Figure 5. Assays for preintegration complexes and purified integrase.

Figure 5

Assays for preintegration complexes and purified integrase. (A) Genetic assay for integration (Brown et al. 1987). Preintegration complexes are isolated from cells infected with an MLV strain carrying the E. coli SupF amber suppressor tRNA. Incubation of these complexes with genomic DNA from a λ strain carrying amber mutations (λgtWES) allows in vitro integration of the MLV supF provirus into the λ DNA. The MLV supF-containing recombinants can make plaques on a lawn of E. coli cells lacking an amber suppressor, which do not allow replication of λgtWES. Each plaque corresponds to an individual integration product. Variations on this genetic assay include the use of alternative genetic markers for the retroviral DNA (see, e.g., Grandgenett et al. 1993) and the use of preintegration complexes assembled in vitro using model, “mini-retroviral” genomes (Fujiwara and Craigie 1989; Vora et al. 1994). (B) Polymerase chain reaction (PCR) assay for mapping integration sites. Following integration in vivo or in vitro, the population of target DNA molecules can include proviruses integrated at diverse sites. Two oligonucleotide primers are used in a PCR to amplify DNA sequences representing the insertion sites, one complementary to a sequence near an end of the viral DNA (color) and the second complementary to a selected site in the target DNA molecule. The lengths of the resulting PCR products are determined by the distances between the two primers, which depend on the locations of integration sites relative to the target DNA sequence selected for the target-specific primer. One of the primers is radioactively labeled, allowing the resulting PCR products to be detected by autoradiography. The size and intensities of the product bands reflect the locations of the integration sites in the target DNA and the relative frequency with which they are used (Pryciak and Varmus 1992b; Kitamura et al. 1992). The example illustrated here shows an analysis of the distribution of sites in a minichromosome substrate used as targets for integration by MLV preintegration complexes in vitro. The two lanes on the left (M) show the distribution of integration events in the intact minichromosome, and the two lanes to their right (D) show the distribution of integration events into the same DNA as a naked DNA molecule. The vertical bars show the position of nucleosomes on the DNA substrate (see also Fig. 1). Assay for 3′-end processing (C) and integration (D) of synthetic oligonucleotide models of a viral DNA end. A duplex oligonucleotide with the sequence of an end of the unintegrated viral DNA molecule is labeled at the 5′-end of the strand representing the 3′-end of viral DNA (colored asterisk) (1). (C) The 3′--end processing reaction results in excision of two bases from the 3′-end of the labeled strand (2). (D) After 3′--end processing, the same oligonucleotide (2) can be integrated into a second oligonucleotide, producing products longer than the original labeled strand (3), as well as shorter products resulting from use of the radioactively labeled oligonucleotide as an integration target (4). The starting material (left lane), and the reaction products (right lane) can be resolved by gel electrophoresis. (E) Disintegration assay. Synthetic oligonucleotides are used to construct a substrate that mimics the product of an in vitro oligonucleotide integration reaction. The 5′-end of the target DNA strand that is interrupted by the integrated viral DNA is radioactively labeled (5). Reversal of the integration reaction results in excision of the duplex oligonucleotide that represents the processed viral DNA end and restoration of the continuity of the target DNA strand (6).

From: Biochemical Activities and Assays

Cover of Retroviruses
Coffin JM, Hughes SH, Varmus HE, editors.
Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.
Copyright © 1997, Cold Spring Harbor Laboratory Press.

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