On the structure, genesis and significance of DNA duplications at the Rous sarcoma proviral insertion sites in Rat-1 cells

Abstract

We have previously shown that most Rous sarcoma proviruses in Rat-1 cells are simple insertions, at apparently random sites, and are not transcribed. A small minority of simple insertions are transcribed and these show a bias to insertion at sites closely 3' to presumptive cellular CG-rich islands. However, most transcribed proviruses are complex, typified by contiguous duplications of proviral DNA 5' to a complete proviral unit. The cellular sites of these complex insertions are unknown and their structure and significance incompletely documented. We report here more extensive analyses of proviral duplications, with the following findings. The 5' duplications predominantly involve proviral regions known to contain enhancer functions, substantiating earlier data. The cellular insertion sites are not biased to CG-rich loci at the level displayed by simple transcribed proviruses. The detailed structure of two duplications, and partial analysis of several others, strongly favours their genesis by illegitimate template transfer at reverse transcription, followed by self-recombination. These findings show that aberrant reverse transcription can generate duplications that dispense with the need for an expressed provirus to be integrated at a favourable cellular site.

A Novel, Immortal, and Multipotent Human Neural Stem Cell Line Generating Functional Neurons and Oligodendrocytes

“…Here, we show that a spontaneous and significant downregulation of v‐myc expression occurs in IhNSC in concomitance with mitogen removal and the onset of cell differentiation. Although inactivation of the Mo‐MLV promoter‐enhancer and of other endogenous proviruses has been previously documented, both ex vivo and in vivo [41, , , , , , , –49], the cellular and molecular mechanisms that underlie this phenomenon in our system remain to be elucidated. In view of the fact that we are analyzing a mixed, bulk population, an effect related to the integration site [44, 46, 50, 51] on v‐myc downregulation is most unlikely.…”

Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses

“…Other examples include endogenous proviruses of mice (2, 17, 29) and chicks (6) that are transcriptionally silent but, once activated, are fully replication competent. Another model for provirus inactivation is based on Rous sarcoma virus in rat fibroblasts, in which the vast majority of integrated proviruses are silent (12,13), although the Rous sarcoma virus promoter-enhancer supports transient expression in rat fibroblasts.…”

“…(16,32). While the use of cell clones selected for LTR-driven expression may preclude analysis of the full range of vector expression levels (13,27), two-promoter vectors may introduce problems associated with promoter interference (10,11).…”