Selective deletion of large segments of Balbiani ring DNA during molecular cloning

Transcription units in Balbiani ring 1 (BR1) and Balbiani ring 2 (BR2) of Chironomus tenans salivary glands are comprised of about 40 kb of repetitive DNA sequences organized in a satellite-like array. Because of this sequence organization, it was possible to prepare 30 to 40-kb target DNA fragments for cloning by performing limit restriction endonuclease digestion of high-Mr genomic DNA. These fragments were ligated to cohesive termini of the linearized cosmid, pHC79, packaged in vitro, and used to transduce Escherichia coli. Alternatively, target fragments were randomly sheared to a mean length of 8-10 kb, annealed to plasmid pBR322 using homopolymeric tails, and used for bacterial transformation. Recombinant cosmids and plasmids generally contained inserts which were proportional to the length of target fragments used in ligation reactions. However, the subset of recombinants that hybridized to 32P-labeled 75S RNA (highly enriched in BR1 + BR2 sequences) had disproportionately smaller inserts. With the exception of one metastable clone with a 2.1-kb insert, all others had inserts of 0.8 or 0.4 kb. Similar results were obtained in host cells that were recA- or recBC-. The most likely conclusion is that repetitive BR sequences are highly unstable during replication in E. coli and are selectively deleted.