Document 0718
 DOCN  M9590718
 TI    Specificity of viral DNA integration in vitro and in vivo.
 DT    9509
 AU    Coffin J; Withers-Ward E; Barnes J; Kitamura Y; Tufts University School
       of Medicine. Department of Molecular; Biology and Microbiology, Boston,
       MA
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session I, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920001
 AB    To understand the role of DNA sequence and structure in targeting
       integration to specific sites or regions of the genome, we have
       developed a highly sensitive PCR-based assay capable of detecting with
       single-base resolution, a single molecule of proviral DNA into the
       cellular genome in 10-6 cells. Using this assay we have investigated the
       use of simple plasmid DNA targets for integration in vitro and natural
       or inserted DNA sequences as integration targets in infected cells. In
       vitro, we found that integration targets are distributed across the
       target regions examined, but highly non-randomly. The pattern of use of
       specific regions seems to be related to local DNA structure more than
       sequence, and is independent of the broader context of the target
       sequence. Specificity can be modified by methylation of C residues in
       alternating G-C regions and as few as 2 methyl groups in (CG)3 are
       sufficient to create a strong target site. In vivo, we have found that
       the majority of integration events are widely distributed in the cell
       genome. Comparison of the usage as integration targets of 8 randomly
       chosen regions with 4 regions preselected by virtue of prior integration
       events indicates little or no regional bias or avoidance of specific
       sequences. As with in vitro integration, there is a highly non-random
       pattern of local preference and aversion for specific sites, with some
       sites being used more than 100-fold greater than expected from a random
       distribution, and others, presumably, correspondingly less. Similar but
       much more intense patterns can be seen with multicopy DNA targets
       suggesting that all or most copies have the same specificity, despite
       potential differences in location. Finally, sequences introduced by
       transfection also serve as integration targets as efficiently as
       endogenous sequences, and display similar patterns of targeting in vitro
       and in vivo. We are currently developing this system to examine directly
       effects of transfection factor binding and transcription per se on the
       selection of integration targets.
 DE    Binding Sites  Cytosine/METABOLISM  DNA, Viral/*GENETICS/METABOLISM
       Genome, Viral  Guanine/METABOLISM  Methylation  Plasmids  Polymerase
       Chain Reaction  Proviruses/*GENETICS  Transcription, Genetic  Virus
       Integration/*GENETICS  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).