three

new EcoRI fragments, S”, 14* and 8’ in place of fragment 4.

132

14 15 17 18

21

23

Fig. 2. Gel electrophoresis pattern of EcoRI restriction digest of two overlapping I-P1 hybrid phage DNAs. Electrophoresis of

EcoRI-digested DNA samples was carried out in 0.80/, agarose gels in Tris . borate-EDTA buffer and DNA stained with ethidium

bromide. Lanes, fromleft to right: 1, PlCmc1.100; 2, I-P1 hybrid 15; 3. I-PI hybrid 143; 4, LD69 and 5, PlCmc1.100 DNAs. a-P1 hybrid

15 contains EcoRI fragments 15, 17, l&21 and 23; I-P1 hybrid 143 contains EcoRI fragments 14 and 21.

pare Fig. 1. These ideas are illustrated in the follow- ing. Consider a 1D69-PI hybrid phage with a PI DNA insert in which there are two EcoRI restriction sites (Fig. 3). Three new EcoRI fragments replace AD69-EcoRI-2 after cleavage; one corresponds to an authentic Pl EcoRI fragment, which locates the general position of the insert on the P 1 map, the other two each contain both Iz and Pl DNA. The unique

1 ii 1 i i AD69 DNA P1 DNA A069 DNA

A B

Fig. 3. Schematic representation of pattern ofEcoR1 restriction

fragments generated in a rlD69-Pl hybrid phage DNA possessing

two EcoRI sites in its Pl DNA insert. Arrows indicate EcoRI

sites, light line represents ID69 DNA, heavy line PI DNA. Four

EcoRI sites define the five EcoRI fragments in AD69 DNA. The

Pl DNA inserts in RD69-P 1 hybrid phages is located in the tlD69

EcoRI-2 fragment at the single BornHI site. Bars A (1.9 kb)

and B (3.8 kb) indicate the location of the 1 DNA regions in the

A-P1 flanking fragments.

BamHI site of AI)69 at which Pl DNA is inserted is not centrally located within ID69-EcoRI-2 and so the 1 DNA content of the two hybrid fragments differs, approx. 1.9 and 3.8 kb (regions A and B, respectively). It is not immediately clear in such a digestion which of the two hybrid fragments contains the 1.9 kb and which the 3.8 kb of A DNA. Hybrid d-P 1 phages with one or more EcoRI sites in their P 1 DNA insert will always have two such I-P1 frag- ments.

Determination which of these hybrid fra~~nts contains the 1.9 kb and which the 3.8 kb of A DNA, and therefore estimation of the amount of PI DNA in each, is straightforward if one of the A-PI frag- ments is less than 3.8 kb so that its ;i DNA content is necessarily 1.9 kb. This information, however, does not fix the orientation of the Pl insert with respect to the P 1 physical map. In cases, as depicted in Fig. 3, where the Pl insert has two (or more) EcoRI restriction sites it was frequently possible to determine the specific Pl DNA content in each

flanking /l-P1 hybrid fragment by digestion with BamHI and Bg/II followed by reference to the Pl restriction map for these sites. This analysis may also rule out certain arrangements of the possible calcu- lated amounts of Pl DNA in the hybrid DNA fragments. Double digestion experiments were used to verify such conclusions. In cases where only a single EcoRI site was found in the Pl insert, informa- tion from other restriction enzyme digestions was used to locate the insert on the Pl physical map, determine which Pl-EcoRI restriction site was present and permit estimation of the amount of Pl DNA extending in either direction from the EcoRI

133

site. Table I provides a compilation of the physical data for the 18 A-PI hybrids. It includes a list of the Pl restriction fragments present in each insert (columns 2-4), the size of each insert (column 5) the coordinates of the definite DNA content of each insert (column 6) as well as the size of the undefined region(s) at the end of the insert (column 7). Table II lists the PI EcoRI, BgZII, and BamHI restriction fragments present in each of the PI DNA inserts of 54 A-PI hybrid phages used in this work.

