Five point mutations in a particular beta-lactamase allele jointly increase bacterial resistance to a clinically important antibiotic by a factor of approximately 100,000. In principle, evolution to this high-resistance beta-lactamase might follow any of the 120 mutational trajectories linking these alleles. However, we demonstrate that 102 trajectories are inaccessible to Darwinian selection and that many of the remaining trajectories have negligible probabilities of realization, because four of these five mutations fail to increase drug resistance in some combinations. Pervasive biophysical pleiotropy within the beta-lactamase seems to be responsible, and because such pleiotropy appears to be a general property of missense mutations, we conclude that much protein evolution will be similarly constrained. This implies that the protein tape of life may be largely reproducible and even predictable.

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Antibiotic resistance can evolve through sequential accumulation of multiple mutations1. To study such gradual evolution, we developed a selection device, the morbidostat, which continuously monitors bacterial growth and dynamically regulates drug concentrations such that the evolving population is constantly challenged. We analyzed evolutionary trajectories of Escherichia coli populations towards resistance to chloramphenicol, doxycycline, and trimethoprim. Over a period of ~20 days, resistance levels increased dramatically, with parallel populations showing similar phenotypic trajectories. Whole-genome sequencing revealed both drug-specific and drug-general genetic changes. Chloramphenicol and doxycycline resistance evolved through diverse combinations of mutations in genes involved in translation, transcription, and transport2. In contrast, trimethoprim resistance evolved in a stepwise manner1,3, through mutations restricted to the target enzyme dihydrofolate reductase (DHFR)4,5. Sequencing DHFR over time revealed that parallel populations not only evolved similar mutations, but also acquired them in similar order6. Uncovering such recurrent genotypic pathways may help the spread of resistance.

The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.chromosomal instability ͉ mitochondrion ͉ mutation rate ͉ mutational spectrum ͉ Saccharomyces cerevisiae D espite its relevance to every aspect of genetics and evolution, our understanding of the mutation process and its bearing on organismal fitness remains quite limited (1-4). Owing to the technical difficulties in directly observing very low-frequency events, most estimates of the per-nucleotide mutation rate are derived either from surveys of visible mutations at reporter loci (to enhance the detectability of mutations) or from nucleotide-sequence comparisons of silent sites in distantly related species (to magnify the accumulation of mutations). Neither approach is without problems, the first requiring assumptions about the fraction of mutations with observable phenotypic effects and the second relying on assumptions about interspecific divergence times, generation lengths, and neutrality of the monitored nucleotide sites.Long-term mutation-accumulation (MA) experiments, whereby replicate lines are taken through regular bottlenecks to minimize the efficiency of selection, have proven to be highly valuable resources for procuring spontaneous mutations in an essentially unbiased fashion (5-8). However, brute-force sequencing of PCR-amplified products constrains the number of mutations that can be detected in a reasonable amount of time. Here, we demonstrate the feasibility of whole-genome sequencing as a means to assay the complete spectrum of mutational effects in a moderately sized eukaryotic genome.Our analyses are based on an examination of parallel MA lines of a key model system, the yeast Saccharomyces cerevisiae. The initially isogenic lines were passed through 200 single-cell bottlenecks on a 3-to 4-day cycle of clonal growth for a total of Ϸ4,800 cell divisions per line [see supporting information (SI) Text]. Although there is some opportunity for the selective elimination of deleterious mutations during daily clonal amplification, this effect is quite small under the imposed bottlenecking procedure. For mutations with a relative selective disadvantage of s ϭ ...

Comparison of the gene-expression profiles between adults of Drosophila melanogaster and Drosophila simulans has uncovered the evolution of genes that exhibit sex-dependent regulation. Approximately half the genes showed differences in expression between the species, and among these, approximately 83% involved a gain, loss, increase, decrease, or reversal of sex-biased expression. Most of the interspecific differences in messenger RNA abundance affect male-biased genes. Genes that differ in expression between the species showed functional clustering only if they were sex-biased. Our results suggest that sex-dependent selection may drive changes in expression of many of the most rapidly evolving genes in the Drosophila transcriptome.