Paul A.Whittaker, Annette J.B.Campbell1, Edwin M.Southern and Noreen E. ..... Olson, M.V., Dutchik, J.E., Graham, M.Y., Brodeur, G.M., Helms, C., Frank, M.,.
Volume 16 Number 14 1988
Nucleic Acids Research
Enhanced recovery and restriction mapping of DNA fragments cloned in a new X vector Paul A.Whittaker, Annette
J.B.Campbell1, Edwin M.Southern and Noreen E.Murrayl
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OXI 3QU and 'Department of Molecular Biology, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EH9 3JR, UK Received May 13, 1988; Accepted June 15, 1988
ABSTRACT In this paper we describe a modification to the A vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector,* EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional A vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of >107pfu/qg of human DNA.
INTRODUCTION The current interest in the isolation and mapping of large regions of various genomes (1, 2, 3) prompted us to set out to improve both the recovery and the restriction mapping of DNA sequences in bacteriophage A. As a cloning vector, A currently affords the most efficient recovery of cloned fragments of DNA and, in common with M13, provides a very sensitive system for screening by DNA hybridisation. However, the large number of A genes necessary for the propagation of the phage not only constrains the size of the DNA fragments that can be cloned, but also complicates their restriction mapping. While the size of the vector genome cannot be reduced significantly if the recombinant derivatives are to remain plaqueforming, restriction analysis of the cloned DNA can be facilitated by changing the genetic organisation of the vector. To this end we have modified the A vector EMBL3 (4) such that the cohesive ends and one polylinker sequence are juxtaposed, thus simplifying the mapping of cloned DNA sequences using cos-labelling (5). The strain of E. coli used when amplifying or screening phage A libraries is important: examples exist where some segments of genomic DNA cannot be propagated in phage A unless certain mutant strains of E. coli are used (6, 7, 8). Moreover, recent studies have shown that the Mcr (modified cytosine restriction) systems of E. coli restrict incoming DNA containing methylcytosine (9). As mammalian DNA is heavily methylated, Mcr restriction could interfere with cloning experiments, although only anecdotal evidence suggesting that this is the case has been presented (10). Therefore, we have investigated this possibility by examining the effect of E. coli Mcr systems on the recovery of recombinant phage containing © I R L Press Limited, Oxford, England.
6725
Nucleic Acids Research human DNA and show that the mcrA and mcrB gene products constitute barriers to the uptake of human DNA by E. coli.
MATERIALS AND METHODS Enzymes Restriction enzymes were purchased from New England Biolabs (FspI, MboI, NaeI, Sall), Boehringer Mannheim (BamHI, EcoRI, EcoRV, HindIll, NotI, PstI, PvuII), or Cambridge Biotechnology (BglII, HincII). T4 DNA ligase and T4 polynucleotide kinase were purchased from Amersham International. Si nuclease, Klenow fragment of E. coli DNA polymerase I and calf intestinal phosphatase were purchased from Boehringer Mannheim.
Bacteria and Phage The bacterial strains used are listed in Table 1. Bacteriophages from our collection, and their new derivatives, are shown in Figures 1 and 2.
Construction of EMBL3cos The 403bp HincII fragment of A DNA that includes the cos sequence has been cloned in pUC9 (11). In this plasmid (pWP14), the restriction targets are not positioned conveniently for the transfer of cos and the polylinker to EMBL3. The HincII fragment was, therefore, excised from pWP14 and inserted at the HindIII site in mp8 to give mpAC5 (Figure 1); this required treating the HindIII-cut vector with Klenow polymerase and dNTPs to generate blunt ends. The orientation of cos with respect to the polylinker was confirmed by determination of
TABLE 1 Bacterial strains used in this work Strain
Relevant Genotype
NM522 (hsd-mcrB) A5 supE (lac-pro) A/F' pro+ lacI° lacZ AM15 ED8689 hsdR514 derivative of W3350, mcr4 P2cox3 lysogen of ED8689 NM534 ER1378* hsdR2 mcrBl supE44 ER1381* hsdR2 supE44 ER1564* hsdR2 mcrA 1272::TnlO supE44 ER1565* hsdR2 mcrA 1272::TnlO mcrBI supE44 hsdR2 merA mcrBl supE K802* NM646* P2cox3 lysogen of K802 ED8654* hsdR514 mcrA supE44 supF NM538* hsdR514 mcrA supF MC1061* hsdR mcrA mcrB hsdR mcrA mcrB supE44 thyA SB204 NM621* recD1009 thy+ P1 transductant of SB204 DL491* sbcC201 TnlO:: phoR derivative of NM621 HL1228* recA (srlR-recA) A derivative of NM621 DB1318* hsdR2 zjj2O2::TnlO mci4 recA ::cam' recD1014 CES200* hsdR mci4 recB21 recC22 sbcl5 sbcC NM519* hsdR A4 mc74 recB21 recC22 sbcA23 * Strains tested for recovery of recombinant phage. mcr genotype not completely known, but Mcr+. 6726
Ref./Source (21) W.J. Brammar N.E. Murray
(9) (9) E.A. Raleigh E.A. Raleigh
(22) This work
(23) (4) (24) D.J. Finnegan This work D.R.F. Leach A. Craig (8) F.W. Stahl N.E. Murray
Nucleic Acids Research the nucleotide sequence. The EcoRI, BamHI and Sall targets are all to one side of cos in this orientation so that the intervening DNA can function as the left arm of A EMBL3cos. Digestion with FspI and EcoRI released the cos sequence within a linker fragment that was used to join the left arm of A sbamI° cI Pam (A NM1265) cut at its single NaeI site to the right arm of A Eam b527 srI3° cIam srI4° srI5° (A NM1266) cut with EcoRI (see Fig. 1). Recombinant phage selected on suppressor-free (Su-) bacteria have a second cos sequence in the same orientation as the original one, but in a DNA segment that replaces the A+ DNA between the NaeI site at 20,040bp and the left-most EcoRI site at 21,226bp. The DNA of one such phage was cut at its single EcoRI site and its left arm including the additional cos sequence was used to replace the left arm of EMBL3 thereby generating a derivative of B°
I
N
777N3
77
Pam
z .: --
XNM1265
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-
Nae I
xA NM 1266
j EcoRI
FM
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A
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PSBMR
t EcoRI EMBL Lig ate
3/EcoRI ~~~RB S
s t
V
A
20 kb
A
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r -
~~~~~~~~~~~~~~~~~~A NM 1274
e=,
=
1275 ~~~~~~~~~~~~~~~~~ANM
- >*--570 bp -
