Notes on cloning maize DNA

The lambda-1059 strain (J. Karn et al., PNAS 77:5172-5176, 1980) and its more recent EMBL derivatives (A.-M. Frischauf, H. Lehrach, A.-M. Poustka and N. Murray, in preparation) are in widespread use as cloning vectors. The E. coli Q358 strain (r-k, m+k, Su+II, 80R) is generally used as the permissive host for phage propagation, and the P2 lysogens Q359 (r-k, m+k, Su+II, 80R, P2) or Q364 (r-k, m+k, Su+II, delta-lac-pro, P2) are used as the selective hosts for detection of recombinant phage (J. Karn et al., 1980). I have consistently found that phage constructed in vitro using purified EcoRI-cut arms of the EMBL4 vector, and either total EcoRI-cut maize DNA or maize EcoRI fragments in the 15-25 kb size range, plate much less efficiently on Q358 or Q364 than on other E. coli strains permissive for lambda replication. The data in Table 1 show the results of such experiments, using the Q358, Q364 and K803 (strain LE392 [F-, hsdR514 (r-k, m-k), supE44, supF58, lacY1, galK2, galT22, metB1, trpR55]; T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982) strains as host bacteria. Although the number of p.f.u.'s is smaller when a total EcoRI digest of maize DNA is ligated into the EMBL4 vector than when 15-25 kb fragments of maize DNA are used, it is consistently observed that the plating efficiency of the newly-packaged phage containing maize DNA is higher on the K803 strain by a factor of 13-18 than it is on the Q358 strain. This is not true of either the parental EMBL4 phage DNA, EMBL4 phage DNA that has been cut with EcoRI and religated, or recombinant phage constructed using Caenorhabditis eleqans DNA (a gift of D. Burke). Phage containing these DNAs plate with approximately equal efficiency on both strains. The plating efficiency of maize DNA-containing phage is slightly higher on the selective host Q364 than on Q358, while the plating efficiency of phage containing C. elegans DNA is somewhat lower on the selective host (Table 1; D. Burke, personal communication).

To determine whether the inability to form plaques on Q358 is an inherent property of recombinant phage containing maize DNA, recombinant phage were propagated on K803 and tested for their ability to grow on the various strains. The results are shown in Table 2 and indicate that once the maize DNA has been "laundered" through E. coli, the recombinant phage grow equally well on Q358, Q364 and K803. Since plant DNA's are known to be more extensively modified than other DNA's, it appears a reasonable conjecture that the difference in plating efficiency is attributable to differences among E. coli strains in the ability to replicate heavily modified DNAs.



Nina Fedoroff

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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