Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone.
A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the YAC. The application of powerful yeast genetic techniques to an infectious adenovirus clone promises to significantly enhance the genetic analysis of adenovirus and to simplify the construction of adenovirus-based vectors for vaccines or for gene transfer to mammalian cells or whole animals. The adenovirus YAC was produced by homologous recombination in vivo between adenovirus 2 virion DNA and YAC vector plasmids carrying segments of the viral left and right genomic termini. This recombinational cloning strategy is generally applicable to the construction of YACs containing other DNA segments, such as the genomes of other viruses. Further, it is very efficient and may permit the targeted cloning of segments of the genomes of higher organisms directly from genomic DNA.
Ketner
G
G
Department of Immunology and Infectious Diseases, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205.
Spencer
F
F
Tugendreich
S
S
Connelly
C
C
Hieter
P
P
D000260:Adenoviruses, Human / Q000235:genetics*; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D018797:Cell Cycle Proteins; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577355:CDC27 protein, S cerevisiae; C577366:CDC27 protein, human; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004279:DNA, Viral; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases