Despite over 10,000 papers in the literature using transient transfection, the location of introduced plasmid DNA has virtually not been addressed. By fluorescence in situ hybridization, we find that transiently transfected plasmids carrying like kinds of transcriptional control regions form self-associating assemblies that localize distinctly. rRNA gene (pol-I) promoter plasmids localize in nucleoli abutting the cell's rRNA genes, and 5S RNA gene (pol-III) promoter plasmids localize at peri-nucleolar sites abutting the cell's 5S RNA genes. Plasmids bearing various transcribed segments or no eukaryotic insert do not localize in this manner, and localization appears affected at the individual plasmid level, dependent on regions where transcription factors bind, not on ability to actually direct transcription. Similarly transfected plasmids bearing pol-II promoter regions also exhibit specific self-associations, which are specific for different pol-II promoters. Importantly, plasmids bearing the promoter region from a MHC class I gene or a MMTV-LCR transgene array preferentially localize with the chromosomal copies of those sequences. Such specific self-association of promoter regions was not expected. These findings seem to synergize with a rapidly expanding body of exciting research on sub-nuclear organization, showing preferential locations of certain chromosomal gene regions, frequently related to transcriptional regulation (1-10), including MHC (11) and other genes important in the immune system (12-14) as well as many other gene regions such as ?-globin (e.g., 15-21). We hypothesize that the self-association and localization seen with transfected plasmids reflect forces that act on chromosomal genes, and thus that the plasmid approach could provide a manipulatable system to complement the study of localization by chromosomal regions.
We aim to learn more about localization directed by the MHC class I promoter region, including identifying which sequence elements mediate the effect, whether they correlate with transcription factor binding sites, and a peri-nucleolar association we have observed. We will also endeavor to extend the plasmid localization system to unique genes, examining genetic elements from the ?-globin locus, one of the best-studied chromosomal regions. Should small plasmids reproduce localization effects shown for this large chromosomal locus, we will examine what sequence elements are needed for the promoter-LCR interaction. Through these studies we anticipate to gain a better understanding of localizing effects that can act on pol-II promoters and hopefully provide a convenient way to study some of the associative forces that act on the cell's chromosomal DNA. ? ? ?
