Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

摘要

The manipulation of large (>10 kb) plasmid systems amplifies problems common totraditional cloning strategies. Unique or rare restriction enzyme recognition sequences areuncommon and very rarely located in opportunistic locations. Making site-specific deletions andinsertions in larger plasmids consequently leads to multiple step cloning strategies that are oftenlimited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies.Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids aresystems that typify such situations.Recombinational cloning techniques based on homologous recombination inSaccharomyces cerevisiae that circumvent many ofthese common problems have beendeveloped. However, these techniques are rarely realistic options for such large plasmid systemsdue to the above mentioned difficulties associated with the addition ofrequired yeast DNAreplication, partitioning and selectable marker sequences.To determine ifrecombinational cloning techniques could be modified to simplify themanipulation of such a large plasmid system, a recombinational cloning system for the creation ofhuman adenovirus EI-deletion rescue plasmids was developed.Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alonesufficient to foster successful recombinational cloning without additional partitioning sequences,using only slight modifications of existing protocols. In addition, we describe conditions forefficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2kb) and insertion of donor fragment DNA using only a single non-unique restriction site.The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinationalcloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative andpositive selection to reduce background transformants to undetectable levels.The modification of existing protocols as described in this report facilitates the use ofrecombinational cloning strategies that are otherwise difficult or impractical for use with largeplasmid systems. Improvement of general recombinational cloning strategies and strategiesspecific to the manipulation ofthe adenovirus genome are considered in light of data presentedherein.