PMID- 19858479
OWN - NLM
STAT- MEDLINE
DCOM- 20100106
LR  - 20211020
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Print)
IS  - 0027-8424 (Linking)
VI  - 106
IP  - 45
DP  - 2009 Nov 10
TI  - Cloning human herpes virus 6A genome into bacterial artificial chromosomes and 
      study of DNA replication intermediates.
PG  - 19138-43
LID - 10.1073/pnas.0908504106 [doi]
AB  - Cloning of large viral genomes into bacterial artificial chromosomes (BACs) 
      facilitates analyses of viral functions and molecular mutagenesis. Previous 
      derivations of viral BACs involved laborious recombinations within infected 
      cells. We describe a single-step production of viral BACs by direct cloning of 
      unit length genomes, derived from circular or head-to-tail concatemeric DNA 
      replication intermediates. The BAC cloning is independent of intracellular 
      recombinations and DNA packaging constraints. We introduced the 160-kb human 
      herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative 
      intermediates with the Sfil enzyme that cleaves the viral genome in a single 
      site. The recombinant BACs contained also the puromycin selection gene, GFP, and 
      LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained 
      stably in puromycin selected 293T cells. In the presence of irradiated helper 
      virus, supplying most likely proteins enhancing gene expression they expressed 
      early and late genes in SupT1 T cells. The method is especially attractive for 
      viruses that replicate inefficiently and for viruses propagated in suspension 
      cells. We have used the fact that the BAC cloning "freezes" the viral DNA 
      replication intermediates to analyze their structure. The results revealed that 
      HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, 
      predicted to arise by circularization of parental genomes with a DR at each 
      terminus. HHV-6A DNA molecules prepared from the infected cells also contained 
      DNA molecules with a single DR. Such forms were not previously described for 
      HHV-6 DNA.
FAU - Borenstein, Ronen
AU  - Borenstein R
AD  - The S. Daniel Abraham Institute for Molecular Virology and The Department of Cell 
      Research and Immunology, Tel Aviv University, Tel Aviv 66978, Israel.
FAU - Frenkel, Niza
AU  - Frenkel N
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20091026
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
SB  - IM
MH  - Blotting, Southern
MH  - Cell Line
MH  - Chromosomes, Artificial, Bacterial/genetics
MH  - Cloning, Molecular/*methods
MH  - DNA Replication/*genetics
MH  - Electrophoresis, Gel, Pulsed-Field
MH  - Genome, Viral/*genetics
MH  - Herpesvirus 6, Human/*genetics
MH  - Humans
MH  - Microscopy, Fluorescence
PMC - PMC2767366
COIS- The authors declare no conflict of interest.
EDAT- 2009/10/28 06:00
MHDA- 2010/01/07 06:00
PMCR- 2009/10/26
CRDT- 2009/10/28 06:00
PHST- 2009/10/28 06:00 [entrez]
PHST- 2009/10/28 06:00 [pubmed]
PHST- 2010/01/07 06:00 [medline]
PHST- 2009/10/26 00:00 [pmc-release]
AID - 0908504106 [pii]
AID - 0169 [pii]
AID - 10.1073/pnas.0908504106 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19138-43. doi: 
      10.1073/pnas.0908504106. Epub 2009 Oct 26.