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Mutation D30N Is Not Preferentially Selected by Human Immunodeficiency Virus Type 1 Subtype C in the Development of Resistance to Nelfinavir. Zehava Grossman, 2004.Differences in baseline polymorphisms between subtypes may result in development of diverse mutational pathways during antiretroviral treatment . We compared drug resistance in patients with human immunodeficiency virus subtype C (referred to herein as "subtype-C-infected patients") versus subtype-B-infected patients following protease inhibitor (PI) therapy . Genotype, phenotype, and replication capacity (Phenosense; Virologic) were determined . We evaluated 159 subtype-C- and 65 subtype-B-infected patients failing first PI treatment . Following nelfinavir treatment, the unique nelfinavir mutation D30N was substantially less frequent in C (7%) than in B (23%; P = 0.03) while L90M was similar (P < 0.5) . Significant differences were found in the rates of M36I (98 and 36%), L63P (35 and 59%), A71V (3 and 32%), V77I (0 and 36%), and I93L (91 and 32%) (0.0001 < P < 0.05) in C and B, respectively . Other mutations were L10I/V, K20R, M46I, V82A/I, I84V, N88D, and N88S . Subtype C samples with mutation D30N showed a 50% inhibitory concentration (IC50) change in susceptibility to nelfinavir only . Other mutations increased IC50 correlates to all PIs . Following accumulation of mutations, replication capacity of the C virus was reduced from 43% ± 22% to 22% ± 15% (P = 0.04) . We confirmed the selective nature of the D30N mutation in C, and the broader cross-resistance of other common protease inhibitor mutations . The rates at which these mutational pathways develop differ in C and subtype-B-infected patients failing therapy, possibly due to the differential impact of baseline polymorphisms . Because mutation D30N is not preferentially selected in nelfinavir-treated subtype-C-infected patients, as it is in those infected with subtype B, the consideration of using this drug initially to preserve future protease inhibitor options is less relevant for subtype-C-infected patients . Identification of an Unknown Promoter, OUTIIp, within the IS10R Element. Esteban Martínez-García, 2003.A novel promoter in IS10R (OUTIIp) has been found in one of its ends in an inverted position relative to promoter pOUT . OUTIIp shows characteristics similar to those of rpoS-dependent promoters such as a gearbox expression pattern . It is under catabolite repression and positively regulated by ppGpp or conditioned media . This opens new challenges in IS10R transposition . Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha. So-Young Kim, 2003.A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution . An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha . Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system . A telomere-originated autonomously replicating sequence (ARS) of H . polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes . In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids . Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants . This newly developed in vivo recombination system of H . polymorpha provides a suitable library for activity-based selection of mutants after directed evolution .
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