Cancer therapy has traditionally focused on eliminating fast-growing populations of cells. Yet, an increasing body of evidence suggests that small subpopulations of cancer cells can evade strong selective drug pressure by entering a ‘persister' state of negligible growth. This drug-tolerant state has been hypothesized to be part of an initial strategy towards eventual acquisition of bona fide drug-resistance mechanisms. However, the diversity of drug-resistance mechanisms that can expand from a persister bottleneck is unknown. Here we compare persister-derived, erlotinib-resistant colonies that arose from a single, EGFR-addicted lung cancer cell. We find, using a combination of large-scale drug screening and whole-exome sequencing, that our erlotinib-resistant colonies acquired diverse resistance mechanisms, including the most commonly observed clinical resistance mechanisms. Thus, the drug-tolerant persister state does not limit—and may even provide a latent reservoir of cells for—the emergence of heterogeneous drug-resistance mechanisms.

DNA microarrays have been widely used in gene expression analysis of biological processes. Due to a lack of sequence information, the applications have been largely restricted to humans and a few model organisms. Presented within this study are results of the cross-species hybridization with Affymetrix human high-density oligonucleotide arrays or GeneChip using distantly related mammalian species; cattle, pig and dog. Based on the unique feature of the Affymetrix GeneChip where every gene is represented by multiple probes, we hypothesized that sequence conservation within mammals is high enough to generate sufficient signals from some of the probes for expression analysis. We demonstrated that while overall hybridization signals are low for cross-species hybridization, a few probes of most genes still generated signals equivalent to the same-species hybridization. By masking the poorly hybridized probes electronically, the remaining probes provided reliable data for gene expression analysis. We developed an algorithm to select the reliable probes for analysis utilizing the match/mismatch feature of GeneChip. When comparing gene expression between two tissues using the selected probes, we found a linear correlation between the cross-species and same-species hybridization. In addition, we validated cross-species hybridization results by quantitative PCR using randomly selected genes. The method shown herein could be applied to both plant and animal research.

We recently published the precise chromosomal localization on chromosome 16p13.1 of the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Here we report the identification of mutations in the gene encoding the transmembrane transporter protein, ABC-C6 (also known as MRP-6), one of the four genes located in the region of linkage, as cause of the disease. Sequence analysis in four independent consanguineous families from Switzerland, Mexico, and South Africa and in one non-consanguineous family from the United States demonstrated several different mis-sense mutations to cosegregate with the disease phenotype. These findings are consistent with the conclusion that PXE is a recessive disorder that displays allelic heterogeneity, which may explain the considerable phenotypic variance characteristic of the disorder.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.DOI: http://dx.doi.org/10.7554/eLife.15802.001