Certain regions of the Pl map (mu 8-15, 33-47 and 72-80) contain very few (or no) restriction sites for the three enzymes used in this study, and conse-

TABLE I

Composition of PI DNA inserts in 18 1D69-Pl hybrid phages

I-PI

hybrid L’

(1)

Restriction fragments’ Size in Pl DNA insert (mu) - Pl mu=

EcoRI BglII BamHI Definite locationd End contribution”

(2) (3) (4) (5) (6) (7)

76 6 8.4 87.2-95.4 o.ojo.2

18 7, 20, 22 7 8, 9 8.7 95.0- 2.6 o.oj1.1

132 19, (24) 10 8.6 0.3- 8.5 o.ojo.4

77 10 6.0 4.5-10s O.O/O‘O

136 10 6, 14 10.5 10.5-18.9 O.Ol2.1

141 10 14 7.4 14.4-21.6 o.ojo.2

53 14* 10.1

31 I6 8, 9 5.3 24.8-30.1 o.o/o.o

42 11 8.0 29.3-37.3 o.o/o.o

75 5 nd 32.2-38.6

27 1 7.7 37.9-42.9 2.7r

95 1 5.6 40.1-44.9 O.OjO.8

139 9, 14, 21(25) 8.9 45.4-53.9 o.ojo.4

15 15, 17, 18, 21, 23 6.2 52.0-58.2 o.o/o.o

91 5 8.7 57.6-66.2 0.0/o. 1

124 8 7.4 65.0-72.1 o.ojo.3

60 8.2 71.4-77.5 0.0/z. 1

51 13 10, II, 12, 13 6.3 78.5-84.8 o.o/o.o

a ir-Pl hybrids listed in clockwise order of their PI DNA inserts with respect to the Pi physical map (see Fig. 1).

’ Numbers refer to fragment designations as in Fig. 1. Numbers in parentheses refer to those fragments, presence of which is inferred

from positions of adjacent fragments; EcoRI fragment 14* is not a true Pl DNA fragment, it derives from the Tn9 tandem insertion

in PI EcoRI-4 (see legend to Fig. 1).

c 1 mu = 0.9 kb; estimated error f 0.25 mu; nd, not determined.

d Coordinates refer to positions in mu on P 1 physical map, Fig. 1; 1-P 1 hybrid 53 covers the tandem Tn9 dimer element in PI Cmc 1.100

(see Fig. 1).

e Terminal region(s) of the Pl DNA insert in Pl mu in the two L-PI flanking segments (see EXPERIMENTAL AND DISCUSSION,

section b) whose DNA content is not known with respect to which end of the fragments they belong; thus 0.0/0.2 denotes that 0.2 mu

of Pi DNA of the insert is not accounted for in the numbers given in column 6 or depicted in Fig. 1; it should be added to either one

of the two defined end point locations of the insert listed in column 6 and illustrated in Fig. 1.

f Distribution at ends unknown.

133

TABLE fl

List oF Pl Ec’caRI, BglIi and BamHI restriction fragments identified in 54 1D69-Pi hybrid phages -

A-PI Restriction fragments b i-P1 Restriction fragments b

hybrid a hybrid a

EC0RI Bg/II BarnHI EcuRI BgIII BamHI

5

Ii

13

15

18

25

26

27

28

30

31

33

35

37

39

42

45

49

50

51

53

55

59

60

62

63

64

8”, 16

14,21

15, 18

15, 17, l& 21, 23

7, 20, 22

10

14”

15, 18

16

a”, 14*

16

14*

15

19

8 ‘?, 16

13

14*

19

10

IS. 18

10

7

7

8, 9

8, 9

9

fl

9

8% 9

6, 14

7

8, 9

10, 11, 12, 13

8, 9 6, 14

6, I4

72

75

76

77

79

86

90

91

93

95

47

99

104

111

116

13s

122

124

126

128

132

136

139

141

143

145

148

14

6

IO

2Q, 22

20,x!

5

17, 18, 21, 23

20,22

15

14

13

15

8

f9,24

9

19, 24

10

9, 14, 21.

IO

14,21

9, 14, 21

19,24

5

IO

6, 14

7

8, 9

5

IO, I!, 12, 13

8

10

6, 14

14

a I-P1 hybrids analysed in this study which contain at least one identifiable ECoRI, BgBI, or BumHI restriction fragment.

