The isolation of restriction fragments containing the att site of bacteriophage lambda
Marini, J.C.; Landy, A.
Virology 76(1): 196-209
1977
ISSN/ISBN: 0042-6822
PMID: 835230
DOI: 10.1016/0042-6822(77)90296-3
Accession: 006707290
Deletion mutants of bacteriophage .lambda. originating at the att site (POP') and extending to the right (PO.DELTA.') or to the left (.DELTA.OP') were used to identify a DNA restriction fragment which contains the phage att site. A single .lambda. Hind restriction fragment [generated by endo R.cntdot.Hind II + III from Haemophilus influenzae] was missing from the gel electrophoresis profiles of both classes of deletion mutants. This att-containing fragment is .apprx. 1370 base pairs in length. Smaller att-containing fragments were obtained by carrying out secondary digests of the purified Hind fragment with endo R.cntdot.Hae III [from H. aegypticus] or with endo R.cntdot.Mbo II. To identify the secondary att-containing fragments, the Hae III digests of the Hind POP' fragment was compared with the Hae III digests of Hind fragments containing the partial att sites .DELTA.OP' or PO.DELTA.'; the Mbo II digest of the POP' Hind fragment was compared with the Mbo II digests of Hind fragments containing the hybrid att sites BOP' and POB' (isolated from gal- and bio-transducing phage, respectively). In each independent set of secondary digestions the POP' secondary fragment was unique to the profile of the Hind att fragment, since only this Hind fragment contains DNA sequences from both sides of the phage att site (POP'). The att site-containing fragment isolated from Hae digestion is 980 base pairs; the Mbo att site fragment is 380 base pairs in length and is contained entirely within the larger Hae fragment. These fragments can be isolated in sufficient quantity for biochemical studies on att site interactions and in sufficient purity for sequence analysis.