Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

Abstract

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of sitespecific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.

Gene disruption by transformation in Neurospora crassa.

“…An unexpected feature of Neurospora transformation is the high degree of nonhomologous integration and the fact that many transformants integrated more than one copy of the transforming DNA. With mammalian cells, site-specific integrations are rarely encountered (18,21). In yeast cells, homologous integration is virtually the rule (14).…”

“…In yeast cells, integration appears to be strictly dependent on interaction of transforming DNA with homologous sequences (14). In mammalian cells, by contrast, integration of a particular gene can occur at many sites, and even increasing the target size does not enhance homologous recombination (e.g., by placing a ribosomal DNA repeated sequence on a plasmid) (18,21). In N. crassa, both homologous and nonhomologous recombination events seem to occur during transformation.…”

UV stimulation of DNA-mediated transformation of human cells.

“…The lack of site specificity-even when ribosomal DNA segments, which have ca. 200 homologous gene copies in the mammalian genome (42), are transfected-strongly argues against homologous recombination as a predominant mode of integration. In our experiments the amount of homology between dominant marker and host genome has no effect on the level of UV stimulation of the transformation process.…”

Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences.

“…Although gene transfer procedures have been used to demonstrate that homologous recombination can occur efficiently between two DNAs (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) introduced at the same time into cells, recombination between added DNA and homologous chromosomal sequence has not been detected (25). The occurrence of such events, however, has been postulated to explain results obtained with the class I genes of the mouse histocompatibility complex (26).…”