Switching an active site helix in dihydrofolate reductase reveals limits to subdomain modularity.

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study subdomain modularity by creating 20 chimeras of an enzyme, Escherichia coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR.

Relevance of Higher-Order Epistasis in Drug Resistance.

We studied five chemically distinct but related 1,3,5-triazine antifolates with regard to their effects on growth of a set of mutants in dihydrofolate reductase. The mutants comprise a combinatorially complete data set of all 16 possible combinations of four amino acid replacements associated with resistance to pyrimethamine in the malaria parasite Plasmodium falciparum. Pyrimethamine was a mainstay medication for malaria for many years, and it is still in use in intermittent treatment during pregnancy or as a partner drug in artemisinin combination therapy.

Biophysical principles predict fitness landscapes of drug resistance.

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency.

Accessible mutational trajectories for the evolution of pyrimethamine resistance in the malaria parasite Plasmodium vivax.

Antifolate antimalarials, such as pyrimethamine, have experienced a dramatic reduction in therapeutic efficacy as resistance has evolved in multiple malaria species. We present evidence from one such species, Plasmodium vivax, which has experienced sustained selection for pyrimethamine resistance at the dihydrofolate reductase (DHFR) locus since the 1970s. Using a transgenic Saccharomyces cerevisiae model expressing the P.

Compensatory mutations restore fitness during the evolution of dihydrofolate reductase.

Whether a trade-off exists between robustness and evolvability is an important issue for protein evolution. Although traditional viewpoints have assumed that existing functions must be compromised by the evolution of novel activities, recent research has suggested that existing phenotypes can be robust to the evolution of novel protein functions. Enzymes that are targets of antibiotics that are competitive inhibitors must evolve decreased drug affinity while maintaining their function and sustaining growth.

Stepwise acquisition of pyrimethamine resistance in the malaria parasite.

The spread of high-level pyrimethamine resistance in Africa threatens to curtail the therapeutic lifetime of antifolate antimalarials. We studied the possible evolutionary pathways in the evolution of pyrimethamine resistance using an approach in which all possible mutational intermediates were created by site-directed mutagenesis and assayed for their level of drug resistance. The coding sequence for dihydrofolate reductase (DHFR) from the malaria parasite Plasmodium falciparum was mutagenized, and tests were carried out in Escherichia coli under conditions in which the endogenous bacterial enzyme was selectively inhibited.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution.

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum.

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites.

Duplication, gene conversion, and genetic diversity in the species-specific acyl-CoA synthetase gene family of Plasmodium falciparum.

While genes encoding antigens and other highly polymorphic proteins are commonly found in subtelomeres, it is unusual to find a small family of housekeeping genes in these regions. We found that in the species Plasmodium falciparum only, a non-subtelomeric acyl-CoA synthetase (ACS) gene has expanded into a family of duplicated genes mainly located in the subtelomeres of the genome. We identified the putative parent of the duplicated family by analysis of synteny and phylogeny relative to other Plasmodium ACS genes.

Gene conversion as a source of nucleotide diversity in Plasmodium falciparum.

Examination of polymorphisms in the Plasmodium falciparum gene for falcipain 2 revealed that this gene is one of two paralogs separated by 10.8 kb in chromosome 11. We designate the annotated gene denoted chr11.

Patterns of insertion and deletion in contrasting chromatin domains.

Transposable elements (TEs) play a fundamental role in the evolution of genomes. In Drosophila they are disproportionately represented in regions of low recombination, such as in heterochromatin. This pattern has been attributed to selection against repeated elements in regions of normal recombination, owing to either (1) the slightly deleterious position effects of TE insertions near or into genes, or (2) strong selection against chromosomal abnormalities arising from ectopic exchange between TE repeats.

Unexpected stability of mariner transgenes in Drosophila.

A number of mariner transformation vectors based on the mauritiana subfamily of transposable elements were introduced into the genome of Drosophila melanogaster and examined for their ability to be mobilized by the mariner transposase. Simple insertion vectors were constructed from single mariner elements into which exogenous DNA ranging in size from 1.3 to 4.