Construction of a cloned library of adenovirus DNA fragments in bacteriophage M13.

F A Eggerding,W C Pierce

doi:10.1016/s0021-9258(17)44609-6

Abstract

The construction of recombinant M13 phages containing adenovirus DNA inserts was undertaken to provide strand-specific hybridization probes for analyses of adenovirus type 2 RNA transcripts. A library of molecular probes was constructed by cloning restriction endonuclease fragments of adenovirus types 2 and 5 DNA in the duplex replicative form DNA of the single-stranded bacteriophage vectors, M13mp7, M13mp8, and M13mp9 (Messing, J., and Vieira, J. (1982) Gene 19,269-276). Adenovirus DNA segments from early, intermediate, and late gene regions, accounting for at least 95% of the adenovirus chromosome, have been cloned in both possible orientations using these M13 derivatives as vectors. DNA cloned into these vectors can readily be obtained in a circular single-stranded form directly from mature phage particles. The cloned DNA fragments have been oriented and further characterized by restriction endonuclease mapping and hybridization with 32P-labeled adenovirus DNA. The polarity and fidelity of the adenovirus DNA in the recombinant phages has been confirmed by hybridization with labeled adenovirus 2 early and late mRNA. Restriction endonuclease analyses of M13 clones containing adenovirus DNA inserts spanning genome coordinates 31.7-56.9 have indicated that the relative locations of some restriction coordinates located within this region do not correspond to the mapped restriction sites in the DNA of adenovirus 2. Potential uses for these M13 clones in studies of adenovirus gene expression are discussed.

Highlights

Further characterizedby restriction endonuclease In contrast to the detailed structural characterization of mapping and hybridization with 32P-labeled adenovi- adenoviralcytoplasmicmRNAspecies,less information is rus DNA
The polarity and fidelity of the adenovirus available on how the mRNA precursorsof these messages are DNA in the recombinant phages bheaesn confirmed by synthesized in thenucleus, processed, and transported to the hybridization withlabeled adenovirus 2 early and late cytoplasm. the Ad2 genome, like that of other mRNA
Relative locations of some restriction coordinates lo- Initial viral transcripts are modified by processing mechacatedwithinthisregion do notcorrespondtothe nisms that include addition of 5’ cap structures, 3’ polyademapped restriction sites in the DNA of adenovirus 2 . nylation,methylation of internaladenosine residues, and Potential uses for these M13 clones in studies of ade- splicing to remove intervening sequences [10,30,31,32]

Summary

Introduction

Further characterizedby restriction endonuclease In contrast to the detailed structural characterization of mapping and hybridization with 32P-labeled adenovi- adenoviralcytoplasmicmRNAspecies,less information is rus DNA. 2-4 mg of single-stranded phage DNA containing a cloned tional early transcription units, designated ElA, ElB, E2A, adenovirusinsertcan be obtained from 1 liter of infected E3, and E4, it hansow been demonstrated that themajor late cells.

Results

Conclusion