Rescue of Functional Replication Origins from Embedded Configurations in a Pl...

青花瓷

Rescue of Functional Replication Origins from Embedded Configurations in a Plasmid Carrying the Adenovirus Genome

青花瓷

研究对象：缺乏EcoRI 酶切位点的一个5型腺病毒突变毒株。实验内容：基因组序列插入到质粒中。u'an
Closed circular forms of the recombinant viral
genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome
released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus
at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the
parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments
demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small
fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to Clal-linearized pXAd,
virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the
wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right
end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the
analyses reported here is that a free end is a necessary configuration for the sequences which make up the
adenovirus origin of replication.