’ Numbers refer to restriction fragment designations as in Fig. 1.

quently these regions proved more difficult to identify

by the procedure described above. To minimize this

difficulty, the number of hybrids used in this analysis

was considerably greater than statistically necessary

to cover the map. Thus we were able to find sufficient

L-P1 hybrids with identifiable Pl sequences (i.e.,

restriction fragments) bracketing the restrictionless

regions and extending into and overlapping these

regions. Although not required for the purpose of the

present work, a more complete coverage of the re-

strictionless regions of the map could be achieved by

plaque hybridization of the A-P1 library using suit-

ably labelled restriction fragments lying adjacent to

or spanning the Pl region of interest, As indicated

above, a majority of the I-P1 hybrids could not be

used in this analysis due to lack of readily identifiable

restriction fragments. Many of these would, how-

ever, be expected to have inserts that cover just these

regions.

In four regions of the map the exact degree of

coverage by the library is uncertain. Three of these

regions (at about mu 10, 45 and 78) are probably

covered in view of the considerations mentioned

above. The fourth {at mu 85-86) is covered by Pt

EcaRI-11 but this fragment was not detected in any

of the l-P1 hybrid phages. Other PI EcclRI frag-

ments not observed were the four largest frag-

ments l-4 and the composite fragment 8’ which is

generated by insertion of tandem TnP elements into

Pl EcoRI-4 (see Fig. 1). The region between

135

mu 72-82 proved difficult to identify due to the lack of restriction sites for each of the three enzymes employed. However, a I-P 1 hybrid phage containing an insert from this region was identified by a genetic test which involved comp~ementation of an E. cofi

dnaB ts mutant, defective in replication at 37°C by the Pl bun gene product. The Pl bun gene codes for a protein which is an analogue of the E. coli dn&-

coded protein. The ban gene is located at approx. mu 75-76 on the physical map (Yarmolinsky, 1987). Lysogenization of the E. coli mutant by A-P1 hybrid 60 (in conjunction with a helper phage) conferred on this strain the ability to grow at 37°C. The size of the Pl DNA insert in this hybrid almost covers the gap in this region of the map (see Fig. 1 and Table I).

A second biological test proved useful for identify- ing &Pl hybrid clones cont~ing the CmR gene of Tn9. Several i-P1 phages, including I-P1 hybrid 53, were found to confer Cm resistance to a sensitive strain of E. cob after lysogenization.

(c) Conclusions

We have shown by use of a combination of physi- cal endonuclease restriction analysis and biological functional tests that a subset of 18 I-P1 hybrid phages contains Pl DNA inserts that cover, in an overlapping way, approx. 98% of the Pl chromo- some. These phages, as well as other hybrid phages that have been similarly characterized (54 in all), will be made available on request.

ACKNOWLEDGEMENTS

We would like to thank Michael Lin for helpful advice in the construction of the genomic library.

REFERENCES

Chattoraj, D.K., Abeles, A.L. and Yarmolinsky, M.: Pl plasmid maintenance: a paradigm of precise control. In Helinski, D.R., Cohen, S.N., Clewell, D.B., Jackson, D. and Hollaender, A. (Eds.), Plasmids in Bacteria. Plenum, New York, 1985, pp. 355-381.

De Bruijn, F.J. and Bukhari, AI.: Analysis of transposable ele- ments inserted in the genomes of bacteriophages Mu and Pl. Gene 3 (1978) 315-331.

Enquist, L.W. and Weisberg, R.A.: The red plaque test: a rapid method for identification of excision defective variants of bacteriophage lambda. Virology 72 (1976) 147-153.

Ikeda, H. and Tomizawa, J.I.: Prophage Pi, an extrachromoso- ma1 replication unit. Cold Spring Harbor Symp. Quant. Biol. 33 (1968) 791-798.

Mizusawa, S. and Ward, D.F.: A bacteriophage lambda vector for cloning with BarnHI and Sau3A. Gene 20 (1982) 317-322.

Yarmolinsky, M.B.: Bacteriophage Pl. In O’Brien, S.J. (Ed.), Genetic Maps, Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY, 1987, pp. 38-47. Yarmolinsky, M.B. and Sternberg, N.L.: Bacteriophage PI. In

Calendar, R. (Ed.), The Bacteriophages. Plenum, New York, 1987, Chapter 10, in press.

Communicated by M. Yarmolinsky