BACKGROUND: Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory. RESULTS: The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana. CONCLUSION: We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.

Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.

Tissue macrophages are an important cellular reservoir for replication of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus. In vitro, the ability of macrophages to support viral replication is differentiation dependent in that precursor monocytes are refractory to infection. There is, however, no consensus as to the exact point at which infection is restricted in monocytes. We have revisited this issue and have compared the efficiencies of early HIV-1 replication events in monocytes and in differentiated macrophages. Although virus entry in monocytes was comparable to that in differentiated macrophages, synthesis of full-length viral cDNAs was very inefficient. Relative to differentiated macrophages, monocytes contained low levels of dTTP due to low thymidine phosphorylase activity. Exogenous addition of D-thymidine increased dTTP levels to that in differentiated macrophages but did not correct the reverse transcription defect. These results point to a restriction in monocytes that is independent of reverse transcription precursors and suggest that differentiation-dependent cellular cofactors of reverse transcription are rate limiting in monocytes.