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Summary Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3β-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5–21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.

Three versions of a commercial human immunodeficiency virus (HIV) type 1 (HIV-1) load test (the AMPLICOR HIV-1 MONITOR Test versions 1.0, 1.0+, and 1.5; Roche Diagnostics, Branchburg, N.J.) were evaluated for their ability to detect and quantify HIV-1 RNA of different genetic subtypes. Plasma samples from 96 patients infected with various subtypes of HIV-1 (55 patients infected with subtype A, 9 with subtype B, 21 with subtype C, 2 with subtype D, 7 with subtype E, and 2 with subtype G) and cultured virus from 29 HIV-1 reference strains (3 of subtype A, 6 of subtype B, 5 of subtype C, 3 of subtype D, 8 of subtype E, 3 of subtype F, and 1 of subtype G) were tested. Detection of subtypes A and E was significantly improved with versions 1.0+ and 1.5 compared to that with version 1.0, whereas detection of subtypes B, C, D, and G was equivalent with the three versions. Versions 1.0, 1.0+, and 1.5 detected 65, 98, and 100% of the subtype A-infected samples from patients, respectively, and 71, 100, and 100% of the subtype E-infected samples from patients, respectively. Version 1.5 yielded a significant increase in viral load for samples infected with subtypes A and E (greater than 1 log 10 HIV RNA copies/ml). For samples infected with subtype B, C, and D and tested with version 1.5, only a slight increase in viral load was observed (10 ). We also evaluated a prototype automated version of the test that uses the same PCR primers as version 1.5. The results with the prototype automated test were highly correlated with those of the version 1.5 test for all subtypes, but were lower overall. The AMPLICOR HIV-1 MONITOR Test, version 1.5, yielded accurate measurement of the HIV load for all HIV-1 subtypes tested, which should allow the test to be used to assess disease prognosis and response to antiretroviral treatment in patients infected with a group M HIV-1 subtype.

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.

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.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells(1,2). In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA)(3). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

We recently reported a high divergence among African subtype F strains. Three well-separated groups (F1, F2, and F3) have been shown based on the phylogenetic analysis of the p24 gag and envelope sequences with genetic distances similar to those observed for known subtypes. In this study, we characterized the near-full-length genomes of two strains from epidemiological unlinked individual belonging to each of the subgroups : F1 (96FR-MP411), F2 (95CM-MP255 and 95CM-MP257), and F3 (96CM-MP535 and 97ZR-EQTB11). Phylogenetic analysis of the near-full-length sequences and for each of the genes separately showed the same three groups, supported by high bootstrap values. Diversity plotting, BLAST subtyping, and bootstrap plotting confirmed that the divergent F strains correspond to nonrecombinant viruses. The divergence between F1 and F2 is consistently lower than that seen in any other intersubtype comparison, with the exception of subtypes B and D. Based on all the different analyses, we propse to divide subtype F into two subclades, with F1 gathering the known subtype strains from Brazil and Finland, and our African strain (96FR-MP411), and F2 containing the 95CM-MP255 and 95CM-MP257 strains from Cameroon. The F3 strains, 97ZR-EQTB11 from the Democratic Republic of Congo and 96CM-MP535 from Cameroon, meet the criteria of a new subtype designated as K. The equidistance of subtype K to the other subtypes of HIV-1 suggests that this subtype existed as long as the others, the lower distance between B and D, and between F1 and F2 suggest a more recent subdivision for these latter strains. (Résumé d'auteur)

In this paper, we studied the variability of HIV-1 subtype F strains in Africa. For 11 viruses, mainly of Central African origin, different parts of the genome were genetically characterized. For all strains the V3–V5 region of the envelope gene was sequenced, and for 7 strains, the entire envelope gene was studied. For 10 strains, the p24 region of the gag gene was also sequenced. For each region studied, three subgroups in the F subtype were identified, F1, F2, and F3. These three subgroups were supported by high bootstrap values and the intra- and inter-subgroup F distances were comparable to those obtained for the known subtypes A, B, C, D, E, G, and H. In subgroup F1, some African strains clustered with previously described strains from Brazil and Romania, suggesting an African origin of the HIV-1 epidemic in these countries. A more detailed analysis of the gag and the envelope sequences allowed the identification of four recombinant viruses. Our data show a high diversity among subtype F strains, suggesting the presence of new subtypes in the regions studied. If biological differences exist among subtypes, it is important that these subtypes be well defined. The data from our study show that there is a need to clearly identify the different subgroups within the F subtype.

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.