RRC ID 28662
Author Fukuda H, Terashima M, Koshikawa M, Kanegae Y, Saito I.
Title Possible mechanism of adenovirus generation from a cloned viral genome tagged with nucleotides at its ends.
Journal Microbiol Immunol
Abstract The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi-quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12-kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal-protein-deoxycytidine complex enters from the enzyme-cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double-stranded DNA, can be activated by two cycles of one-directional full-length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only Pac I but also Bst BI for construction of an adenovirus vector, without reducing construction efficiency.
Volume 50(8)
Pages 643-54
Published 2006-1-1
DOI 10.1111/j.1348-0421.2006.tb03829.x
PII JST.JSTAGE/mandi/50.643
PMID 16924150
MeSH Adenoviruses, Human / genetics* Adenoviruses, Human / physiology* Cell Line Cosmids DNA Replication / genetics DNA Restriction Enzymes / metabolism DNA, Viral / genetics Genetic Vectors Genome, Viral* Green Fluorescent Proteins Humans Oligonucleotides / metabolism Replication Origin Transfection Virus Replication / genetics
IF 1.566
Times Cited 18
DNA material pAxcwit (RDB03120) pAxCAwtit (RDB03121) pAxCAiLacZit (RDB03380) pAxcwit2 (RDB05212) pAxCAwtit2 (RDB05213) pAxCALNLwtit2 (RDB05214) pAxEFwtit2 (RDB05215)