2 resultados para reverse genetics
em DRUM (Digital Repository at the University of Maryland)
Resumo:
Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+. HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions.
Resumo:
During ecological speciation, divergent natural selection drives evolution of ecological specialization and genetic differentiation of populations on alternate environments. Populations diverging onto the same alternate environments may be geographically widespread, so that divergence may occur at an array of locations simultaneously. Spatial variation in the process of divergence may produce a pattern of differences in divergence among locations called the Geographic Mosaic of Divergence. Diverging populations may vary in their degree of genetic differentiation and ecological specialization among locations. My dissertation examines the pattern and evolutionary processes of divergence in pea aphids (Acyrthosiphon pisum) on alfalfa (Medicago sativa) and clover (Trifolium pretense). In Chapter One, I examined differences among North American aphid populations in genetic differentiation at nuclear, sequence-based markers and in ecological specialization, measured as aphid fecundity on each host plant. In the East, aphids showed high host-plant associated ecological specialization and high genetic differentiation. In the West, aphids from clover were genetically indistinguishable from aphids on alfalfa, and aphids from clover were less specialized. Thus, the pattern of divergence differed among locations, suggesting a Geographic Mosaic of Divergence. In Chapter Two, I examined genomic heterogeneity in divergence in aphids on alfalfa and clover across North America using amplified fragment length polymorphisms (AFLPs). The degree of genetic differentiation varied greatly among markers, suggesting that divergent natural selection drives aphid divergence in all geographic locations. Three of the same genetic markers were identified as evolving under divergent selection in the eastern and western regions, and additional divergent markers were identified in the East. In Chapter Three, I investigated population structure of aphids in North America, France, and Sweden using AFLPs. Aphids on the same host plant were genetically similar across many parts of their range, so the evolution of host plant specialization does not appear to have occurred independently in every location. While aphids on alfalfa and clover were genetically differentiated in most locations, aphids from alfalfa and clover were genetically similar in both western North America and Sweden. High gene flow from alfalfa onto clover may constrain divergence in these locations.